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Blood, 1 April 2004, Vol. 103, No. 7, pp. 2847-2849. Prepublished online as a Blood First Edition Paper on December 4, 2003; DOI 10.1182/blood-2003-09-3300. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-09-3300v1 103/7/2847    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Miranda, C. J. Articles by Santos, M. M. Search for Related Content PubMed PubMed Citation Articles by Miranda, C. J. Articles by Santos, M. M. Related Collections Brief Reports Immunobiology Red Cells Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  RED CELLS Brief report Contributions of 2-microglobulin–dependent molecules and lymphocytes to iron regulation: insights from HfeRag1-/- and 2mRag1-/- double knock-out mice Carlos J. Miranda, Hortence Makui, Nancy C. Andrews, and Manuela M. Santos From the Centre de recherche, Centre Hospitalier de l'Université de Montréal and the Université de Montréal, Montreal, QC, Canada; Howard Hughes Medical Institute, Department of Pediatrics, Harvard Medical School, Children's Hospital, Boston, MA; UnIGENe, Instituto de Biologia Molecular e Celular, Porto, Portugal.    Abstract Top Abstract Introduction Study design Results and discussion References   Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, coding for a 2-microglobulin (2m)-associated major histocompatibility complex class I-like protein. However, iron accumulation in patients with HH can be highly variable. Previously, analysis of 2mRag1-/- double-deficient mice, lacking all 2m-dependent molecules and lymphocytes, demonstrated increased iron accumulation in the pancreas and heart compared with 2m single knock-out mice. To evaluate whether the observed phenotype in 2mRag1-/- mice was due solely to the absence of Hfe or to other 2m-dependent molecules, we generated HfeRag1-/- double-deficient mice. Our studies revealed that introduction of Rag1 deficiency in Hfe knock-out mice leads to heightened iron overload, mainly in the liver, whereas the heart and pancreas are relatively spared compared with 2mRag1-/- mice. These results suggest that other 2m-interacting protein(s) may be involved in iron regulation and that in the absence of functional Hfe molecules lymphocyte numbers may influence iron overload severity. (Blood. 2004;103: 2847-2849)    Introduction Top Abstract Introduction Study design Results and discussion References   The protein involved in hereditary hemochromatosis (HH), HFE, has been identified as a 2-microglobulin (2m)-dependent, major histocompatibility complex class I (MHC-I)-like molecule,1 complementing earlier findings that 2m deficiency in mice leads to iron overload.2 Significantly, knock-out of the Hfe gene clearly confers the HH phenotype to mice.3,4 Although HFE mutations are clearly associated with iron overload, the marked variability of the phenotype among homozygous individuals for the same HFE mutations indicates that other environmental and genetic factors modify disease severity.5 Earlier reports show that defective numbers of peripheral lymphocytes and liver lymphocyte populations are associated with a more severe clinical expression of iron overload and damage in HH.6,7 Previously, we investigated the effect of total lymphocyte absence in iron overload by crossing 2m-/- mice, lacking all 2m-dependent MHC-I molecules,2 with mice deficient in recombinase activator gene 1 (Rag1) required for normal B- and T-lymphocyte development.8 Immunodeficient 2mRag1-/- mice develop iron overload and, unlike their single knock-out counterparts, when challenged with dietary iron loading, iron accumulation occurs not only in the liver but also massively in the pancreas and heart myocytes.9 In this report, we further investigate the possible involvement of 2m-dependent molecules and lymphocytes in iron regulation. We examine changes in iron metabolism in compound mutant HfeRag-/- and 2mRag1-/- mice placed on a standard diet and after dietary iron challenge.    Study design Top Abstract Introduction Study design Results and discussion References   All procedures were performed in accordance with guidelines of Canadian Council on Animal Care and approved by the Animal Care Committee of the Centre Hospitalier de l'Université de Montréal. Hfe-/- mice have been described previously.3 2m-/- and Rag1-/- mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mutant mice have been back-crossed a minimum of 5 times onto the C57BL/6 (B6) background. Compound mutants 2mRag1-/- and HfeRag1-/- were obtained as described.9 Mouse peripheral blood cells were screened by flow cytometry in a Coulter Epics Elite counter (Coulter, Hialeah, FL) for the absence of T and B lymphocytes, using anti-TCR (T-cell marker) and anti-CD45R/B220 (B-cell marker) antibodies (PharMingen, San Diego, CA), and were genotyped by polymerase chain reaction (PCR) for 2m and Hfe mutations. All animals were 8 weeks old at the beginning of the experiments. They were given a commercial diet (Teklad Global 18% protein rodent diet; Harlan Teklad, Madison, WI), or, when indicated, the same standard diet supplemented with 2.5% (wt/wt) carbonyl iron (Sigma Immunochemicals, St Louis, MO). Iron levels were measured by acid digestion of tissue samples followed by iron quantification by atomic absorption spectroscopy.9 For histology, tissue sections were stained with Prussian blue for ferric iron detection (iron stain kit; Sigma Immunochemicals).    Results and discussion Top Abstract Introduction Study design Results and discussion References   To investigate whether the exacerbation of iron loading in 2mRag1-/- mice is due to the absence of only Hfe or to other 2m-dependent molecules, we generated mice lacking both Hfe and Rag1 (HfeRag1-/- mice). It was expected that if the only 2m-dependent molecule involved in iron regulation was Hfe, the 2mRag1-/- phenotype would be recapitulated in HfeRag1-/- double mutants. Surprisingly, hepatic iron rose dramatically in HfeRag1-/- double mutants, whereas iron levels in the heart and pancreas were not statistically different from Hfe-/- single knock-out mice (Table 1). The hepatic iron increment in HfeRag1-/- was significant (P < .01) when compared with Hfe-/- single knock-out mice. In contrast, liver iron stores remained similar in 2mRag1-/- compared with 2m-/- mice. View this table: [in this window] [in a new window]   Table 1.. Tissue iron concentration in mice fed a standard or iron-enriched diet (2.5% wt/wt carbonyl iron) for 3 months   In the heart, iron concentration in HfeRag1-/- double mutants was similar to Hfe single knock-out mice (Table 1). In marked contrast, cardiac iron was significantly higher in 2mRag1-/- compared with 2m-/- mice (P < .01) or to the remaining mouse strains (P < .0005). Similarly, in the pancreas, HfeRag1-/- did not differ from Hfe-/- mice, whereas 2mRag1-/- mice showed a 3.7-fold increase in iron concentration over 2m-/- single knock-out mice (P < .0001) and all other mouse strains. Thus, on a standard diet, the phenotype of HfeRag1-/- mice is strikingly different from that of 2mRag1-/-, the main difference being the affected organs where introduction of the Rag1 mutation leads to augmented iron loading. To further investigate the responses to dietary iron loading in compound mutants, mice were challenged with a 2.5% wt/wt carbonyl iron-supplemented diet for 3 months. In the liver, the highest iron concentration was found in HfeRag1-/- mice, followed by Hfe-/-, 2mRag1-/-, and 2m-/- mice (Table 1). In the heart and pancreas, 2mRag1-/- mice accumulated more iron than any other mouse strains. These differences were highly significant (P < .0001 and P < .01, respectively, in the heart and pancreas). Iron accumulation was visible in 2mRag1-/- cardiac myocytes and pancreas sections stained with Prussian blue but remained undetectable in HfeRag1-/- mice (Figure 1). View larger version (105K): [in this window] [in a new window]   Figure 1.. Storage of excess iron in the heart and pancreas of mice after 3 months on a diet supplemented with 2.5% wt/wt carbonyl iron (Prussian blue staining, n = 5). The animals were 5 months old at the end of the experiment. Heart section from representative 2mRag1-/- (A) and HfeRag1-/- mice (B). Pancreas section from representative 2mRag1-/- (C) andHfeRag1-/- mice (D). Original magnification, x 600.   Thus, the observed phenotype differences between mice lacking both Hfe and Rag1 and mice lacking 2m and Rag1 persist and are even enhanced after dietary iron challenge. The differences in phenotype are likely due to the lack of other molecules that interact with 2m in 2m-deficient mice. Involvement of other 2m-dependent molecules in iron regulation has previously been suggested by the demonstration of increased hepatic iron in Hfe2m double-mutant mice compared with single knock outs10 and by the finding that MHC-I-deficient mice have elevated hepatic iron.11 Intriguingly, the phenotype of 2mRag1 double-deficient mice considerably resembles juvenile hemochromatosis (JH) in humans12 and hepcidin-deficient mice13 in the severity of iron overload and the involvement of the heart and pancreas. JH is an autosomal recessive disorder, which leads to early-onset, severe iron overload. Heart failure and endocrine manifestations are the most prominent clinical features of JH, whereas liver involvement, although present, is clinically less relevant. Iron accumulation in the liver of hepcidin-deficient mice is not particularly different from Hfe-/- or 2m-/- mice but rises dramatically in the pancreas and heart.13 In Hfe-deficient mice, hepatic hepcidin mRNA is inappropriately low, and the mice show a blunted response to iron loading.14-16 However, 2m-deficient mice have higher hepcidin levels than healthy mice, which is an appropriate, although insufficient response to iron loading.17 A possible explanation for this difference may reside in the fact that 2m-/- mice are known to be functionally "leaky," as residual functional MHC-I expression can still be detected.18,19 By analogy, residual Hfe expression may occur in 2m-deficient mice, giving them limited control over iron-induced hepcidin expression. This finding may explain why Hfe-/- and HfeRag1-/- mice accumulate more iron in the liver than do 2m-/- and 2mRag1-/- mice, respectively. Accumulating evidence suggests that iron does not directly regulate hepcidin expression in hepatocytes17,20 and that hepcidin levels may be indirectly regulated by other cells. In fact, hepcidin expression can be induced by interleukin 6 (IL-6),20 a cytokine synthesized predominantly by macrophages and lymphocytes. Thus, lymphocytes may interfere with hepcidin regulation either directly, by secreting IL-6, or indirectly, through effects on macrophage differentiation and function.21 Alternatively, the fact that phenotype differences between Hfe- and 2m-deficient mice are evident only after lymphocyte depletion may indicate that lymphocytes represent an important iron storage compartment. Phenotype differences between HfeRag1-/- and 2mRag1-/- mice may be attributable to the existence of other 2m-dependent, MHC-I-like molecules involved in iron regulation. Hfe is predominantly expressed in the liver, with the highest levels of Hfe mRNA found in hepatocytes.22,23 This is consistent with the finding that Hfe deletion primarily affects the liver. Presumably other 2m-dependent molecules might be more relevant in the heart and pancreas, which also express hepcidin.24 In conclusion, the work presented here suggests that alterations in the expression of 2m-dependent, MHC-I-like molecules and lymphocyte numbers may relate to phenotype variation in HH patients.    Acknowledgements   We thank Christian Dallaire for his help with atomic absorption spectroscopy, F. Arosa and B. Fournier for valuable discussions, and Ovid Da Silva for his editorial assistance.    Footnotes   Submitted September 25, 2003; accepted November 21, 2003. Prepublished online as Blood First Edition Paper, December 4, 2003; DOI 10.1182/blood-2003-09-3300. Supported by a grant from the Canadian Institutes of Health Research (CIHR; grant no. MOP44045, and the Fundaço para a Ciência e a Tecnologia (FCT; grant no. CBO/33485/99-00). M.M.S. is the recipient of a CIHR New Investigator award. N.C.A. is an Associate Investigator of the Howard Hughes Medical Institute. 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. Reprints: Manuela Santos, Centre de recherche, CHUM—Hôpital Notre-Dame, Pavillon De Sève Y5608, 1560 rue Sherbrooke est, Montréal (Québec) H2L 4M1, Canada; e-mail: msantos{at}istar.ca.    References Top Abstract Introduction Study design Results and discussion References   Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet. 1996;13: 399-408.[CrossRef][Medline] [Order article via Infotrieve] Santos M, Schilham MW, Rademakers LH, et al. Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man. J Exp Med. 1996;184: 1975-1985.[Abstract/Free Full Text] Levy JE, Montross LK, Cohen DE, Fleming MD, Andrews NC. The C282Y mutation causing hereditary hemochromatosis does not produce a null allele. Blood. 1999;94: 9-11.[Abstract/Free Full Text] Zhou XY, Tomatsu S, Fleming RE, et al. HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci U S A. 1998;95: 2492-2497.[Abstract/Free Full Text] Olynyk JK, Cullen DJ, Aquilia S, et al. A population-based study of the clinical expression of the hemochromatosis gene. N Engl J Med. 1999;341: 718-724.[Abstract/Free Full Text] Porto G, Vicente C, Teixeira MA, et al. Relative impact of HLA phenotype and CD4-CD8 ratios on the clinical expression of hemochromatosis. Hepatology. 1997;25: 397-402. Cardoso EM, Hagen K, de Sousa M, Hultcrantz R. Hepatic damage in C282Y homozygotes relates to low numbers of CD8+ cells in the liver lobuli. Eur J Clin Invest. 2001;31: 45-53.[CrossRef][Medline] [Order article via Infotrieve] Mombaerts P, Iacomini J, Johnson RS, et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 1992;68: 869-877.[CrossRef][Medline] [Order article via Infotrieve] Santos MM, de Sousa M, Rademakers LH, et al. Iron overload and heart fibrosis in mice deficient for both beta2-microglobulin and Rag1. Am J Pathol. 2000;157: 1883-1892.[Abstract/Free Full Text] Levy JE, Montross LK, Andrews NC. Genes that modify the hemochromatosis phenotype in mice. J Clin Invest. 2000;105: 1209-1216.[Medline] [Order article via Infotrieve] Cardoso EM, Macedo MG, Rohrlich P, et al. Increased hepatic iron in mice lacking classical MHC class I molecules. Blood. 2002;100: 4239-4241.[Abstract/Free Full Text] Camaschella C, Roetto A, De Gobbi M. Juvenile hemochromatosis. Semin Hematol. 2002;39: 242-248.[CrossRef][Medline] [Order article via Infotrieve] Nicolas G, Bennoun M, Devaux I, et al. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci U S A. 2001;98: 8780-8785.[Abstract/Free Full Text] Ahmad KA, Ahmann JR, Migas MC, et al. Decreased liver hepcidin expression in the Hfe knockout mouse. Blood Cells Mol Dis. 2002;29: 361-366.[CrossRef][Medline] [Order article via Infotrieve] Bridle KR, Frazer DM, Wilkins SJ, et al. Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet. 2003;361: 669-673.[CrossRef][Medline] [Order article via Infotrieve] Muckenthaler M, Roy CN, Custodio AO, et al. Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat Genet. 2003;34: 102-107.[CrossRef][Medline] [Order article via Infotrieve] 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.[Abstract/Free Full Text] Raulet DH. MHC class I-deficient mice. Adv Immunol. 1994;55: 381-421.[Medline] [Order article via Infotrieve] Schell TD, Mylin LM, Tevethia SS, Joyce S. The assembly of functional beta(2)-microglobulin-free MHC class I molecules that interact with peptides and CD8(+) T lymphocytes. Int Immunol. 2002;14: 775-782.[Abstract/Free Full Text] Nemeth E, Valore EV, Territo M, et al. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood. 2003;101: 2461-2463.[Abstract/Free Full Text] Doherty TM. T-cell regulation of macrophage function. Curr Opin Immunol. 1995;7: 400-404.[CrossRef][Medline] [Order article via Infotrieve] Holmstrom P, Dzikaite V, Hultcrantz R, et al. Structure and liver cell expression pattern of the HFE gene in the rat. J Hepatol. 2003;39: 308-314.[CrossRef][Medline] [Order article via Infotrieve] Zhang AS, Xiong S, Tsukamoto H, Enns CA. Localization of iron metabolism-related mRNAs in rat liver indicate that HFE is expressed predominantly in hepatocytes. Blood. 2004;103: 1509-1514.[Abstract/Free Full Text] Ilyin G, Courselaud B, Troadec MB, et al. Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload. FEBS Lett. 2003;542: 22-26.[CrossRef][Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: M. Constante, D. Wang, V.-A. Raymond, M. Bilodeau, and M. M. Santos Repression of Repulsive Guidance Molecule C during Inflammation Is Independent of Hfe and Involves Tumor Necrosis Factor-{alpha} Am. J. Pathol., February 1, 2007; 170(2): 497 - 504. [Abstract] [Full Text] [PDF] C. Chaudhury, J. Kim, S. Mehnaz, M. A. Wani, T. M. Oberyszyn, C. L. Bronson, S. Mohanty, W. L. Hayton, J. M. Robinson, and C. L. Anderson Accelerated Transferrin Degradation in HFE-Deficient Mice Is Associated with Increased Transferrin Saturation J. Nutr., December 1, 2006; 136(12): 2993 - 2998. [Abstract] [Full Text] [PDF] M. Constante, W. Jiang, D. Wang, V.-A. Raymond, M. Bilodeau, and M. M. Santos Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation Am J Physiol Gastrointest Liver Physiol, August 1, 2006; 291(2): G229 - G237. [Abstract] [Full Text] [PDF] H. Makui, R. J. Soares, W. Jiang, M. Constante, and M. M. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2003-09-3300v1 103/7/2847    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Miranda, C. J. Articles by Santos, M. M. Search for Related Content PubMed PubMed Citation Articles by Miranda, C. J. Articles by Santos, M. M. Related Collections Brief Reports Immunobiology Red Cells Social Bookmarking                   What's this?

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