Iron Overload and Heart Fibrosis in Mice Deficient for Both β2-Microglobulin and Rag1
2000; Elsevier BV; Volume: 157; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64827-4
ISSN1525-2191
AutoresMiguel M. Santos, Maria de Sousa, Luke H.P.M. Rademakers, Hans Clevers, J. J. M. Marx, Marco W. Schilham,
Tópico(s)Trace Elements in Health
ResumoGenetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, a β2-microglobulin (β2m)-associated major histocompatibility complex class I-like protein. Accordingly, mutant β2m−/− mice have increased intestinal iron absorption and develop parenchymal iron overload in the liver. In humans, other genetic and environmental factors have been suggested to influence the pathology and severity of HH. Previously, an association has been reported between low numbers of lymphocytes and the severity of clinical expression of the iron overload in HH. In the present study, the effect of a total absence of lymphocytes on iron overload was investigated by crossing β2m−/− mice (which develop iron overload resembling human disease) with mice deficient in recombinase activator gene 1 (Rag1), which is required for normal B and T lymphocyte development. Iron overload was more severe in β2m Rag1 double-deficient mice than in each of the single deficient mice, with iron accumulation in parenchymal cells of the liver, in acinar cells of the pancreas, and in heart myocytes. With increasing age β2m Rag1−/− mice develop extensive heart fibrosis, which could be prevented by reconstitution with normal hematopoietic cells. Thus, the development of iron-mediated cellular damage is substantially enhanced when a Rag1 mutation, which causes a lack of mature lymphocytes, is introduced into β2m−/− mice. Mice deficient in β2m and Rag1 thus offer a new experimental model of iron-related cardiomyopathy. Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, a β2-microglobulin (β2m)-associated major histocompatibility complex class I-like protein. Accordingly, mutant β2m−/− mice have increased intestinal iron absorption and develop parenchymal iron overload in the liver. In humans, other genetic and environmental factors have been suggested to influence the pathology and severity of HH. Previously, an association has been reported between low numbers of lymphocytes and the severity of clinical expression of the iron overload in HH. In the present study, the effect of a total absence of lymphocytes on iron overload was investigated by crossing β2m−/− mice (which develop iron overload resembling human disease) with mice deficient in recombinase activator gene 1 (Rag1), which is required for normal B and T lymphocyte development. Iron overload was more severe in β2m Rag1 double-deficient mice than in each of the single deficient mice, with iron accumulation in parenchymal cells of the liver, in acinar cells of the pancreas, and in heart myocytes. With increasing age β2m Rag1−/− mice develop extensive heart fibrosis, which could be prevented by reconstitution with normal hematopoietic cells. Thus, the development of iron-mediated cellular damage is substantially enhanced when a Rag1 mutation, which causes a lack of mature lymphocytes, is introduced into β2m−/− mice. Mice deficient in β2m and Rag1 thus offer a new experimental model of iron-related cardiomyopathy. The most relevant iron overload diseases in humans are primary, genetically determined, for example, hereditary hemochromatosis (HH. and secondary, transfusional and hemolysis related siderosis (eg, β-thalassemia). HH is an autosomal recessive disease, characterized by a defect in regulation of iron absorption, an increase of transferrin saturation, and progressive iron deposition predominantly in parenchymal cells of several organs.1Bothwell TH MacPhail AP Hereditary hemochromatosis: etiologic, pathologic and clinical aspects.Semin Hematol. 1998; 35: 55-71PubMed Google Scholar Toxicity resulting from iron accumulation in selective target organs leads to the development of liver cirrhosis, cardiomyopathy, diabetes mellitus, hypogonadism, and arthritis.1Bothwell TH MacPhail AP Hereditary hemochromatosis: etiologic, pathologic and clinical aspects.Semin Hematol. 1998; 35: 55-71PubMed Google Scholar, 2Bottomley SS Secondary iron overload disorders.Semin Hematol. 1998; 35: 77-86PubMed Google Scholar The study of the mechanisms of selective tissue accumulation and damage in which iron excess is believed to play a role has been difficult in part as a result of the lack of adequate experimental models of iron overload. Recently, a novel gene of the major histocompatibility complex class I family, HFE, has been found to be mutated in a large proportion of HH patients.3Feder JN Gnirke A Thomas W Tsuchihashi Z Ruddy DA Basava A Dormishian F Domingo Jr, R Ellis MC Fullan A Hinton LM Jones NI Kimmel BE Kronmal GS Lauer P Lee VK Loeb DB Mapa FA McClelland E Meyer NC Mintier GA Moeller N Moore T Morkang E Prass CE Quintana L Starnes SM Schatzman RC Brunke KJ Drayne DT Risch NJ Bacon BR Wolff RK A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.Nat Genet. 1996; 13: 399-408Crossref PubMed Scopus (3314) Google Scholar Previously, we characterized iron metabolism in major histocompatibility complex class I-deficient, β2-microglobulin knockout mice (β2m−/−), an animal model of HH.4De Sousa M Reimão R Lacerda R Hugo P Kaufmann SEH Porto G Iron overload in β2-microglobulin-deficient mice.Immunol Lett. 1994; 39: 105-111Crossref PubMed Scopus (178) Google Scholar, 5Santos M Schilham MW Rademakers LHPM Marx JJM De Sousa M Clevers H Defective iron homeostasis in β2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar Intestinal absorption of iron in β2m−/− mice is inappropriately increased, and transferrin saturation is abnormally high.6Santos M Clevers H De Sousa M Marx JJM Adaptive response of iron absorption to anemia, increased erythropoiesis, iron deficiency, and iron loading in β2-microglobulin knockout mice.Blood. 1998; 91: 3059-3065Crossref PubMed Google Scholar Pathological iron depositions occur predominantly in liver parenchymal cells, indicating defective iron storage in Kupffer cells.5Santos M Schilham MW Rademakers LHPM Marx JJM De Sousa M Clevers H Defective iron homeostasis in β2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar, 7Rothenberg BE Volan JR β2m knockout mice develop parenchymal iron overload: a putative role for class I genes of the major histocompatibility complex in iron metabolism.Proc Natl Acad Sci USA. 1996; 93: 1529-1534Crossref PubMed Scopus (196) Google Scholar In hemochromatosis patients, defective numbers of peripheral blood and liver lymphocyte populations are associated with a more severe clinical expression of iron overload.8Reimão R Porto G De Sousa M Stability of CD4/CD8 ratios in man: new correlation between CD4/CD8 profiles and iron overload in idiopathic haemochromatosis patients.CR Acad Sci Paris. 1991; 313: 481-487PubMed Google Scholar, 9Porto G Vicente C Teixeira MA Martins O Cabeda JM Lacerda R Gonçalves C Fraga J Macedo G Silva BM Alves H Justiça B De Sousa M Relative impact of HLA phenotype and CD4/CD8 ratios on the clinical expression of hemochromatosis.Hepatology. 1997; 25: 397-402Crossref PubMed Google Scholar, 10Arosa FA Oliveira L Porto G da Silva BM Kruijer W Veltman J De Sousa M Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28- T cells.Clin Exp Immunol. 1997; 107: 548-554Crossref PubMed Scopus (66) Google Scholar Correction of the iron overload does not correct the reported anomalies in lymphocyte numbers, and patients with abnormally low numbers of lymphocytes reach high transferrin saturations at a faster rate than those with normal lymphocyte numbers after completion of the phlebotomy treatment.9Porto G Vicente C Teixeira MA Martins O Cabeda JM Lacerda R Gonçalves C Fraga J Macedo G Silva BM Alves H Justiça B De Sousa M Relative impact of HLA phenotype and CD4/CD8 ratios on the clinical expression of hemochromatosis.Hepatology. 1997; 25: 397-402Crossref PubMed Google Scholar Together these observations indicate that the lymphocyte abnormalities precede and are not the consequence of the iron overload. To investigate the hypothesis that lymphocytes influence the development of iron overload, we introduced a deficiency in the recombinase activator gene 1 (Rag1) onto a β2m−/− genetic background. Rag1 deficiency results in total deficiency of B and T lymphocytes.11Mombaerts P Iacomini J Johnson RS Herrup K Tonegawa S Papaioannou VE Rag-1-deficient mice have no mature B and T lymphocytes.Cell. 1992; 68: 869-877Abstract Full Text PDF PubMed Scopus (2290) Google Scholar We report here the generation of double-deficient β2m Rag1−/− mice, which develop spontaneous iron overload. Challenge with dietary iron loading was obtained by placing mice on an iron-enriched diet containing 2.5% (w/w) carbonyl iron. Iron burden was substantially aggravated by the additional absence of Rag1, with massive iron accumulation in liver parenchymal cells, acinar cells of the pancreas, and heart myocytes. Surprisingly, β2m Rag1 double-knockout mice develop heart fibrosis, which could be prevented by reconstitution with normal hematopoietic cells. The β2m- and Rag1-deficient mice provide an interesting model to define the modifying influence of lymphocytes in iron homeostasis. In addition, this mouse model will facilitate investigation into the pathogenesis of iron-mediated myocardial failure. C57BL/6 mice aged 6 to 8 weeks were purchased from the IFFA Credo (Brussels, Belgium) and used as controls. The β2-microglobulin knockout (β2m−/−) mice were purchased from Jackson Immuno Research Laboratories (West Grove, PA), and Rag1−/−11Mombaerts P Iacomini J Johnson RS Herrup K Tonegawa S Papaioannou VE Rag-1-deficient mice have no mature B and T lymphocytes.Cell. 1992; 68: 869-877Abstract Full Text PDF PubMed Scopus (2290) Google Scholar were obtained from Dr. S. Tonegawa (Massachusetts Institute of Technology, Cambridge, MA). Both mutant mice have been back-crossed onto the C57BL/6 background. β2m−/− mice were bred to Rag1−/− to generate F1 offspring that were heterozygous for both genes. Because the β2m and Rag1 genes are closely linked, homozygous double knockout could only be obtained through recombination by breeding. Recombinants were detected as follows: the F2 offspring of the F1 interbreeding were screened by flow cytometry analysis (FACS) for the absence of T and B lymphocytes in peripheral blood samples. Mice identified as Rag1−/− were screened for recombination events by Southern blotting, using a β2m-specific probe, as described.5Santos M Schilham MW Rademakers LHPM Marx JJM De Sousa M Clevers H Defective iron homeostasis in β2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar Identified Rag1−/−β2m+/−mice were further intercrossed, and the F3 offspring were screened by FACS and Southern blotting. Double-deficient Rag1−/−β2m−/− mice were further bred in our animal facility. For all strains, both males and females were studied. All animals were 8 weeks old at the beginning of the experiments. All animals were given a commercial diet (RMH-B; Hope Farms, Woerden, The Netherlands), or, when indicated, an iron supplemented diet containing 2.5% (w/w) carbonyl iron (Sigma Immunochemicals, St. Louis, MO). For all animal experiments, written approval was obtained from the local Animal Experiments Committee of Utrecht University (Utrecht, The Netherlands). Organ samples were weighed wet, then dried overnight at 106°C and weighed again. The dried samples were ashed in an oven at 500°C for 17 hours, then fully solubilized in 6 mol/L HCl, and the final solution was adjusted with demineralized water to a final HCl concentration of 1.2 mol/L. Iron concentration of the samples was determined by flame atomic absorption spectrometry (Varian SpectrAA 250 Plus; Varian, Mulgrave, Victoria, Australia). Heparinized blood was obtained by orbital puncture under diethylether anesthesia. Hemoglobin, hematocrit, and mean corpuscular volume were determined using a Coulter-S counter (Coulter Electronics, Hialeah, FL). Plasma iron and total iron-binding capacity were determined by the ferrozine method (Iron FZ Test; Roche, Basel, Switzerland) with the COBAS-BIO autoanalyzer (Hoffman-La Roche BV, Mijdrecht, The Netherlands). Transferrin saturation was calculated from the total iron-binding capacity and plasma iron values. Samples of liver, spleen, kidney, lung, heart, and pancreas were fixed in buffered 4% formaldehyde. After routine histology processing, the paraffin sections were stained with hematoxylin and eosin and with azan for demonstration of fibrosis. Ferric iron, Fe(III), was detected by Perl's blue staining. Small pieces of pancreas and heart were fixed in a modified Karnovsky fixative consisting of 2.5% glutaraldehyde and 2. paraformaldehyde in 0.8 mol/L Na-cacodylate buffer, supplemented with 0.25 mmol/L CaCl2, and 0.5 mmol/L MgCl2 for at least 24 hours at 4°C. The tissue was washed twice with the same buffer, postfixed in 1. OsO4 and embedded in Epon 812. Semithin sections (1 μm) were stained with methylene blue and pararosanilin. Ultrathin sections (60 nm) were cut and contrasted with 3% uranyl magnesium acetate for 45 minutes at 63°C followed by Reynolds' lead citrate for 10 minutes. Stained and unstained sections were viewed in a Jeol JEM 1010 electron microscope (Joel LTD, Tokyo, Japan). For iron absorption tests the mice were fasted for 6 hours and housed for 3 days in cages equipped with grates to minimize coprophagy. All test doses were freshly prepared and were administered in aqueous solution using demineralized water. Measurement of iron absorption was performed as previously described.6Santos M Clevers H De Sousa M Marx JJM Adaptive response of iron absorption to anemia, increased erythropoiesis, iron deficiency, and iron loading in β2-microglobulin knockout mice.Blood. 1998; 91: 3059-3065Crossref PubMed Google Scholar Ferric-citrate (Sigma Immunochemicals) was added to 59Fe(III) citrate to obtain a total of 5 μg per mouse, with a 20-fold molar excess of sodium citrate dihydrate (Sigma Immunochemicals) to maintain mononuclear ferric-citrate complexes and to prevent precipitation. Each mouse received ∼50 kBq of 59Fe. The test dose was orally applied with the use of an olive-tipped oroesophageal needle. Total body radioactivity was measured with a whole-body γ counter (Automatic Scanner DS4/4S; Tracelab Ltd., Weybridge, Surrey, UK). The values were corrected for radioisotope decay and day-to-day fluctuations of the scanner with the use of a radium source. 59Fe absorption was determined by whole-body counting 7 days after administration of the test dose. When the animals were tested twice for iron absorption, background values of the first test dose were corrected for radioisotope decay. Recipient animals aged 8 weeks were lethally irradiated (9.5 Gy. and reconstituted with 5 × 106 fetal liver (embryonic day E13.5) cells by intravenous injection. Chimeras were sacrificed at 28 to 36 weeks after reconstitution and chimerism was monitored by flow cytometry analysis using αβTCR, B220, Mac-1, CD4, CD8, and H141.31.10 (anti-Kb) mAb (PharMingen, San Diego, CA). Expression of cell surface proteins was assayed by direct immunofluorescence. Samples of blood and spleen were stained with fluorescein isothiocyanate-conjugated or phycoerythrin-conjugated mAbs. Samples were then treated with FACS Lysing Solution (Becton Dickinson, Mountain View, CA) and washed in phosphate-buffered saline containing 2.5% fetal calf serum and 0.05% sodium azide. Fluorescence intensities were measured on a FACScan flow cytometer (Becton Dickinson). Results are presented as mean ± SEM. Student's t-test was used for comparison between the control and knockout mouse groups. For individual comparisons between two measurements, the paired t-test was used. The level of significance was preset at P < 0.05. β2m Rag1−/− double-knockout mice obtained from β2m−/− and Rag1−/− crossings were screened by Southern blot analysis on DNA extracted from tail samples. The absence of T and B lymphocytes was confirmed by FACS analysis. The majority (>95%) of the peripheral blood mononuclear cells and spleen cells expressed Mac-1 (CD11b), which stains macrophages, natural killer cells, and granulocytes, and were negative for αβTCR (T lymphocytes), B220 (B lymphocytes), and Kb (β2m-dependent, major histocompatibility complex class I) (data not shown). Determination of organ iron concentration, transferrin saturation, histochemical visualization of the cellular distribution of iron, and pathological examination of the extent of injury provide essential information about the type and degree of iron loading. To characterize iron homeostasis in β2m Rag1 double-knockout mice these parameters were analyzed and compared to single-knockout and wild-type (B6) mice. The responses to iron overloading were studied by feeding animals with a carbonyl-iron-supplemented diet (2.5% w/w). No significant differences were found between males and females, and hence the results for both genders were pooled. To determine iron distribution in different organs from mice fed with a standard diet (n = 9 to 12 per group), iron content was measured by flame atomic absorption spectrometry. All mice were sacrificed at 5 months of age. β2m single and β2m Rag1 double-knockout mice had significantly higher hepatic iron levels than B6 wild-type and Rag1−/− mice (Table 1 and Figure 1a; P < 0.0001). In contrast, splenic total iron levels of β2m−/−, Rag1−/−, and double-knockout mice were lower than those seen in B6 wild-type mice (Figure 1b; P < 0.01), a finding confirmed histologically. Noteworthy, β2m Rag1 double-knockout mice fed the standard diet had significantly higher iron levels in the heart than Rag1 single, β2m single-knockout, and B6 wild-type mice (Table 1 and Figure 1c; P < 0.0001). Plasma iron and transferrin saturation, as early markers of iron overload, were significantly higher in β2m-single and β2m Rag1 double-knockout mice (plasma iron >40 μmol Fe/ml; transferrin saturation >80%) when compared to B6 control or Rag1−/− mice (plasma iron <25 μmol Fe/ml; transferrin saturation <60%; P < 0.001).Table 1Tissue Iron Concentration in Mice Fed a Standard Dietμg Fe/g dry weightPOrganB6β2m−/−Rag1−/−β2m Rag1−/−versus B6versusβ2m−/−versusRag1−/−Liver258 ± 61552 ± 101256 ± 52682 ± 313<0.0001NS<0.0001Heart331 ± 56341 ± 74383 ± 104561 ± 138<0.0001<0.0001<0.0001Pancreas151 ± 33168 ± 32136 ± 26604 ± 731NSNSNSSpleen3688 ± 12991512 ± 5002130 ± 11051458 ± 605<0.003NSNSKidney287 ± 30303 ± 68254 ± 86283 ± 70NSNSNSLungs393 ± 32456 ± 68309 ± 99417 ± 59NSNSNSData are presented as mean ± SD. Animals were analyzed at 5 months of age. P = Student's t-test for comparison of β2m Rag1−/−mice with B6 control, β2m−/−, and Rag1−/− mice. Open table in a new tab Data are presented as mean ± SD. Animals were analyzed at 5 months of age. P = Student's t-test for comparison of β2m Rag1−/−mice with B6 control, β2m−/−, and Rag1−/− mice. Overall, when comparing mice kept on a standard diet, body iron levels were the highest in β2m Rag1 double-knockout mice, followed by β2m single-knockout mice, whereas no significant differences in body iron levels were found between Rag1 single-knockout and B6 wild-type mice. After feeding the animals an iron-enriched diet for 12 weeks (n = 12 to 16 per group), both β2m-single and β2m Rag1 double-knockout mice were unable to increase iron levels in spleens (Figure 1b). This inability to store excess iron in spleens was most evident in the β2m Rag1 double-knockout mice, which had only half the total iron content (44 ± 8 μg Fe) of that in wild-type mice (96 ± 8 μg Fe). On the other hand significantly higher amounts of iron were found in the heart (Figure 1c; P < 0.0001) and pancreas of double-knockout, but not single-knockout mice (Figure 1d; P < 0.001) after dietary iron loading. Total iron levels in lungs and kidneys were not significantly different between mouse strains and treatments (data not shown). Transferrin saturation after feeding the iron-enriched diet, increased in B6 control and Rag1−/− mice to >80%, reaching levels similar to those seen in β2m- and β2m Rag1 double-knockout mice kept on a standard diet. Plasma iron concentration in iron-loaded animals was significantly lower in B6 control mice compared to all of the other strains (B6: plasma iron 50 μmol Fe/ml in all other strains; P < 0.01). Taken together, these results show that iron burden is accentuated in dietary iron-loaded β2m Rag1 double-knockout mice when compared to the respective single knockout mice. A typical feature of pathological iron overload in humans is the cellular distribution of storage iron, which has been particularly difficult to mimic in rodents. Therefore, we determined histologically the cellular distribution of storage iron in liver, pancreas, and heart in mice fed a standard diet and in dietary iron-loaded animals. Perl's blue-staining of liver sections from β2m-single and β2m Rag1 double-knockout mice kept on a standard diet revealed the presence of excess iron, which was predominantly in parenchymal cells (data not shown).4De Sousa M Reimão R Lacerda R Hugo P Kaufmann SEH Porto G Iron overload in β2-microglobulin-deficient mice.Immunol Lett. 1994; 39: 105-111Crossref PubMed Scopus (178) Google Scholar, 5Santos M Schilham MW Rademakers LHPM Marx JJM De Sousa M Clevers H Defective iron homeostasis in β2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar Moderate deposits were also observed in the pancreas and the heart of 24- to 30-week-old β2m Rag1 double-knockout mice, but not in the β2m-single and Rag1 single-knockout mice or B6 wild-type mice (data not shown). As previously reported for shorter loading periods,5Santos M Schilham MW Rademakers LHPM Marx JJM De Sousa M Clevers H Defective iron homeostasis in β2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar iron deposition in the liver of B6 wild-type mice fed an iron-enriched diet up to 12 weeks was particularly prominent in Kupffer cells, and was also present in parenchymal cells (Figure 2a). Surprisingly, Rag1 single-knockout mice, that supposedly have normal Kupffer cells, develop hepatic iron overload on dietary iron loading exclusively in parenchymal cells (data not shown), like HH patients and β2m−/− mice.5Santos M Schilham MW Rademakers LHPM Marx JJM De Sousa M Clevers H Defective iron homeostasis in β2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar Dietary iron-loaded β2m Rag1 double-knockout mice show heavy iron depositions in the livers that corresponded to the appearance of hepatocyte clusters (Figure 2b). A remarkable iron loading was present in the pancreas and the heart of β2m Rag1 double-knockout mice (Figure 2, d and f), which was not observed in control B6 (Figure 2, c and e), and β2m single-knockout mice (data not shown). Importantly, in the pancreas this prominent iron deposition was present in acinar cells (Figure 2d), and in the heart it was present in myocytes and in the interstitial tissue (Figure 2f). Examination of hearts from β2m Rag1 double-knockout mice by electron microscopy revealed frequent lysosomal structures containing granular electron-dense material in the cytoplasm of myocytes (Figure 3, a and b). Similar lysosomal iron deposition was observed in mesenchymal perivascular cells. In the pancreas, the acinar cells contained large lysosomes of moderate electron density (Figure 3c). In these lysosomes, scattered ferritin particles were present (Figure 3d). Ferritin accumulation was also evident in the cytoplasm of acinar cells. Overall, dietary iron-loaded β2m Rag1 double-knockout mice develop a more severe iron burden in multiple organs than each of the single-knockout mice, indicating an additive effect of the two mutations. To exclude the possibility that anemia could account for the abnormal iron storage defect in β2m Rag1 double-knockout mice, several erythroid parameters were determined. The results demonstrated that hemoglobin, hematocrit, and mean corpuscular volume were even higher in β2m-single and in β2m Rag1 double-knockout mice when compared to B6 and Rag1−/− mice fed a standard diet (Table 2). We observed an increase of hemoglobin, hematocrit, and mean corpuscular volume values to a similar extent when B6 and Rag1−/− mice were fed the iron-enriched diet for 12 weeks. Thus, the excess storage iron found in β2m Rag1 double-knockout mice could not be attributed to defective erythropoiesis or hemoglobin synthesis.Table 2Erythroid ParametersStrainTreatmentnRBC, ×1012/LHb, mmol/LHCT, %MCV, flB6−89.0 ± 0.58.7 ± 0.539 ± 343 ± 2Carbonyl-iron89.4 ± 0.710.4 ± 0.344 ± 446 ± 1βm−/−−69.2 ± 0.69.9 ± 0.944 ± 448 ± 2Carbonyl-iron89.9 ± 0.110.8 ± 0.249 ± 249 ± 2RAG1−/−−78.9 ± 0.88.9 ± 0.539 ± 342 ± 2Carbonyl-iron89.4 ± 0.49.9 ± 0.543 ± 346 ± 2β2mRAG1−/−−89.6 ± 0.510.2 ± 0.744 ± 446 ± 1Carbonyl-iron69.4 ± 0.110.5 ± 0.247 ± 247 ± 2Data are presented as mean ± SD. n, number of animals. Animals were 5 months old. RBC, red blood cell; Hb, hemoglobin; HCT, hematocrit; MCv, mean corpuscular volume. Open table in a new tab Data are presented as mean ± SD. n, number of animals. Animals were 5 months old. RBC, red blood cell; Hb, hemoglobin; HCT, hematocrit; MCv, mean corpuscular volume. To investigate the effect of the Rag1 mutation on the absorption of iron, ferric iron, Fe(III), absorption6Santos M Clevers H De Sousa M Marx JJM Adaptive response of iron absorption to anemia, increased erythropoiesis, iron deficiency, and iron loading in β2-microglobulin knockout mice.Blood. 1998; 91: 3059-3065Crossref PubMed Google Scholar was measured before and after feeding an iron-enriched diet for 14 days (Figure 4). Ferric iron absorption after this treatment significantly decreased in all mouse strains (P < 0.0001). However, iron absorption in β2m-single and β2m Rag1 double-knockout mice was persistently higher, before and after treatment, when compared to wild-type (B6) or Rag1 single-knockout mice (P < 0.0001,Figure 4). No significant differences were found between iron absorption in β2m-single and β2m Rag1 double-knockout mice, indicating that the Rag1 mutation has no further influence on iron absorption in the gut. Iron deposition in the heart deserves special interest, because heart failure is a frequent cause of death in untreated HH and posttransfusional secondary hemochromatosis.12Finch SC Finch CA Idiopathic hemochromatosis, an iron storage disease.Medicine. 1955; 34: 381-430Crossref PubMed Scopus (319) Google Scholar, 13Buja LM Roberts WC Iron in the heart. Etiology and clinical significance.Am J Med. 1971; 51: 209-221Abstract Full Text PDF PubMed Scopus (382) Google Scholar, 14Cutler DJ Isner JM Bracey AW Hufnagel CA Conrad PW Roberts WC Kerwin DM Weintraub AM Hemochromatosis heart disease: an unemphasized cause of potentially reversible restrictive cardiomyopathy.Am J Med. 1980; 69: 923-928Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 15MacDonald RA Mallory GK Hemochromatosis and hemosiderosis: study of 211 autopsied cases.Arch Intern Med. 1960; 105: 686-697Crossref PubMed Scopus (109) Google Scholar, 16Tuomainen T-P Punnonen K Nyyssönen K Salonen JT Association between body iron stores and the risk of acute myocardial infarction in men.Circulation. 1998; 97: 1461-1466Crossref PubMed Scopus (238) Google Scholar Remarkably, 17 out of 21 β2m Rag1 double-knockout mice aged between 20 and 28 weeks and kept on a standard diet developed heart fibrosis, as detected by azan staining, which was never seen in β2m- and Rag1-single-knockout mice or control mice of the same age and kept on the standard diet (Figure 5, a and b). Only after feeding an iron-enriched diet for 3 months, heart fibrosis was additionally observed in Rag1 single-knockout mice, but not in β2m single-knockout or B6 wild-type mice (data not shown). Previously we have demonstrated that reconstitution of β2m−/− mice with normal hematopoietic cells, redistributes the iron from parenchymal to Kupffer cells in the liver.5Santos M Schilham MW Rademakers LHPM Marx JJM De Sousa M Clevers H Defective iron homeostasis in β2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.J Exp Med. 1996; 184: 1975-1985Crossref PubMed Scopus (191) Google Scholar To further investigate the influence of hematopoietic cells in the development of iron-related heart fibrosis, we reconstituted lethally irradiated 8-week-old β2m Rag1 double-knockout mice with fetal liver-derived hematopoietic progenitor cells from normal mice. All reconstituted β2m Rag1 double-knockout mice (n = 4) showed a normal histology up to 36 weeks of age (Figure 5 c). The control β2m Rag1 double-knockout mice reconstituted with β2m Rag1−/−-derived cells (n = 5) were sacrificed between 20 to 28 weeks when they became ill and had developed extensive fibrosis in the heart (Figure 5d). Thus, wild-type hematopoietic cell transfer prevents the development of heart fibrosis in β2m Rag1 double-knockout mice. The aim of this study was to investigate the modifying influence of lymphocytes in the pathology of iron overload. Such a modifying role has been suggested by the association between low numbers of T lymphocytes in patients with HH and a more severe clinical expression of iron overload.8Reimão R Porto G De Sousa M Stability of CD4/CD8 ratios in man: new correlation between CD4/CD8 profiles and iron overload in idiopathic haemo
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