Inappropriate expression of hepcidin is associated with iron refractory anemia implications for the anemia of chronic disease_FullText
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Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease
David A. Weinstein, Cindy N. Roy, Mark D. Fleming, Massimo F. Loda, Joseph I. Wolfsdorf, and Nancy C. Andrews
The anemia of chronic disease is a preva-We noted that patients with large hepatic trolling the release of iron from cells. We lent, poorly understood condition that adenomas had severe iron refractory ane-conclude that hepcidin plays a major, afflicts patients with a wide variety of mia similar to that observed in anemia of causative role in the anemia observed in diseases, including infections, malignan-chronic disease. This anemia resolved our subgroup of patients with hepatic cies, and rheumatologic disorders. It is spontaneously after adenoma resection adenomas, and we speculate that it is characterized by a blunted erythropoietin or liver transplantation. We investigated important in the pathogenesis of the aneresponse by erythroid precursors, de-the role of the adenomas in the pathogen-mia of chronic disease in general. (Blood. creased red blood cell survival, and a esis of the anemia and found that they 2002;100:3776-3781) defect in iron absorption and macro-produce inappropriately high levels of phage iron retention, which interrupts hepcidin mRNA. Hepcidin is a peptide iron delivery to erythroid precursor cells. hormone that has been implicated in con-© 2002 by The American Society of Hematology
Introduction
The erythroid bone marrow requires a large, continuous supply of iron to make normal red blood cells. This iron comes from 2 sources. A small portion enters the body each day through intestinal absorption of dietary iron. A much larger fraction becomes available through recycling of iron from senescent red blood cells. This iron recycling is carried out by specialized reticuloendothelial macrophages that engulf aged erythrocytes, lyse them, and catabolize their hemoglobin. The macrophages store part of the recovered iron and export the rest to the plasma, where it is taken up by transferrin.
The anemia of chronic disease is an acquired disorder seen in patients with a variety of inflammatory disorders. The pathogenesis of this anemia has been attributed to deficiencies at multiple steps in erythropoiesis, including a blunted response to erythropoietin, decreased survival of mature red blood cells, and decreased iron availability.1 The low serum iron and accumulation of iron in the reticuloendothelial cells of these patients is thought to result from the retention of iron by reticuloendothelial macrophages and decreased intestinal iron absorption.2,3 This impairment of iron delivery by macrophages and enterocytes is thought to be part of a host defense mechanism to fight infection and cancer.4,5 However, it also leads to a deficiency of iron available for erythropoiesis.6 Early in their course, patients with the anemia of chronic disease have normal body iron stores and a mild, normocytic anemia that results from impaired iron recycling. Too much iron is retained in
From Children’s Hospital, Harvard Medical School; Brigham and Women’s Hospital and Harvard Medical School; Dana-Farber Cancer Institute; and Howard Hughes Medical Institute, Children’s Hospital, Harvard Medical School; all of Boston, MA.
Submitted April 26, 2002; accepted June 16, 2002. Prepublished online as Blood First Edition Paper, June 28, 2002; DOI 10.1182/blood-2002-04-1260.
D.A.W. and C.N.R. contributed equally to this work.
Supported in general by a Clinical Research Center Grant from the Public Health Service Division of Research Resources (NIH M01RR02172), a grant from the Association for Glycogen Storage Disease (USA), and the Children’s Hospital Glycogen Storage Disease Research Fund. D.A.W. was supported by
reticuloendothelial macrophages and, consequently, serum iron and serum transferrin saturation values are low. Over time the impaired intestinal iron absorption associated with the anemia of chronic disease leads to frank iron deficiency, and the anemia becomes microcytic.
The molecular basis of the anemia of chronic disease has not been elucidated. Cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), and interferons are hypothesized to be involved in the maintenance of red blood cell production or stability.1 Many studies have shown a correlation between inflammation, elevated circulating cytokines, and anemia in patients and in mice, but whether these inflammatory cytokines act alone or regulate other pathways that are important for red blood cell production is unclear. In vitro studies have shown that treatment of cultured cells with cytokines can alter ferritin and transferrin receptor expression and iron-responsive protein activity in macrophages,7 but direct evidence for a role of those molecules in regulating iron egress is lacking.2,8
Recently, it was speculated that a small peptide hormone might be involved in the pathogenesis of this disorder.9 Hepcidin is a 20–, 22–, or 25–amino acid peptide that is cleaved from a larger precursor. It is produced in the liver and detectable in serum and urine.10,11 Hepcidin has intrinsic antimicrobial activity, and its expression increases in response to inflammatory stimuli.12,13 Animals that inadvertently lost expression of hepcidin as a result of
the Clinical Investigator Training Program: Beth Israel Deaconess Medical Center-Harvard/Massachusetts Institute of Technology (MIT) Health Sciences and Technology in collaboration with Pfizer. C.N.R. is supported by the Hematology Training Grant T32-HL07623-15. M.D.F. is supported in part by National Institutes of Health (NIH) Grant K08-03600.
Reprints: Nancy C. Andrews, Division of Hematology/Oncology, Enders 720, Children’s Hospital Boston, 300 Longwood Ave, Boston, MA 02115; 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.
© 2002 by The American Society of Hematology
BLOOD, 15 NOVEMBER 2002 · VOLUME 100, NUMBER 10
BLOOD, 15 NOVEMBER 2002 · VOLUME 100, NUMBER 10 HEPCIDIN-ASSOCIATED IRON REFRACTORY ANEMIA 3777
targeted disruption of USF2, a neighboring gene, developed hepatic iron overload associated with decreased iron in tissue macrophages,14 whereas animals that retained hepcidin despite loss of USF2 did not.13 Hepcidin mRNA expression is markedly increased in some mouse models of iron overload, raising the possibility that it might be part of a compensatory response to limit iron absorption.12 This conclusion is strongly supported by the recent finding that transgenic mice that constitutively express hepcidin develop severe iron deficiency anemia.13 Together, these observations make hepcidin a very attractive candidate for a direct mediator in the pathogenesis of the anemia of chronic disease by acting as a negative regulator of intestinal iron absorption and macrophage iron release.9However, there was previously little experimental data to support this notion.
We have found evidence of a role for hepcidin in a remarkable iron refractory anemia observed in a cohort of type 1a glycogen storage disease (GSD1a) patients. GSD1a is caused by deficiency of glucose-6-phosphatase, which catalyzes the terminal reactions of both glycogenolysis and gluconeogenesis, converting glucose-6phosphate to glucose. Lack of this enzyme causes an inability to maintain normal glucose homeostasis; severe hypoglycemia occurs if glucose is not provided continuously.15 Continuous provision of a glucose source ameliorates the biochemical abnormalities, allowing patients to survive into adulthood. Prolonged survival has led to the emergence of serious long-term complications, including chronic hepatic inflammation, hepatic adenomas, focal segmental glomerulosclerosis, nephrocalcinosis, and anemia.16,17
Although many of our older patients with GSD1a have mild anemia, 6 patients (16%) have developed severe, unremitting anemia. These individuals did not respond to oral iron supplementation and showed a delayed, partial response to replacement doses of intravenous iron dextran,17 as seen in the anemia of chronic disease. Their anemia is unrelated to metabolic control of GSD1a but is associated with the presence of large hepatic adenomas. In fact, those patients with severe anemia had the greatest tumor burdens of all of our patients with GSD1a.
We observed spontaneous resolution of anemia in an 18-yearold patient who underwent resection of her large, isolated hepatic adenoma. Studies of her liver showed inappropriately high expression of hepcidin mRNA in adenoma tissue. Most adenomas in GSD1a patients are not resectable. However, we observed similar, inappropriate hepcidin mRNA expression in adenoma tissue from another GSD1a patient who experienced resolution of his anemia following an allogeneic liver transplantation. We propose that aberrant high level hepcidin mRNA expression played a direct role in causing the anemia in these patients.
Materials and methods
Tissue preparation and analysis
Tissue samples from the adenoma and the unaffected liver were frozen in liquid nitrogen immediately after resection. Samples were also fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. We isolated total RNA using Ultraspec (Biotecx Laboratories, Houston, TX) per the manufacturer’s instructions. We obtained formalin-fixed and paraffin-embedded tissue from another patient with GSD1a who underwent a liver transplantation for complications of multiple, large hepatic adenomas. The investigative review board of Children’s Hospital Boston approved the use of all tissue samples.
Human cytokine expression array
We prepared a33P deoxycytidine triphosphate (dCTP)–labeled (Amersham Pharmacia Biotech, Piscataway, NJ) cDNA using 5 fg total RNA from adenoma and unaffected liver tissue using human cytokine-specific primers (R&D Systems, Minneapolis, MN). We used the labeled cDNA samples to probe duplicate nylon human cytokine expression arrays (R&D Systems) containing cDNA molecules representing 180 different cytokine genes according to the manufacturer’s instructions. We quantified gene expression by phosphorimager analysis and normalized to cyclophilin or L19.
Northern blot analysis
We purchased control liver RNA from a 27-year-old male subject (Clontech, Palo Alto, CA) and prepared Northern blots according to standard procedures using 5 fg total RNA per lane. We probed blots with a32PdCTP–labeled polymerase chain reaction (PCR) products representing human hepcidin, mouse hepcidin, human l-actin, or mouse l-actin. We quantified the relative expression of hepcidin in each RNA sample by phosphorimager analysis and normalized to l-actin. Nonheme iron was quantified using a colorimetric assay as described previously.18
In situ hybridization
We prepared digoxigenin-labeled probes for in situ hybridization as described previously.19 We performed in situ hybridization essentially as described,19 with minimal modifications. We prehybridized samples at 56°C for 3 hours and then added 1.5 ng riboprobe to hybridize at 55°C overnight. The highest stringency of posthybridization washes was 60°Cin
0.1 X sodium citrate sodium chloride (SSC) (1 X SSC is 15 mM trisodium citrate, 150 mM NaCl) with l-mercaptoethanol and ethylenediaminetetraacetic acid (EDTA) for 2 hours. We applied antibody to digoxigenin for 2 hours at room temperature.
Results
GSD1a patients with large hepatic adenomas have severe iron refractory anemia
Severe anemia in older GSD1a patients is characterized by microcytosis, increased red cell distribution width, low serum iron concentrations, and very low transferrin saturations (Table 1). Oral iron administration did not correct this anemia in any of the patients, and intravenous iron dextran administration allowed for only a partial correction of the anemia over an extended time. Taken together, our findings indicate that the GSD1a patients with large adenomas have a severe defect in reticuloendothelial iron recycling accompanied by systemic iron deficiency.
Patient A presented at 8 months of age (February 1982) with a hypoglycemic coma. A diagnosis of GSD1a was made by liver biopsy and subsequently confirmed by mutation analysis. She was managed on a regimen of frequent feeds during the day and nocturnal nasogastric feeds until June 1998, when uncooked cornstarch replaced overnight nasogastric feeds. At that time, she had a normal hematocrit level and normal iron studies. An abdominal ultrasound showed no evidence of focal hepatic lesions. A year later, at the time of a routine follow-up evaluation, she had a microcytic anemia, and a single, large hepatic adenoma was discovered by ultrasound examination. The anemia persisted, and follow-up magnetic resonance imaging (MRI) obtained 3 months later demonstrated further enlargement of the hepatic adenoma (Figure 1A).
The adenoma was resected in December 2000. The anemia resolved spontaneously within 6 weeks even though no blood
3778 WEINSTEIN et al BLOOD, 15 NOVEMBER 2002 · VOLUME 100, NUMBER 10
Table 1. Hematologic and iron parameters in patients with GSD1a and anemia
| Subject | Sex | Age, y | Hgb, g/dL | Hct, % | MCV, fL | RDW, % | Fe, fg/dL | TIBC, fg/dL | Saturation, % | Ft, ng/mL |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Female | 19 | 9.8 | 29.4 | 80.2 | 15.3 | 20 | 301 | 6.6 | 67.2 |
| 2 | Male | 22 | 8.5 | 26.6 | 64.0 | N/A | 20 | 330 | 6.1 | N/A |
| 3 | Male | 27 | 4.6 | 16.6 | 50.4 | 20.7 | 6 | 349 | 1.7 | 23.7 |
| 4 | Male | 13 | 8.4 | 26.7 | 61.8 | 16.4 | 13 | 411 | 3.2 | 47.9 |
| 5 | Male | 24 | 7.7 | 24.4 | 70.8 | 18.3 | N/A | N/A | N/A | 15.3 |
We obtained hematologic data from 5 patients with GSD1a on oral iron therapy prior to intravenous iron dextran infusions. All patients had large adenomas at the time of these studies. One representative set of data is shown for each patient. Subjects 1 and 2 are patients A and B, respectively. Data were not available for the sixth patient because he was treated at another institution and was not covered by our human studies protocol. Hgb indicates hemoglobin (normal value ranges for adult females, 12-16 g/dL; adult males, 13-18 g/dL; 13-year-old males, 13-16 g/dL); Hct, hematocrit (normal value ranges for adult females, 37%-48%; adult males, 42%-52%; 13-year-old males, 37%-49%); MCV, mean corpuscular volume (normal value range for adults, 90 ± 10 fL; 13-year-old males, 88 ± 10 fL); RDW, red blood cell distribution width (normal, 13%-15%); Fe, serum iron concentration (normal, 50-150 fg/dL); TIBC, total iron-binding capacity (normal, 250-370 fg/dL); saturation, the ratio of iron to total iron-binding capacity (normal, 20%-45%); Ft, serum ferritin (normal values for 19-year-old females, 6-40 ng/mL; 13-year-old males, 23-70 ng/mL; adult males, 20-300 ng/mL, increasing with age); N/A, not available.
transfusions, bone marrow modulators, or iron had been administered (Figure 1B). Over the ensuing months, patient A’s iron studies normalized and have remained normal for 1 year.
GSD1a adenoma tissue does not sequester iron
Removal of patient A’s large adenoma promptly corrected her anemia, which suggested that the adenoma had been hindering iron
Figure 1. Laboratory findings in patient A before and after adenoma resection.
(A) An MRI scan performed prior to resection (August 30, 2000) shows a 13 X 14 cm adenoma in the left lobe of A’s liver (arrow). (B) Individual panels illustrate changes in hematocrit, serum ferritin concentration, serum iron concentration, and erythrocyte sedimentation rate for approximately 1 year before and 6 months after resection (performed December 15, 2000). The serum ferritin was initially above the normal range but fell to subnormal levels after the tumor was resected, and erythroid iron utilization returned to normal.
utilization. To determine whether the adenoma was storing iron at the expense of the bone marrow, we used Perl Prussian blue stain to compare the nonheme iron load of the adenoma versus the unaffected liver. Neither unaffected liver nor adenoma tissue contained significant stainable iron, indicating that liver iron stores were depleted and that the adenoma was not sequestering the metal.
GSD1a adenoma tissue and unaffected liver tissue have comparable cytokine expression profiles
We considered the possibility that the adenoma secreted a soluble factor that inhibited iron absorption and macrophage iron release. Cytokines have been postulated to play a direct role in the pathogenesis of the anemia of chronic disease.8 To evaluate cytokine expression by the adenoma, we probed a 180-gene cytokine cDNA array with labeled RNA samples from unaffected liver or adenoma tissue. We looked for evidence of genes that were differentially expressed between the 2 tissue types and for differential expression of genes that have been suggested to be involved in the anemia of chronic disease (Table 2). We found that no cytokine gene showed more than a 2-fold increase in expression in the adenoma tissue when compared with unaffected liver tissue. There was a slight increase in expression of several genes consistent with a host inflammatory response against the adenoma. IL-13 and IL-16 were slightly increased in the adenoma. Importantly, there was no induction of TNF, IL-1, IL-6, or interferons in the adenoma
Table 2. Expression of cytokines and related genes by unaffected liver tissue and liver adenoma from patient A
Unaffected Adenoma Unaffected liver vs vs liver vs Adenoma mRNA cyclophilin cyclophilin L19 vs L19
IL-1 BBBB IL-6 BBBB IL-13 18.6 24.9 7.1 13.0 IL-16 13.4 17.8 5.1 9.3 “-Interferon B B B B TNF-a B BBB HLA-H 36.1 67.4 13.8 35.1 l2-Microglobulin 182.9 239.8 70.1 124.9 l-Actin 83.2 86.9 31.9 45.3 Cyclophillin 100.0 100.0 38.3 52.1 L19 261.0 192.1 100.0 100.0
We quantified the relative expression of each gene in the array by phosphorimager analysis. This table shows results for molecules that were differentially expressed and for cytokines that have been implicated in the pathogenesis of the anemia of chronic disease. We normalized values for each gene in the array to cyclophilin or L19. Results are shown in arbitrary units. B indicates the transcript was not detectable above background.