Hepcidin inhibits in vitro erythroid colony formation at reduced erythropoietin concentrations

The anemia of chronic disease (ACD) is one of the most common clinical syndromes encountered in the practice of medicine.¹  This disorder typically manifests itself as a hypoproliferative anemia accompanied by a low serum iron concentration despite adequate reticuloendothelial iron stores. ACD has generally been considered to result from a combination of pathologic processes which can be linked to the cytokine mediators of inflammation. However, the diagnostic importance of altered iron metabolism in ACD has led many to consider that these features represent the dominant pathophysiologic contributor to this syndrome. Hepcidin is an antibacterial protein that is produced in the liver, circulated in the blood, and excreted in the urine. It is a type II acute-phase protein, the expression of which is induced by interleukin 6 (IL-6), and down-regulated by tumor necrosis factor.²  The specific iron regulatory functions of hepcidin, as well as the anemia syndromes observed in patients and transgenic animals with abnormalities of hepcidin gene expression,^3,4  have led many to consider that it is the key to unlocking the “mysteries” of ACD.^5,6 

However, the pathogenesis of ACD is not purely a matter of iron, but rather involves 3 major processes. A slight shortening of red cell survival creates a demand for a small increase in red cell production by the bone marrow.⁷  The marrow cannot respond adequately to this demand due to impaired erythropoiesis and impaired mobilization of reticuloendothelial system iron stores.⁷  The impairment of erythropoiesis, in turn, consists of both a blunted erythropoietin (Epo) response to anemia^8,9  and a decreased response of erythroid progenitors to Epo.^10-12  A potential role for hepcidin in the iron anomalies of ACD is well supported by data currently available. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. In this study, the effects of hepcidin on erythroid colony formation in vitro are examined.

Bone marrow aspirates were collected from paid healthy volunteers under protocols approved by the institutional review boards of the investigators' institutions. Informed consent was provided in accordance with the Declaration of Helsinki.

Marrow light density mononuclear (LDMN) cells were cultured for CFU-Es in 0.2 mL plasma clots as previously described,^11,12  with recombinant human (rh) Epo and 25 amino acid (aa) human hepcidin. After incubation for 7 days in a humidified 95% air/5% CO₂ incubator, clots were collected, fixed with 5% glutaraldehyde, and stained with hematoxylin-benzidine.¹³  CFU-Es were identified.¹⁴  In order to compare the results of different experiments, colony formation was expressed as a percentage of control (CFU-E colony formation at rhEpo 1 U/mL without added hepcidin).

Hepcidin was purchased from Alpha Diagnostics (San Antonio, TX). The preparation was reported by the manufacturer to be more than 95% pure. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) showed a major peak at 2790, consistent with the 25 aa hepcidin species, a shoulder at 2807 consistent with methionine oxidation of 25 aa hepcidin, and a lower molecular weight peak at approximately 2691, suggestive of loss of a single amino acid from 25 aa hepcidin. Biologic activity was confirmed by demonstrating that exposure to 100 ng/mL hepcidin for 24 hours decreased expression of ferroportin in marrow LDMN cells.

The possibility of nonspecific peptide effects on CFU-E colony formation or on phosphorylated Bad (pBad) expression by HCD57 cells was evaluated using aa 1-24 of human adrenocorticotropic hormone (ACTH(1-24)).

Cell proliferation was evaluated by cell counting; viability was determined by trypan blue exclusion. Cells were lysed in RIPA buffer and the protein extracted for Western blot analysis. Each lane on the gel had 6.54 μg total protein applied. Gels were probed with antibodies against Bad (CalBiochem, La Jolla, CA), phosphorylated Bad (pBad), and Bcl-x_L (Upstate Cell Signalling Solutions, Charlottesville, VA). Antiferroportin was purchased from Alpha Diagnostics, and was generated against a 19 aa ferroportin peptide.

At the standard in vitro rhEpo concentration used for CFU-E colony assays in vitro (1 U/mL), hepcidin had no significant effects on colony formation (Figure 1A). However, ACD is a syndrome associated with relative reductions in Epo concentration.^8,9  The effects of 100 ng/mL hepcidin on CFU-E colony formation were evaluated at lower rhEpo concentrations (0.1 U/mL-0.5 U/mL). Colony formation was significantly decreased in the presence of hepcidin (Figure 1B). This effect was not reversed by the addition of 500 μg/mL iron-saturated transferrin to the culture medium.

The regulatory pathways controlling apoptosis and proliferation of HCD57 erythroleukemia cells replicate those observed in primary human erythroid progenitors.^15-17  This cell line was studied in order to more fully define the effects of hepcidin on erythropoiesis in a uniform erythroid-cell population. HCD57 cells were incubated in Iscove-modified Dulbecco medium (IMDM) with 10% fetal calf serum (FCS) with 0.3 U/mL rhEpo at 37°C for 24 hours, with or without 100 ng/mL hepcidin. No difference in cell proliferation was seen.

Apoptosis in HCD57 cells is regulated by the Bad/Bcl-x_L pathway.¹⁷  Bad, pBad, and Bcl-x_L are also expressed by primary human CFU-Es.¹⁸  Total Bad and Bcl-x_L protein expression were unchanged by exposure to hepcidin; however, the proportion of the antiapoptotic protein pBad was decreased approximately 50% (Figure 2). CFU-E colony formation (as in Figure 1) reflects a combination of both differentiation and proliferation over a 7-day period. The demonstration of a proapoptotic effect of hepcidin does not rule out the possibility of a later antiproliferative hepcidin effect also contributing to the observed results.

There is considerable evidence to support the contention that hepcidin is a major, and possibly the major, contributor to the hypoferremia and increased reticuloendothelial iron stores associated with inflammation, particularly (though not exclusively) that associated with IL-6.^2,19  A recent report has demonstrated that hepcidin reduces macrophage iron efflux by binding to the iron transporter ferroportin, causing its internalization and subsequent degradation.²⁰  It is unlikely that the inhibition of CFU-E colony formation reported here results from this effect. Although marrow macrophages are present in the LDMN-cell population, and iron is required for erythropoiesis in vitro,²¹  the amount of iron-saturated transferrin contributed to the culture medium by FCS is comparable to the amount required for maximal CFU-E colony formation in serum-free medium in the absence of macrophages (300 μg/mL).²¹  In addition, supplementation of the culture medium by 500 μg/mL iron-saturated transferrin failed to reverse inhibition by hepcidin. The studies reported here do not disprove the possibility that hepcidin-induced impairment of iron flow to erythroid cells contributes to ACD; rather, they suggest an additional mechanism by which hepcidin can inhibit erythropoiesis.

It is important to recognize that not all of the pathogenetic processes for ACD are equally implicated in all clinical scenarios. In a study of anemic children with cancer, the Epo concentrations were appropriate; the major process in these patients was considered to be an impaired erythropoietic response to Epo.²²  A similar study in adult patients with lung cancer found that the primary contributor to anemia was impaired erythropoiesis, but also observed a blunted Epo response.²³  In contrast, it has been suggested that inavailability of iron is the dominant factor in the anemia of juvenile rheumatoid arthritis.²⁴  A complete picture of the contribution of hepcidin or any other mediator to the pathogenesis of ACD requires identification of its contributions to these different processes.

The authors thank Dr Jack Goodman, University of Kentucky Advanced Science & Technology Commercialization Center, for performing MALDI-TOF/MS on hepcidin. The HCD57 murine erythroleukemia cell line was a generous gift from Dr Stephen Sawyer, Department of Pharmacology and Toxicology, Medical College of Virginia/Virginia Commonwealth University, Richmond.