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From the Departments of Immunology,*
Pathology,
and Internal
Medicine,§
University Hospital Utrecht,
Utrecht, The Netherlands; the Molecular Immunology
Laboratory,
Instituto de Biologia Molecular e
Celular, Porto, Portugal; and the Department of
Pediatrics, Leiden University Medical Center,
Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.3 Previously, we characterized iron metabolism in major histocompatibility complex class I-deficient, ß2-microglobulin knockout mice (ß2m-/-), an animal model of HH.4,5 Intestinal absorption of iron in ß2m-/- mice is inappropriately increased, and transferrin saturation is abnormally high.6 Pathological iron depositions occur predominantly in liver parenchymal cells, indicating defective iron storage in Kupffer cells.5,7
In hemochromatosis patients, defective numbers of peripheral blood and liver lymphocyte populations are associated with a more severe clinical expression of iron overload.8-10 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.9 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 ß2m-/- genetic background. Rag1 deficiency results in total deficiency of B and T lymphocytes.11 We report here the generation of double-deficient ß2mRag1-/- 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, ß2mRag1 double-knockout mice develop heart fibrosis, which could be prevented by reconstitution with normal hematopoietic cells. The ß2m- 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice aged 6 to 8 weeks were purchased from the IFFA Credo (Brussels, Belgium) and used as controls. The ß2-microglobulin knockout (ß2m-/-) mice were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA), and Rag1-/-11 were obtained from Dr. S. Tonegawa (Massachusetts Institute of Technology, Cambridge, MA). Both mutant mice have been back-crossed onto the C57BL/6 background. ß2m-/- mice were bred to Rag1-/- to generate F1 offspring that were heterozygous for both genes. Because the ß2m 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 ß2m-specific probe, as described.5 Identified Rag1-/-ß2m+/-mice were further intercrossed, and the F3 offspring were screened by FACS and Southern blotting. Double-deficient Rag1-/-ß2m-/- 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).
Measurement of Tissue Iron Levels
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).
Transferrin Saturation and Hematological Measurements
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.
Histology
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.
Electron Microscopy
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).
Gastrointestinal Iron Absorption
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.6
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 
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.
Fetal Liver Cell Transfer
Recipient animals aged 8 weeks were lethally irradiated (9.5 Gy)
and reconstituted with 5 x 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 
ßTCR, B220, Mac-1, CD4,
CD8, and H141.31.10 (anti-Kb) mAb (PharMingen,
San Diego, CA).
Flow Cytometry
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).
Statistical Analysis
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.
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Results |
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ß2mRag1-/-
double-knockout mice obtained from
ß2m-/- 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 ßTCR (T lymphocytes), B220 (B lymphocytes), and
Kb (ß2m-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 ß2mRag1 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.
Total Iron in Organs, Plasma Iron, and Plasma Transferrin Saturation
Spontaneous Iron Overload
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. ß2m single
and ß2mRag1 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
ß2m-/-,
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, ß2mRag1 double-knockout
mice fed the standard diet had significantly higher iron levels in the
heart than Rag1 single, ß2m
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
ß2m-single and ß2mRag1
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).
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Dietary Iron Overload
After feeding the animals an iron-enriched diet for 12 weeks
(n = 12 to 16 per group), both
ß2m-single and ß2mRag1
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 ß2mRag1
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 ß2m- and ß2mRag1 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 <29 versus >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 ß2mRag1 double-knockout mice when compared to the respective single knockout mice.
Cellular Distribution of Storage Iron
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.
Spontaneous Iron Overload
Perl’s blue-staining of liver sections from ß2m-single and ß2mRag1 double-knockout mice kept on a standard diet revealed the presence of excess iron, which was predominantly in parenchymal cells (data not shown).4,5 Moderate deposits were also observed in the pancreas and the heart of 24- to 30-week-old ß2mRag1 double-knockout mice, but not in the ß2m-single and Rag1 single-knockout mice or B6 wild-type mice (data not shown).
Dietary Iron Overload
As previously reported for shorter loading periods,5
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
ß2m-/- mice.5
Dietary iron-loaded ß2mRag1
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
ß2mRag1 double-knockout mice (Figure 2, d
and f), which was not observed in control B6 (Figure 2, c
and e), and ß2m 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
).
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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.
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Erythroid Parameters
To exclude the possibility that anemia could account for the
abnormal iron storage defect in ß2mRag1
double-knockout mice, several erythroid parameters were determined. The
results demonstrated that hemoglobin, hematocrit, and mean corpuscular
volume were even higher in ß2m-single and in
ß2mRag1 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
ß2mRag1 double-knockout mice could not
be attributed to defective erythropoiesis or hemoglobin synthesis.
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To investigate the effect of the Rag1 mutation on
the absorption of iron, ferric iron, Fe(III), absorption6
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
ß2m-single and ß2mRag1
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 ß2m-single and
ß2mRag1 double-knockout mice,
indicating that the Rag1 mutation has no further
influence on iron absorption in the gut.
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Iron deposition in the heart deserves special interest, because
heart failure is a frequent cause of death in untreated HH and
posttransfusional secondary hemochromatosis.12-16
Remarkably, 17 out of 21 ß2mRag1
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 ß2m- 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
ß2m single-knockout or B6 wild-type mice (data not
shown).
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c). The control
ß2mRag1 double-knockout mice
reconstituted with
ß2mRag1-/--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
ß2mRag1 double-knockout mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the ß2m-deficient mice that develop a progressive iron overload similar to that seen in HH patients,4-7 we introduced the Rag1 mutation,11 to create a total absence of mature lymphocytes.
When kept on a standard diet, the double-knockout mice develop a more severe phenotype than the ß2m-deficient mice, involving increased iron accumulation in the liver, heart, and pancreas. The ß2mRag1 double-knockout mice have visible iron depositions specifically in parenchymal cells of the liver and significantly higher iron levels in the heart than single-knockout and control mice. This indicates that the additional absence of lymphocytes, in the ß2m model of iron overload, exacerbates the accumulation of iron in target organs, especially the heart. Moreover, the double-deficient mice spontaneously develop fibrosis in the heart.
The observed phenotype in the double-deficient mice is also an accentuation of the phenotype of the Rag1 single-knockout mice, which can normally regulate iron absorption and storage, and do not develop heart fibrosis under standard conditions. Rag1 single-knockout mice will develop heart fibrosis after very long periods of dietary iron loading of at least 12 weeks. Thus, dietary iron loading in combination with the lack of lymphocytes leads to cardiomyopathy. Altered cellular distribution of the iron in the heart may be a contributing factor in the development of cardiomyopathy, and may change in the absence of lymphocytes, as was observed in the liver of Rag1 single-knockout mice after dietary overloading. In the ß2mRag1 double-knockout mice, dietary iron loading is not necessary because the ß2m mutation leads to iron overload already under normal conditions.
When fed an iron-supplemented diet, ß2mRag1 double-knockout mice, like ß2m-single and HFE-/- mice,17 have a significantly lower capacity to store iron in the spleens when compared with B6 control mice on the same diet. This is partially because of the absence of a functional HFE-ß2m complex, which could lead to defective storage of iron in reticuloendothelial cells. Importantly, HH patients have been reported to have a defect in iron storage in reticuloendothelial cells.18-20 The lower capacity to store iron may be aggravated by the lack of lymphocytes.21,22 The lack of lymphocytes alone in Rag1-deficient mice leads to an aberrant storage of iron exclusively in parenchymal cells on dietary iron overload, indicating that lymphocytes may influence the iron storage capacity of reticuloendothelial cells.
As a consequence of the deficient iron metabolism in the double-mutant mice, excess iron is progressively deposited in the liver, heart, and pancreas. Thus, dietary iron overload in double-mutant mice leads to an exacerbation of the pattern of tissue iron deposition observed when the mice are kept on a standard diet.
Iron deposition in the hearts of ß2mRag1 double-knockout mice, presumably leading to fibrosis, deserves special attention because heart failure is the most important life-threatening situation in untreated HH and in secondary hemochromatosis.12-16 To our knowledge, experimentally induced iron-related cardiomyopathy has never been reported before in mice.
Cardiac manifestations are apparent in 20% to 30% of patients
presenting with clinical manifestations of HH. In younger patients they
are often the presenting feature and almost always the cause of early
death unless the iron is removed.1
In both HH and
secondary hemochromatosis the iron is found predominantly within
myocytes, leading to degeneration and fibrosis, with disturbances of
cardiac rhythm and eventually death.12-16,23
The typical
deposition of iron in myocytes and the associated tissue damage has
been difficult to mimic in animal models. In rats, after regular
feeding of carbonyl iron24
or the more efficient
trimethylhexanoyl-ferrocene,25
modest iron deposits are
found in endothelial cells and perivascular macrophages. In these
animal models, no stainable iron is found in myocytes and cellular
damage does not occur.
The mechanism by which excess iron in myocytes causes damage may involve oxidative stress and the consecutive alteration of myocyte functions, through the iron-catalyzed Fenton chemistry.26,27 The reason why the heart is the first organ to be affected may relate to the fact that the anti-oxidant enzyme equipment varies among tissues.28 It is interesting to note that in several other instances related to oxidative stress the heart also seems to be a major target organ involved.29,30
A ß2m-deficient mouse lacks appropriate surface expression of the HFE gene product. The introduction of the Rag1 mutation leads to the additional absence of T and B cells. In HH patients, a correlation between T lymphocytes and the severity of the iron overload has been reported.8 In these patients the numbers of B lymphocytes are normal and do not change after phlebotomy treatment.9 No direct influence of B lymphocytes on iron metabolism or iron-binding proteins has been suggested. Thus, it is unlikely that the absence of B cells leads to the reported effects on iron metabolism in ß2mRag1 double-deficient mice.
On the other hand, T lymphocytes are major regulators of cytokine
production, either directly or indirectly via regulation of macrophage
function. The lack of iron storage in the Kupffer cells of dietary
overloaded Rag1 mutant mice may be an illustration of
such an indirect mechanism. Cytokines produced locally by T lymphocytes
and macrophages, namely interleukins, tumor necrosis factor-, and
interferon-, are powerful modifiers of iron
homeostasis.31,32
For example, after dietary iron
overload, wild-type mice respond with an increase in tumor necrosis
factor- production, which in turn down-regulates intestinal iron
absorption via increase in ferritin expression in intestinal epithelial
cell.33
Such cytokine-induced alterations in iron
metabolism are also clearly illustrated in the pathogenesis of anemia
of chronic disease, the most frequent anemia found in hospitalized
patients, often occurring in patients with chronic infectious,
inflammatory, and neoplastic disorders.34,35
In anemia of
chronic disease, associated disturbances of iron homeostasis include
withdrawal of the metal from the sites of erythropoiesis and the
circulation to the storage compartment in the reticuloendothelial
system.
Cytokines have also been associated with cardiomyocyte loss in other
studies.36
Cytokines secreted by T-helper type 1
lymphocytes, such as interleukin-1, interleukin-2, and interferon-
can induce tumor necrosis factor production from target cells,
including myocytes. Tumor necrosis factor and several other cytokines
are able to induce nitric oxide production, which depresses cardiac
function and can induce apoptosis.37
The effect of dietary iron overload on the heart of Rag1 single-mutant mice and the exacerbated phenotype in the double-mutant mice suggest that dysregulation of cytokine production may be responsible for the specific cellular iron storage in the heart and the loss of cardiomyocytes. The cytokines may either be released by lymphocytes or be locally produced and regulated by lymphocytes.
The cardiac phenotype was prevented by transfer of normal hematopoietic cells into double-deficient mice, indicating that the combined effect of both mutations on the heart during the first 8 weeks of life could be reversed by the combined introduction of normal reticuloendothelial cells expressing a functional HFE molecule and mature lymphocytes expressing a functional antigen receptor. This reversal may reflect a redistribution of iron under the influence of normal hematopoietic cells.
In conclusion, the present study shows that the development of iron overload pathology is substantially enhanced when a Rag1 mutation, which causes a lack of mature lymphocytes, is introduced into ß2m-/- mice. The ß2mRag1 double-knockout mouse model represents an ideal animal model of iron-mediated cardiomyopathy, and will be a useful model to evaluate therapeutic strategies not only for prevention and correction of iron overload, but also for the treatment of iron-related tissue damage. In addition, mice deficient in both ß2m and Rag1 offer a new experimental model for defining in vivo which lymphocytes play a role in iron-related pathological processes and by what mechanism. This model may contribute to the understanding of the heterogeneity of the pathology of HH in man.38,39
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Acknowledgements |
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Footnotes |
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Supported by a grant from Junta Nacional de Investigação Científica e Tecnológica-PRAXIS XXI (BD/2866/94).
Accepted for publication August 21, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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