Blood, 1 December 2002, Vol. 100, No. 12, pp. 4239-4241
BRIEF REPORT
Increased hepatic iron in mice lacking classical MHC class I
molecules
Elsa M. Cardoso,
Maria G. Macedo,
Pierre Rohrlich,
Eduarda Ribeiro,
Manuel T. Silva,
François A. Lemonnier, and
Maria de
Sousa
From Molecular Immunology and Immunobiology, Institute
for Molecular and Cell Biology; Instituto Superior de Ciências da
Saúde-Norte; Molecular Immunology and Pathology, Instituto de
Ciências Biomédicas Abel Salazar, Oporto, Portugal;
Unité d'Immunité Cellulaire Antivirale, Institut Pasteur,
Paris, France.
 |
Abstract |
Iron accumulation in the liver in hereditary hemochromatosis (HH)
has been shown to be highly variable. Some studies point to the
importance of major histocompatibility complex (MHC) class I
(MHC-I) and CD8+ cells as modifiers of iron overload. In
this report, using mice knockout for H2Kb
/
and H2Db/ genes, it is demonstrated that
lack of classical MHC-I molecules results in a spontaneous increase of
nonheme iron content in the liver (mainly located in the hepatocytes)
when compared to wild-type mice. In CD8/
and Rag2/ mice, no spontaneous hepatic iron
accumulation was observed. These results demonstrate for the first time
that classical MHC-I molecules could be involved in the regulation of
iron metabolism and contribute to the established genotype/phenotype
discrepancies seen in HH.
(Blood. 2002;100:4239-4241)
© 2002 by The American Society of Hematology.
| |
Introduction |
Since the discovery of the HFE
gene,1 several molecules implicated in heritable defects
of iron metabolism have been identified, giving new insights into the
molecular control of cellular pathways of iron balance.2
Despite these advances, a considerable unexplained variability in the
amount of iron loading in HFE hemochromatosis still
persists.3,4 Thus, the finding of new molecular regulators to further improve our understanding of iron homeodynamics continues to
be pertinent. Recently, Andrews and collaborators made an important contribution in this area by characterizing genes that modify the
hemochromatosis phenotype in mice. They reported that mice double
knockout for Hfe and 
2-microglobulin
(2m) accumulate more tissue iron than mice lacking
Hfe only.4 This finding suggests that other(s)
2m-interacting protein(s), such as classical major
histocompatibility complex (MHC) class I molecule(s) (MHC-I), may be
involved in iron regulation. Alternatively, MHC-dependent cells such as
CD8+ T lymphocytes or others could play a role in iron
metabolism.5 To further explore other candidate molecular
and cellular regulators of iron balance, we investigated the
spontaneous iron status of mice with disrupted classical
Mhc-I genes and of mice knockout for CD8 and
Rag2 genes.
| |
Study design |
C57BL/6J (B6), H2Kb/,
H2Db/ single-knockout and
H2Kb/Db/ double-knockout
mice were raised at the Pasteur Institute animal facilities and are
reported elsewhere.6 All H2 knockout mice were
backcrossed onto the B6 background for 12 generations. The
CD8 knockout mice (CD8
/)7 that had
been backcrossed to B6 for 13 generations were purchased from The
Jackson Laboratory (Bar Harbor, ME) at 8 to 9 weeks and aged at the
Institute for Molecular and Cell Biology (IBMC) animal
facility. Rag2/8 were
backcrossed 9 times onto the B6 background; additional B6 mice were
bred in IBMC. All mice used in this study were male, aged 4 to
5 months, and were maintained on standard mouse diet. Iron status was
evaluated as previously described.9 Iron staining in the
liver was performed by using the Perls Prussian blue method, and evaluation of ferritin accumulation was done by electron microscopy in liver samples processed as described.10 The blood
samples were obtained by cardiac puncture in mice under anesthesia or by retro-orbital bleeding. Serum transferrin saturation was calculated by dividing serum iron by total iron-binding capacity (TIBC) and multiplying by 100. For the hematocrit, fresh peripheral blood was
harvested in EDTA (ethylenediaminetetraacetic acid) tubes and
assessed on a Pentra 120 counter (ABX, Montpellier, France). Flow
cytometry analysis of CD4+ and CD8+ T cells was
performed on splenocytes by direct immunofluorescence. Red blood cells
were removed by osmotic lysis, and splenic cells were stained with
anti-T-cell receptor (anti-TCR)-CyChrome (H57-597; PharMingen Europe, Heidelberg, Germany),
CD8-fluorescein isothiocyanate (FITC), and CD4-phycoerythrin
(PE) (Caltag Laboratories, Burlingame, CA) monoclonal
antibodies. Cells were passed on a FACSCalibur flow cytometer, and data
were analyzed by using CellQuest (Becton Dickinson, Aalst, Belgium).
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Results and discussion |
To investigate whether classical MHC-I molecules are involved in
iron regulation, the iron status of mice totally devoid of classical
MHC-I molecules
(H2Kb/Db/)6 was
evaluated. These mice are more adequate than other models that have
been used as MHC-I deficient, such as mice knockout for
2m, or for the transporter associated with
antigen processing (TAP), in which low levels of classical MHC-I may
still be expressed on the cell surface.11
Interestingly, H2Kb/Db/ mice
had significantly higher (P = .0009) hepatic nonheme iron
content (mean ± SD, 481 ± 120 µg/g dry wt) than the B6
control mice (299 ± 54 µg/g dry wt) (Figure
1). Single H2Kb
and H2Db knockout mice had a phenotype
intermediate between the double-knockout and the wild-type (mean ± SD, 358 ± 65 and 392 ± 41 µg/g dry wt, respectively,
Figure 1).
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| Figure 1.
Increased hepatic nonheme iron concentration in mice
lacking classical MHC class I molecules.
Liver samples from B6, H2Kb/,
H2Db/,
H2Kb/Db/,
CD8/, and Rag2/
male mice aged 4 to 5 months were analyzed for nonheme iron
concentration. Individual values are represented. n indicates the
number of mice analyzed; the mean and the standard deviation (SD)
values are indicated. P values were calculated by the
unpaired Student 2-tailed t test; values of
P < .05 were considered significant. As indicated, mean
values from H2Kb/,
H2Db/, and
H2Kb/Db/ mice were
significantly different from B6 control mice; not indicated, but also
significantly different (P < .05), were the mean values
for these groups: H2Kb/ versus
H2Kb/Db/;
H2Kb/ versus
CD8/; H2Kb/
versus Rag2/;
H2Db/ versus
CD8/; H2Db/
versus Rag2/;
H2Kb/Db/ versus
CD8/; and
H2Kb/Db/ versus
Rag2/.
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In agreement with the higher quantitative hepatic iron concentration of
the H2Kb/Db/ mice, positive
Perls Prussian blue iron staining in a vast majority of hepatocytes was
seen (Figure 2A). Occasional Kupffer
cells also were positive. On the contrary, no stainable iron was
observed in B6 mice (Figure 2B). In addition, numerous ferritin
lysosomes with abundant iron-containing ferritin molecules were
observed by electron microscopy in hepatocytes of the
H2Kb/Db/ mice. Moreover, in
these mice, frequent cytosolic ferritin molecules also were observed
(Figure 2C). Conversely, in wild-type mice (B6) lysosomal ferritin was
scarce, and no ferritin accumulation was observed in the cytoplasm
(Figure 2D).
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| Figure 2.
Iron ferritin in the liver.
Representative sections from the liver of a double-knockout mouse for
classical MHC-I molecules
(H2Kb/Db/) (A,C) and from a
control B6 mouse (B,D). (A-B) Histological sections stained for ferric
iron (Perls); (C-D) ultrathin section of the same liver sample,
contrasted with lead citrate. The
H2Kb/Db/ and the B6 mice
shown had a hepatic iron concentration of 517 µg/g dry wt and 252 µg/g dry wt, respectively. In the picture representative of the
results seen in H2Kb/Db/
mice, the iron was detected in hepatocytes (A), while in B6 mice no
stainable iron could be found (B). Although in B6 mice, lysosomes
containing some iron ferritin could be visualized (arrows, D),
H2Kb/Db/ mice had many
lysosomes, with abundant iron ferritin, as well as iron ferritin in the
cytoplasm (arrowheads, C). Original magnification × 1000 (A-B); EM
magnification × 90 000 (C-D).
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The increased hepatic iron content observed could be associated with
the reduction in CD8+ T-lymphocyte numbers seen in these
mice. The mice with the H2Kb,
H2Db, or both genes disrupted had a
significantly lower percentage of CD8+ T lymphocytes in the
spleen (mean ± SD, 13% ± 2%, 20% ± 5%, and 3% ± 1%,
respectively, P < .05) compared to the B6 control mice (34% ± 4%). To control for this possibility, additional controls were examined. As shown in Figure 1, livers of
CD8/ and Rag2/
mice (both with less than 0.5% of splenic CD8+ T
lymphocytes) had a hepatic iron concentration comparable to B6 mice
(261 ± 51 µg/g dry wt and 272 ± 42 µg/g dry wt,
respectively). Similar results were reported
previously.12,13
The increased iron deposits in the liver were observed without a
corresponding increase in serum transferrin saturation
(32% to 40%) or in the hematocrit (44% to 47%). No significant
differences were found between groups. The finding of hepatic iron
overload, without changes in the transferrin saturation or in the
hematocrit, may indicate that MHC class I molecules are important for
the regulation of iron import/export by hepatocytes. Iron accumulation before the circulating transferrin is fully saturated has been seen by
others in both humans14 and mice.15 Studies of
iron absorption are needed to define further the mechanism leading to
hepatic iron load presented in this first report. However, in
comparison with published reports of liver iron overload in 2m/ and
Hfe/ mice, the values found in
H2Kb/Db/ mice are similar or
slightly higher than those seen in heterozygous 2m+/16 and
Hfe+/.17
The present data indicate that the reported spontaneous increase in
hepatic iron content is specific of MHC class I genotype. Earlier
studies have shown that hepatic magnesium and zinc content in mice were
associated with the H2 genotype.18,19 Even
though many of these associations could be due to genes within the MHC locus, there also is evidence indicating that classical MHC antigens themselves may regulate physiological processes by interacting physically on the cell surface with different
ligands.20-22 HFE itself, a nonclassical MHC-I protein,
was shown to interact with the transferrin receptor.23
Recently, a study in 2m/ mice
demonstrated that MHC-I molecules are functionally required for the
development and plasticity of the central nervous
system.24 Thus, besides the well-characterized role of
MHC-I molecules in immune responses, they may play other regulatory
functions. Classical MHC-I molecules could associate with molecules
critical for iron transport within or on the cell surface. In
conclusion, the present results indicate that MHC class I expression
may be an additional contributory factor to the clinical heterogeneity
found in iron overload.
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Acknowledgments |
We thank Luisa Mariano (Molecular Immunology and Pathology, ICBAS)
and Ana do Vale (Immunobiology, IBMC) for assistance during the
preparation of samples for electron microscopy, and Júlia Carvalho (Clinical Chemistry, Santo António General Hospital) for
serum iron analysis. We thank Jean Luc Decourt, Marie Françoise Hurtaud (Hematologie, Hopital Robert Debré, Paris), F. Arosa (IBMC, Oporto) and C. S. Cardoso for their valuable help.
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Footnotes |
Submitted May 29, 2002; accepted July 8, 2002.
Prepublished online as
Blood First Edition Paper, August 8, 2002; DOI
10.1182/blood-2002-05-1565.
Supported by the EU (QLG1-CT-1999-00665) and the American
Portuguese Biomedical Research Fund.
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: Maria de Sousa, Molecular Immunology,
Institute for Molecular and Cell Biology (IBMC), Campo Alegre 823, 4150-180 Oporto, Portugal; e-mail: mdesousa{at}ibmc.up.pt and
ecardoso{at}ibmc.up.pt.
| |
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Abstract
Introduction
