Investigation of the role of interleukin-6 and hepcidin antimicrobial peptide in the development of anemia with age.

Anemia is common in older adults and associated with adverse health outcomes in epidemiological studies. A thorough understanding of the complex pathophysiological mechanisms driving anemia in the elderly is lacking; but inflammation, iron restriction, and impaired erythroid maturation are thought to influence the phenotype. We hypothesized that interleukin-6 contributes to this anemia, given its pro-inflammatory activities, its ability to induce hepcidin antimicrobial peptide, and its negative impact on several tissues in older adults. We tested this hypothesis by comparing changes in indices of inflammation, iron metabolism and erythropoiesis in aged C57BL/6 mice to aged mice with targeted deletions of interleukin-6 or hepcidin antimicrobial peptide. Circulating neutrophil and monocyte numbers and inflammatory cytokines increased with age. Decline in hemoglobin concentration and red blood cell number indicated that C57BL/6, interleukin-6 knockout mice, and hepcidin antimicrobial peptide knockout mice all demonstrated impaired erythropoiesis by 24 months. However, the interleukin-6 knock out genotype and the hepcidin antimicrobial peptide knock out genotype resulted in improved erythropoiesis in aged mice. Increased erythropoietic activity in the spleen suggested that the erythroid compartment was stressed in aged C57BL/6 mice compared to aged interleukin-6 knockout mice. Our data suggest C57BL/6 mice are an appropriate mammalian model for the study of anemia with age. Furthermore, although interleukin-6 and hepcidin antimicrobial peptide are not required, they can participate in the development of anemia in aging mice, and could be targeted, pre-clinically, with existing interventions to determine the feasibility of such agents for the treatment of anemia in older adults.

In anemia of multiple myeloma, hepcidin is induced by increased bone morphogenetic protein 2.

Hepcidin is the principal iron-regulatory hormone and a pathogenic factor in anemia of inflammation. Patients with multiple myeloma (MM) frequently present with anemia. We showed that MM patients had increased serum hepcidin, which inversely correlated with hemoglobin, suggesting that hepcidin contributes to MM-related anemia. Searching for hepcidin-inducing cytokines in MM, we quantified the stimulation of hepcidin promoter-luciferase activity in HuH7 cells by MM sera. MM sera activated the hepcidin promoter significantly more than did normal sera. We then examined the role of bone morphogenetic proteins (BMPs) and interleukin-6 (IL-6), the major transcriptional regulators of hepcidin. Mutations in both BMP-responsive elements abrogated the activation dramatically, while mutations in the IL-6-responsive signal transducer and activator of transcription 3-binding site (STAT3-BS) had only a minor effect. Cotreatment with anti-BMP-2/4 or noggin-Fc blocked the promoter induction with all MM sera, anti-IL-6 blocked it with a minority of sera, whereas anti-BMP-4, -6, or -9 antibodies had no effect. BMP-2-immunodepleted MM sera had decreased promoter stimulatory capacity, and BMP-2 concentrations in MM sera were significantly higher than in normal sera. Our results demonstrate that BMP-2 is a major mediator of the hepcidin stimulatory activity of MM sera.

Animal models of anemia of inflammation.

Anemia of inflammation (AI) is a complex multi-organ response to inflammatory disorders. Because AI can result from many infectious and non-infectious inflammatory diseases, multiple mechanisms may contribute to its pathogenesis, including iron restriction, direct erythropoietic suppression, shortened red blood cell survival, and frank hemolysis. Animal models have been helpful in the study of the mechanisms of AI and its potential treatments, but each model reflects distinct aspects of this heterogeneous syndrome. It is therefore important to study a variety of models of AI. This review focuses on the use of infectious and noninfectious mouse models of inflammation that have been shown to manifest anemia. We review many of the models reported in the literature or developed in our laboratory, and discuss their respective merits and drawbacks.

IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.

Hypoferremia is a common response to systemic infections or generalized inflammatory disorders. In mouse models, the development of hypoferremia during inflammation requires hepcidin, an iron regulatory peptide hormone produced in the liver, but the inflammatory signals that regulate hepcidin are largely unknown. Our studies in human liver cell cultures, mice, and human volunteers indicate that IL-6 is the necessary and sufficient cytokine for the induction of hepcidin during inflammation and that the IL-6-hepcidin axis is responsible for the hypoferremia of inflammation.

Dopamine D2R DNA transfer in dopamine D2 receptor-deficient mice: effects on ethanol drinking.

Dopamine (DA) signals are transmitted via specific receptors including the D2 receptors (D2R). Previous studies have shown that D2R upregulation in the nucleus accumbens (NAc) attenuated alcohol consumption. We hypothesized that upregulation of D2R in the NAc would significantly influence alcohol drinking. We tested this hypothesis by determining the effect that D2R upregulation has on alcohol intake in genetically altered mice lacking D2Rs. After a steady baseline of drinking behavior was established for all mice, a null vector or a genetically modified adenoviral vector containing the rat D2R cDNA was infused into the NAc of wild-type (Drd2+/+), heterozygous (Drd2+/-), and receptor-deficient mice (Drd2-/-). Ethanol intake and preference were then determined using the two-bottle choice paradigm. Our results indicated that Drd2+/+ mice treated with the D2R vector significantly attenuated (58 %) their ethanol intake as well as reduced preference. Drd2+/- and mutant mice showed a similar attenuation, although the change was not as marked (12 %) and did not last as long. In contrast, Drd2-/- mice treated with the D2R vector displayed a temporary but significant increase (46 %) in ethanol intake and preference (consumption). These results supported the notion that the D2R plays an important role in alcohol consumption in mice and suggest that a key threshold range of D2R levels is associated with elevated alcohol consumption. Significant deviations in D2R levels from this range could impact alcohol consumption, and could help to explain possible individual variations in alcohol response, metabolism, sensitivity and consumption.

Mouse models of anemia of cancer.

Anemia of cancer (AC) may contribute to cancer-related fatigue and impair quality of life. Improved understanding of the pathogenesis of AC could facilitate better treatment, but animal models to study AC are lacking. We characterized four syngeneic C57BL/6 mouse cancers that cause AC. Mice with two different rapidly-growing metastatic lung cancers developed the characteristic findings of anemia of inflammation (AI), with dramatically different degrees of anemia. Mice with rapidly-growing metastatic melanoma also developed a severe anemia by 14 days, with hematologic and inflammatory parameters similar to AI. Mice with a slow-growing peritoneal ovarian cancer developed an iron-deficiency anemia, likely secondary to chronically impaired nutrition and bleeding into the peritoneal cavity. Of the four models, hepcidin mRNA levels were increased only in the milder lung cancer model. Unlike in our model of systemic inflammation induced by heat-killed Brucella abortus, ablation of hepcidin in the ovarian cancer and the milder lung cancer mouse models did not affect the severity of anemia. Hepcidin-independent mechanisms play an important role in these murine models of AC.

Hepcidin--a potential novel biomarker for iron status in chronic kidney disease.

BACKGROUND AND OBJECTIVES: Hepcidin is a key regulator of iron homeostasis, but its study in the setting of chronic kidney disease (CKD) has been hampered by the lack of validated serum assays. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study reports the first measurements of bioactive serum hepcidin using a novel competitive ELISA in 48 pediatric (PCKD2-4) and 32 adult (ACKD2-4) patients with stages 2 to 4 CKD along with 26 pediatric patients with stage 5 CKD (PCKD5D) on peritoneal dialysis. RESULTS: When compared with their respective controls (pediatric median = 25.3 ng/ml, adult = 72.9 ng/ml), hepcidin was significantly increased in PCKD2-4 (127.3 ng/ml), ACKD2-4 (269.9 ng/ml), and PCKD5D (652.4 ng/ml). Multivariate regression analysis was used to assess the relationship between hepcidin and indicators of anemia, iron status, inflammation, and renal function. In PCKD2-4 (R(2) = 0.57), only ferritin correlated with hepcidin. In ACKD2-4 (R(2) = 0.78), ferritin and soluble transferrin receptor were associated with hepcidin, whereas GFR was inversely correlated. In PCKD5D (R(2) = 0.52), percent iron saturation and ferritin were predictors of hepcidin. In a multivariate analysis that incorporated all three groups (R(2) = 0.6), hepcidin was predicted by ferritin, C-reactive protein, and whether the patient had stage 5D versus stages 2 to 4 CKD. CONCLUSIONS: These findings suggest that increased hepcidin across the spectrum of CKD may contribute to abnormal iron regulation and erythropoiesis and may be a novel biomarker of iron status and erythropoietin resistance.

Persistent dopamine functions of neurons derived from embryonic stem cells in a rodent model of Parkinson disease.

The derivation of dopamine neurons is one of the best examples of the clinical potential of embryonic stem (ES) cells, but the long-term function of the grafted neurons has not been established. Here, we show that, after transplantation into an animal model, neurons derived from mouse ES cells survived for over 32 weeks, maintained midbrain markers, and had sustained behavioral effects. Microdialysis in grafted animals showed that dopamine (DA) release was induced by depolarization and pharmacological stimulants. Positron emission tomography measured the expression of presynaptic dopamine transporters in the graft and also showed that the number of postsynaptic DA D(2) receptors was normalized in the host striatum. These data suggest that ES cell-derived neurons show DA release and reuptake and stimulate appropriate postsynaptic responses for long periods after implantation. This work supports continued interest in ES cells as a source of functional DA neurons.

Suppression of hepcidin during anemia requires erythropoietic activity.

Hepcidin, the principal iron regulatory hormone, regulates the absorption of iron from the diet and the mobilization of iron from stores. Previous studies indicated that hepcidin is suppressed during anemia, a response that would appropriately increase the absorption of iron and its release from stores. Indeed, in the mouse model, hepcidin-1 was suppressed after phlebotomy or erythropoietin administration but the suppression was reversed by inhibitors of erythropoiesis. The suppression of hepcidin necessary to match iron supply to erythropoietic demand thus requires increased erythropoiesis and is not directly mediated by anemia, tissue hypoxia, or erythropoietin.

Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs.

Hepcidin is the principal iron regulatory hormone and its overproduction contributes to anemia of inflammation (AI). In vitro, hepcidin binds to and induces the degradation of the exclusive iron exporter ferroportin. We explored the effects and distribution of synthetic hepcidin in the mouse. A single intraperitoneal injection of hepcidin caused a rapid fall of serum iron in a dose-dependent manner, with a 50-microg dose resulting in iron levels 80% lower than in control mice. The full effect was seen within only 1 hour, consistent with a blockade of iron export from tissue stores and from macrophages involved in iron recycling. Serum iron remained suppressed for more than 48 hours after injection. Using radiolabeled hepcidin, we demonstrated that the serum concentration of hepcidin at the 50-microg dose was 1.4 microM, consistent with the inhibitory concentration of 50% (IC50) of hepcidin measured in vitro. Radiolabeled hepcidin accumulated in the ferroportin-rich organs, liver, spleen, and proximal duodenum. Our study highlights the central role of the hepcidin-ferroportin interaction in iron homeostasis. The rapid and sustained action of a single dose of hepcidin makes it an appealing agent for the prevention of iron accumulation in hereditary hemochromatosis.

Hepcidin excess induces the sequestration of iron and exacerbates tumor-associated anemia.

The iron-regulatory hormone hepcidin has been proposed as the mediator of anemia of inflammation (AI). We examined the acute and chronic effects of hepcidin in the mouse. Injections of human hepcidin (50 microg/mouse), but not of its diluent, induced hypoferremia within 4 hours. To examine the chronic effects of hepcidin, we implanted either tumor xenografts engineered to overexpress human hepcidin or control tumor xenografts into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Despite abundant dietary iron, mice with hepcidin-producing tumors developed more severe anemia, lower serum iron, and increased hepatic iron compared with mice with control tumors. Hepcidin contributes to AI by shunting iron away from erythropoiesis and sequestering it in the liver, predominantly in hepatocytes.

Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response.

Infection and inflammation produce systemic responses that include hypozincemia and hypoferremia. The latter involves regulation of the iron transporter ferroportin 1 by hepcidin. The mechanism of reduced plasma zinc is not known. Transcripts of the two zinc transporter gene families (ZnT and Zip) were screened for regulation in mouse liver after turpentine-induced inflammation and LPS administration. Zip14 mRNA was the transporter transcript most up-regulated by inflammation and LPS. IL-6 knockout (IL-6(-/-)) mice did not exhibit either hypozincemia or the induction of Zip14 with turpentine inflammation. However, in IL-6(-/-) mice, LPS produced a milder hypozincemic response but no Zip14 induction. Northern analysis showed Zip14 up-regulation was specific for the liver, with one major transcript. Immunohistochemistry, using an antibody to an extracellular Zip14 epitope, showed both LPS and turpentine increased abundance of Zip14 at the plasma membrane of hepatocytes. IL-6 produced increased expression of Zip14 in primary hepatocytes cultures and localization of the protein to the plasma membrane. Transfection of mZip14 cDNA into human embryonic kidney cells increased zinc uptake as measured by both a fluorescent probe for free Zn(2+) and (65)Zn accumulation, as well as by metallothionein mRNA induction, all indicating that Zip14 functions as a zinc importer. Zip14 was localized in plasma membrane of the transfected cells. These in vivo and in vitro experiments demonstrate that Zip14 expression is up-regulated through IL-6, and that this zinc transporter most likely plays a major role in the mechanism responsible for hypozincemia that accompanies the acute-phase response to inflammation and infection.

Dopamine D2 receptor binding, Drd2 expression and the number of dopamine neurons in the BXD recombinant inbred series: genetic relationships to alcohol and other drug associated phenotypes.

BACKGROUND: It has not been established to what extent the natural variation in dopamine systems contribute to the variation in ethanol response. The current study addresses this issue by measuring D2 dopamine (DA) receptor binding, the expression of Drd2, the number of midbrain DA neurons in the BXD recombinant inbred (RI) series and then compares these strain means with those previously reported for a variety of ethanol and other drug-related phenotypes. METHODS: Data were collected for 21 to 23 of the BXD RI strains and the parental strains. D2 DA receptor autoradiography was performed using 125I-epidepride as the ligand [ Kanes S, Dains K, Cipp L, Gatley J, Hitzemann B, Rasmussen E, Sanderson S, Silverman S, Hitzemann R (1996) Mapping the genes for haloperidol-induced catalepsy. J Pharmacol Exp Ther 277:1016-1025]. Drd2 expression was measured using the Affymetrix oligoarray system. Immunocytochemical techniques were used to determine the number of midbrain DA neurons [Hitzemann B, Dains K, Hitzemann R (1994) Further studies on the relationship between dopamine cell density and haloperidol response. J Pharmacol Exp Ther 271:969-976]. RESULTS AND CONCLUSIONS: The range of difference in receptor binding for the RI strains was approximately 2-fold in all regions examined, the core, the shell of the nucleus accumbens (NAc) and the dorsomedial caudate-putamen (CPu); heritability in all regions was moderate--(h2 approximately 0.35). Drd2 expression in forebrain samples from the RI and parental strains ranged 1.5- to h2-fold and was moderate-0.47. Variation in the number of tyrosine hydroxylase (TH) positive neurons was moderate, 41% and 26% and h2 was low--0.19 and 0.15 for the ventral tegmental area (VTA) and substantia nigra compacta (SNc), respectively. Significant correlations were found between D2 DA receptor binding and the low dose (1.33 g/kg) ethanol stimulant response. (p < 0.002) and between expression and conditioned place preference (CPP) (p < 0.0005). No significant correlations were detected between ethanol preference and either receptor binding or Drd2 expression; however, a significant correlation was found between preference and Ncam expression. Ncam is approximately 0.2 Mb from Drd2. Overall, the data suggest ethanol preference and CPP are associated with the expression of Drd2 or closely linked genetic loci.

DRD2 gene transfer into the nucleus accumbens core of the alcohol preferring and nonpreferring rats attenuates alcohol drinking.

BACKGROUND: Transient overexpression of the dopamine D2 receptor (DRD2) gene in the nucleus accumbens (NAc) using an adenoviral vector has been associated with a significant decrease in alcohol intake in Sprague Dawley rats. This overexpression of DRD2 reduced alcohol consumption in a two-bottle-choice paradigm and supported the view that high levels of DRD2 may be protective against alcohol abuse. METHODS: Using a limited access (1 hr) two-bottle-choice (water versus 10% ethanol) drinking paradigm, we examined the effects of the DRD2 vector in alcohol intake in the genetically inbred alcohol-preferring (P) and -nonpreferring (NP) rats. In addition, micro-positron emission tomography imaging was used at the completion of the study to assess in vivo the chronic (7 weeks) effects of ethanol exposure on DRD2 levels between the two groups. RESULTS: P rats that were treated with the DRD2 vector (in the NAc) significantly attenuated their alcohol preference (37% decrease) and intake (48% decrease), and these measures returned to pretreatment levels by day 20. A similar pattern of behavior (attenuation of ethanol drinking) was observed in NP rats. Analysis of the [C]raclopride micro-positron emission tomography data after chronic (7 weeks) exposure to ethanol revealed clear DRD2 binding differences between the P and NP rats. P rats showed 16% lower [C]raclopride specific binding in striatum than the NP rats. CONCLUSIONS: These findings further support our hypothesis that high levels of DRD2 are causally associated with a reduction in alcohol consumption and may serve as a protective factor against alcoholism. That this effect was seen in P rats, which are predisposed to alcohol intake, suggests that they are protective even in those who are genetically predisposed to high alcohol intake. It is noteworthy that increasing DRD2 significantly decreased alcohol intake but did not abolish it, suggesting that high DRD2 levels may specifically interfere with the administration of large quantities of alcohol. The significantly higher DRD2 concentration in NP than P rats after 7 weeks of ethanol therefore could account for low alcohol intake.