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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 2963-2970
Different Roles of Glycosylphosphatidylinositol in Various
Hematopoietic Cells as Revealed by a Mouse Model of Paroxysmal
Nocturnal Hemoglobinuria
By
Y. Murakami,
T. Kinoshita,
Y. Maeda,
T. Nakano,
H. Kosaka, and
J. Takeda
From Departments of Immunoregulation and Molecular Cell Biology,
Research Institute for Microbial Diseases, Osaka University, and
Departments of Dermatology and Environmental Medicine, Osaka University
Medical School, Suita, Osaka, Japan.
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ABSTRACT |
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have one or
a few clones of mutant hematopoietic stem cells defective in
glycosylphosphatidylinositol (GPI) synthesis as a result of somatic
mutation in the X-linked gene PIG-A. The mutant stem cell clone
dominates hematopoiesis by a mechanism that is unclear. To
test whether a lack of multiple GPI-anchored proteins results in
dysregulation and expansion of stem cells, we generated mice in which
GPI-anchor negative cells are present only in the hematopoietic system.
We transplanted lethally irradiated mice with female fetal liver cells
bearing one allele of the Piga gene disrupted by conditional gene targeting. Because of the X-chromosome inactivation, a significant fraction of the hematopoietic stem cells in fetal livers was GPI-anchor negative. In the transplanted mice, cells of all hematopoietic lineages
contained GPI-anchor negative cells. The percentage of GPI-anchor
negative cells was much higher in T lymphocytes including immature
thymocytes than in other cell types, suggesting a regulatory role for
GPI-anchored proteins at an early stage of T-lymphocyte development.
However, the proportions of GPI-anchor negative cells in various blood
cell lineages were stable over a period of 42 weeks, indicating that
Piga mutation alone does not account for the dominance of the
mutant stem cells and that other phenotypic changes are involved in
pathogenesis of PNH.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SOMATIC MUTATION and subsequent clonal
expansion are critical events in the pathogenesis of somatically
acquired genetic diseases. Paroxysmal nocturnal hemoglobinuria (PNH) is
an acquired hematopoietic stem cell disorder associated with somatic
mutation of the PIG-A gene,1-3 but the mechanism of
clonal expansion has not been elucidated.4,5 PNH is
characterized by intravascular hemolysis, venous thrombosis, and
cytopenia caused by bone marrow failure.6,7 The defect
lasts for many years. Patients with PNH have abnormal cells of various
hematopoietic lineages that are defective in the biosynthesis of
glycosylphosphatidylinositol (GPI), which serves as a membrane anchor
for many cell surface proteins.8 The activation of the
complement leads to the lysis of red blood cells deficient in the
GPI-anchored proteins CD59 and CD55, which protect host cells from an
attack by complement.8,9
The gene responsible for PNH, PIG-A, is involved in the first
step of GPI anchor biosynthesis.1 In all patients reported to date, a somatic mutation occurred in
PIG-A.2,3,10,11 It is hypothesized that this
uniformity is caused by the X-linkage of PIG-A and autosomal
localizations of all other GPI-synthesis genes,12-15
because a single hit of somatic mutation is sufficient for knocking out
an X-linked gene, whereas two hits are required for inactivation of
autosomal genes.
Although a somatic mutation of PIG-A appears to occur in one or
a few of the large number of pluripotent hematopoietic stem cells, GPI
negative (GPI ) cells dominate in the bone marrow and
the peripheral blood.8,16 Because affected stem cells are
defective in the expression of various GPI-anchored proteins and
because many GPI-anchored proteins are involved in cell-to-cell or
cell-to-stroma interactions,8 GPI stem
cells might escape the negative regulation provided by the bone marrow
environment, resulting in clonal dominance. To test whether
GPI stem cells have an intrinsic ability to expand,
we17 and Rosti and colleagues18 disrupted the
mouse Piga gene, a homologue of PIG-A, in embryonic
stem cells and raised chimeric mice bearing GPI
cells. Because GPI-anchored proteins are essential for mouse development, mice with high chimerism did not survive to birth. Among
the mice with low chimerism, only a few had GPI
blood cells. The analysis of this limited number of mice showed that
percentages of GPI red blood cells remained constant
for several months to 1 year, suggesting that Piga mutation
alone does not immediately cause the clonal dominance of
GPI stem cells. However, the chimeric mice had
GPI cells in nonhematopoietic tissues including bone
marrow stromal cells, a situation different from that in patients with
PNH. Moreover, because of embryonic lethality, it was practically very
difficult to obtain a reasonable number of chimeric mice for analysis.
To solve these problems, we have made chimeric mice bearing
GPI cells only of hematopoietic lineage by means of
the Cre/loxP system and fetal liver transplantation.
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MATERIALS AND METHODS |
Generation of chimeric mice by transplantation of
Piga-disrupted hematopoietic stem cells.
Mice transgenic with human cytomegalovirus-Cre
(hCMV-Cre) have been previously reported. A
promoter of hCMV drives expression of Cre recombinase in most
cell types beginning at the preimplantation stage.19 Mice
with a Piga gene bearing loxP sites within intron 5 and the
3' flanking region (termed
Pigaflox) were also reported
previously.20 We crossed Pigaflox male
mice with heterozygous or homozygous Cre transgenic female mice
(Fig 1). Female embryos would receive the
Pigaflox allele that would become functionally
inactive as a result of Cre-mediated elimination of exon 6. In
contrast, males would be normal because Piga is X-linked. A
Piga-disrupted female embryo would be heterozygous for
Piga disruption and would become mosaic of PIGA-expressing and
PIGA-nonexpressing cells as a result of random X-inactivation (Fig 1).
More than 70% of those female embryos showed abnormality in the
cephalic portion. They died soon after birth or during
development.21 We collected fetal liver cells of day 14 fetuses showing the morphological abnormalities and pooled them to
transfer to the lethally irradiated C57BL/6 hosts (Fig 1). In some
experiments, we used a portion of fetal liver cells for in vitro
methylcellulose colony assay (see below), along with transplantation.
To distinguish donor-derived cells from host-derived cells, we used
mice whose Ly-5 allelic locus was 5.1 for the donor and 5.2 for the
host.22
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| Fig 1.
Generation of PNH model mice.
Pigaflox male mice were crossed with
heterozygous or homozygous Cre transgenic female mice.
Piga-disrupted female embryos would be heterozygous for
Piga disruption and would become mosaic of PIG-A-expressing
and PIG-A-nonexpressing cells because of random X-inactivation. Fetal
liver cells of 14 days post coitum (d.p.c.) fetuses
showing the morphological abnormalities were collected and pooled to
transfer to the lethally irradiated C57BL/6 hosts. Shaded cells
indicate GPI fetal liver cells bearing inactivated
normal Piga allele (black+) and activated,
disrupted-Piga allele (white ). Unshaded cells are
GPI+ because of inactivation of disrupted-Piga
allele (black). A portion of fetal liver cells was used for in vitro
methylcellulose colony assay along with transplantation. To distinguish
donor-derived cells from host-derived cells, mice with the Ly5.1 allele
were used for the donors, and those with Ly5.2 were used for the hosts.
Floxed indicates Piga allele bearing loxP
sites.
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Antibodies.
FITC-conjugated anti-Ly5.1, -CD4, and -CD8; phycoerythrin
(PE)-conjugated anti-Thy1.2, -heat stable antigen (HSA), and -CD25; biotinylated anti-CD44, -IAb, and -HSA; and alophycocyanin
(APC)-conjugated anti-Thy1.2 and streptavidin were purchased from
Pharmingen (San Diego, CA). Anti-B220 (RA3-6B2),
anti-Mac-1 (M1/70), anti-CD4 (GK1.5), anti-CD8 (53-6.7), and
anti-TER119 were purified from ascites of mice injected with each
antibody-producing hybridoma (gifts from Dr T. Kina, Kyoto University, Kyoto, Japan) and biotinylated.
Fetal liver and bone marrow transplantations.
Single-cell suspensions were prepared from day 14 fetal livers and bone
marrow in Dulbecco's modified Eagle's medium (DMEM). Six- to
9-month-old female C57BL/6 mice were lethally irradiated (900 cGy) by a
137Cs source. On the same day, they were injected
intravenously with 2 × 106 to 2 × 107 fetal liver cells or bone marrow cells suspended in 0.2 mL of DMEM. After injections, mice were maintained on aqueous
antibiotics (103 U/mL of polymyxin B sulfate and 1 mg/mL of
neomycin sulfate).
Methylcellulose colony assay.
Fetal liver and bone marrow cells (105) were plated in 1 mL
of complete methylcellulose medium (MethoCult GF M3434; Stem Cell Technologies Inc, Vancouver, Canada). Hematopoietic
colonies were identified according to the standard
criteria.23 Granulocyte-erythroid-macrophage-megakaryocyte (GEMM) colonies were counted, picked up, and individually
stained with PE-anti-HSA for flow cytometric analysis.
Flow cytometric analysis of the peripheral blood cells.
The contribution of GPI cells to hematopoiesis in
chimeric mice was assessed by fluorescence-activated cell sorting
(FACS) analysis of the peripheral blood cells. For the
analysis of red blood cells, we stained them first for a lineage marker
with biotinylated anti-TER119, and second with a mixture of PE-anti-HSA
and APC-streptavidin. For the three-color analysis of white blood
cells, we stained them first with one of the biotinylated
lineage-specific marker antibodies (anti-B220 for B lymphocytes,
anti-Mac1 for granulocytes and monocytes, and anti-CD4 and anti-CD8 for
T lymphocytes), and second with a mixture of a donor-specific antibody
(fluorescein isothiocyanate (FITC)-conjugated anti-Ly5.1),
PE-conjugated anti-GPI-linked surface antigen (anti-HSA for B
lymphocytes, granulocytes, and monocytes, and anti-Thy1.2 for T
lymphocytes), and APC-streptavidin, as a second-step reagent for the
lineage markers. The stained cells were analyzed with a dual laser FACS
caliber (Becton Dickinson, San Jose, CA).
Flow cytometric analysis of thymocytes.
Thymocytes were stained in four colors with biotinylated anti-Ly5.1
followed by a mixture of FITC-anti-CD4, APC-anti-CD8, PE-anti-Thy1.2,
and a second-step reagent, peridinin chlorophyll protein
(PerCP)-streptavidin. To study CD4,CD8 double negative cells,
thymocytes were incubated with a mixture of
biotinylated-anti-CD4, -anti-CD8, -anti-B220, and
-anti-IAb, and negatively selected with streptavidin beads
and a magnetic cell sorting (MACS) system. The unbound
cells were further stained for four-color FACS analysis with
biotinylated anti-CD44 followed by a mixture of FITC-anti-Ly5.1,
PE-anti-CD25, APC-anti-Thy1.2, and PerCP-streptavidin.
Acidified serum lysis test.
To obtain GPI red blood cells, peripheral blood
cells sampled from a chimeric mouse were stained with biotinylated
anti-HSA and subjected to a negative selection with streptavidin beads and a MACS system. GPI+ red blood cells were obtained from
a chimeric mouse that was transplanted with fetal liver cells of a
Piga-nondisrupted littermate. Erythrocytes (3 × 108) were washed three times and suspended in 1 mL of
gelatin Veronal buffered saline.24 Human
serum was acidified with a 10% volume of 2N HCl and serially diluted
to 1 in 214 with gelatin Veronal buffered saline. A sample
of 250 µL of each diluted serum was mixed with 25 µL of erythrocyte
suspension and incubated for 1 hour at 37°C. For 100% lysis, water
was used in place of serum, and for 0% lysis, heat-inactivated serum
was used. After incubation, each sample was centrifuged, and the
optical density of hemoglobin in the supernatant was measured at 412 nm.24
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RESULTS |
Generation of GPI-anchor deficient hematopoietic stem cells.
To study the characteristics of GPI hematopoietic
stem cells, we crossed Pigaflox males (bearing the
Pigaflox gene on the X-chromosome) with hCMV-Cre
females (expressing Cre by means of the hCMV promoter) and prepared
fetal liver cells from day 14 embryos. All TER119-positive liver cells
from a morphologically normal embryo expressed GPI-anchored protein HSA
(Fig 2A). In contrast, TER119-positive
liver cells from morphologically abnormal female embryos were mosaic
for the surface expression of HSA (Fig 2B), indicating that disruption
of Piga occurred in the erythroid lineage. Percentages of
HSA-negative cells varied among fetuses from 20% to 60%, presumably
because of varying efficiencies of Cre-mediated Piga disruption
or skewed X-chromosome inactivation or both. Because disruption of one
Piga allele may have also occurred in the hematopoietic stem
cells and in turn may have led, together with the X-chromosome
inactivation, to a mosaic phenotype, we transplanted these fetal liver
cells into lethally irradiated adult mice to compare characteristics of
GPI and GPI+ hematopoietic stem cells in
vivo.
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| Fig 2.
FACS analysis of fetal liver cells mosaic for the
expression of GPI. A Pigaflox male mouse was
crossed with hCMV-Cre female, and liver cells were prepared from day 14 embryos. (A) Liver cells from morphologically normal embryos. (B) Liver
cells from morphologically abnormal female embryos. Fetal liver cells
were stained for TER119 and HSA. Numbers in upper quadrants show
percentages of HSA+ and HSA
TER+ cells.
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Generation of PNH model mice by transplantation of fetal liver cells
mosaic for the expression of GPI.
After transplantation, we examined the peripheral blood cells every 6 to 12 weeks to see whether they contain GPI cells.
We stained them for three-color FACS analysis with antibodies to a
donor marker Ly 5.1 (except for red blood cells), a lineage marker, and
a GPI-anchored protein. Figure 3 shows FACS
profiles of red blood cells and the Ly5.1-positive blood cells 34 weeks after transplantation. The surface expressions of marker GPI-anchored proteins were mosaic in red blood cells, granulocytes, monocytes, B
cells, and CD4 and CD8 T cells from a mouse transplanted with mosaic
fetal liver cells (Fig 3A-F, right panels), whereas they were positive
in most of the corresponding cells from a control mouse transplanted
with liver cells of a normal littermate embryo (Fig 3A-F, left panels).
The GPI cells were seen even at 42 weeks after
transplantation (see below). These results indicated that
GPI hematopoietic stem cells with a long-term
repopulating capability differentiated into various mature blood cells.
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| Fig 3.
FACS profiles of the blood cells 34 weeks after
transplantation. The peripheral blood cells were stained in three
colors for Ly5.1 (except for red blood cells), a lineage marker, and a
GPI-anchored protein. Except for red blood cells, cells were first
gated by Ly5.1 expression. Right panels show the surface expressions of
GPI-anchored proteins in various lineages of cells from a mouse
transplanted with mosaic fetal liver cells. Left panels show those from
a mouse transplanted with liver cells of a normal littermate embryo.
(A) Erythrocytes stained for TER119 and HSA. (B) Granulocytes stained
for Mac-1 and HSA. (C) Monocytes stained for Mac-1 and HSA.
Granulocytes and monocytes were distinguished according to light
scattering profiles. More than 99% of granulocytes and monocytes were
Ly5.1-positive, donor-derived cells. (D) B cells stained for B220 and
HSA. More than 99% of B cells were of donor origin. (E) CD4 T cells
stained for CD4 and Thy1. Ninety-five percent of CD4 T cells were of
donor origin. (F) CD8 T cells stained for CD8 and Thy1. Ninety percent
of CD8 T cells were of donor origin.
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PNH patients suffer from hemolysis caused by a deficiency of complement
regulatory GPI-anchored proteins. The chimeric mice had normal blood
counts and had no such episodes. This may be because complement
activity in mice, especially of the C57BL/6 strain, is low and because
mouse erythrocytes have transmembrane complement regulatory molecules,
such as Crry25 and transmembrane type decay-accelerating
factor (DAF).26 GPI
erythrocytes were three times more sensitive to complement than GPI+ erythrocytes, as determined by the concentration of
serum required for 50% lysis (Fig 4). It
was reported that PNH erythrocytes with complete DAF/CD59-deficiency
are 10 to 15 times more sensitive to complement than normal
erythrocytes.27 This difference may be a result of the
presence of transmembrane complement regulatory molecules on mouse
erythrocytes.
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| Fig 4.
Acidified serum lysis test. HSA+ ( ) and
HSA ( ) erythrocytes were incubated with acidified
human serum, and percent lysis was determined.
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GPI hematopoietic stem cells have no intrinsic
ability to dominate GPI+ cells.
Proportions of GPI cells in red blood cells,
granulocytes, monocytes, and B cells were usually similar (Fig 3A-D).
They were much higher in CD4 and CD8 T cells (Fig 3E and F). These
differences were not caused by experimental variations, because when
more than 3 million fetal liver cells per mouse were transplanted into a group of mice, percentages of GPI cells in a
particular blood cell type were very similar among mice
(Fig 5A). Under these conditions,
hematopoiesis would have been reconstituted by the large number of
hematopoietic stem cells that eliminated mouse-to-mouse variations. We
monitored percentages of GPI cells from 6 to 42 weeks after transplantation (Fig 5). Panel A in Fig 5 shows results for
a group of six mice that received aliquots of the same pool of mosaic
fetal liver cells. Percentages of GPI cells changed
little. Panel B in the same figure shows results for a group of 22 mice
who received another pool of cells. In this case, liver cells from
mosaic embryos were mixed with those from normal embryos to reduce
proportions of GPI cells. Again, percentages of
GPI cells changed little for up to 42 weeks in red
blood cells, granulocytes, monocytes, and B cells. In T cells,
percentages of GPI cells slightly increased at the
beginning, but were stable after 18 weeks. We performed seven other
transplantation experiments using 40 recipient mice and obtained
similar results. These results together indicate that
GPI hematopoietic stem cells did not have an
intrinsic ability to dominate GPI+ cells.
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| Fig 5.
Percentages of GPI cells from 6 to 42 weeks after transplantation. (A) Mean percentages of GPI
cells in various blood cells from a group of six mice who received
aliquots of the same pool of mosaic fetal liver cells. (B) Those from a
group of 22 mice who received another pool of cells consisting of a
mixture of mosaic and normal embryos. Bars indicate standard deviation
(SD).
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When fewer numbers of fetal liver cells were transplanted, the result
was different. Figure 6 shows the result
with four mice that received 5 × 105 cells of the
same cell pool. Percentages of GPI cells varied
among mice under these conditions, most likely because of the small
number of transplanted hematopoietic stem cells. In two mice,
percentages of GPI cells were very high in various
lineages (Fig 6A and B). Moreover, the percentage of
GPI cells varied with time in individual mice. In
one of the mice, the proportion of GPI monocytes was
80% at 12 weeks after transplantation, but was 20% at 30 weeks
(Fig 6B). Therefore, when the number of hematopoietic stem
cells was small, the contribution of each stem cell to hematopoiesis varied, and sometimes GPI stem cells supported most
of the hematopoiesis.
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| Fig 6.
Percentages of GPI cells in mice who
received a smaller number of fetal liver cells. (A-D) Four mice who
received 0.5 million cells of the same pool of mosaic fetal liver cells
were monitored for up to 27 weeks after transplantation. Percentages of
GPI cells in various blood cells are shown. Percentages
of Ly5.1-positive cells were higher than 90%.
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GPI-anchored proteins play roles early in T lymphopoiesis.
Because GPI cells exceeded GPI+ cells in
both peripheral blood CD4 and CD8 T lymphocytes, we analyzed T cells in
lymph nodes and spleens. We found that proportions of
GPI cells in both CD4 and CD8 T lymphocytes in these
lymphoid organs were similarly high (Table
1), indicating that GPI cells dominate entire
compartments of mature CD4 and CD8 T lymphocytes.
To determine the stage of T-cell differentiation at which
GPI cells acquire dominance, we analyzed thymocytes
of chimeric mice bearing GPI cells. The numbers of
thymocytes from mice bearing GPI cells and normal
mice were similar, and proportions of four populations defined by
expressions of CD4 and CD8 were similar. In all four populations, the
GPI cell number was similarly high as in the
peripheral T cells (Fig 7A), indicating
that GPI cells are already dominant at the
CD4, CD8 stage. Percentages of
GPI cells were similar in later developmental
stages, namely CD4+, CD8+ cells and CD4 single
and CD8 single positive cells. Therefore, GPI cells
developed to mature T cells at similar efficiency to GPI+
cells.
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| Fig 7.
Analysis of thymocytes from chimeric mice bearing
GPI cells. (A) Thymocytes were stained in four colors
for Ly5.1, CD4, CD8, and Thy1. CD4+ and
CD4 cells were separately examined for the expression of
CD8 and Thy1. (B) The selected CD4,CD8 double negative thymocytes were
further stained in four colors for Ly5.1, CD44, CD25, and Thy1.
CD25+ and CD25 cells were separately
examined for the expression of CD44 and Thy1.
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To further dissect developmental stages within the
CD4, CD8 stage, we sorted
CD4, CD8 thymocytes and stained
them with antibodies to CD44 and CD25 together with anti-Thy1.
GPI cells were already dominant in the most immature
CD44+CD25 stage of thymocytes (Fig 7B).
It seems, therefore, that GPI cells become dominant
at this stage within the thymus or before the lodging in thymus, or
that a lack of GPI facilitates homing into the thymus.
GPI B cells were very rare in the peripheral
lymphoid organs.
We compared B lymphocytes in the peripheral blood and those in lymph
nodes and spleens. Percentages of GPI B
lymphocytes were much lower in the peripheral lymphoid
organs than in the peripheral blood (Table 1), suggesting that
GPI B lymphocytes have some difficulty in homing
into these lymphoid tissues. This was unique to B lymphocytes
because percentages of GPI granulocytes and
monocytes/macrophages were similar in the peripheral blood and spleen
(data not shown).
Homing of the GPI hematopoietic stem cells was as
efficient as that of the GPI+ counterparts.
To test whether the lack of GPI-anchored proteins has any effect on the
homing of the hematopoietic stem cells after transplantation, we
recovered bone marrow cells from mice transplanted with mosaic fetal
liver cells and retransplanted them into lethally irradiated mice. We
determined percentages of GPI cells in the
peripheral blood of donor and recipient mice in the secondary
transplantation. We also cultured the bone marrow cells in vitro to
estimate the percentage of GPI colony-forming units
(CFU)-GEMM. Percentages of GPI
CFU-GEMM correlated well with those of GPI
granulocytes in the donor mice (Table 2).
They also correlated well with those of GPI
granulocytes in the recipient mice (Table 2), indicating that GPI and GPI+ hematopoietic stem cells
have similar bone-marrow homing efficiencies.
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Table 2.
Correlations Between Percentages of GPI
CFU-GEMM in Donor Bone Marrow and GPI Granulocytes
in the Donor and Recipient Blood
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DISCUSSION |
We have established a mouse model to study the problem of clonal
expansion of GPI hematopoietic stem cells, a
critical event in the pathogenesis of PNH. The model mimics PNH in two
important ways. First, the mice generated in this study had
GPI cells only in the hematopoietic system. For
this, we obtained GPI hematopoietic stem cells from
fetal livers by means of conditional gene knockout and transplanted
them into lethally irradiated adult mice. Second, like patients with
PNH, the mice bore GPI and GPI+
hematopoietic stem cells with exactly the same genetic background. As a
source of hematopoietic stem cells for transplantation, we used female
embryos in which one of the two alleles of the Piga gene was
disrupted in the whole body. As a result of the X-chromosome inactivation, these embryos were mosaic for the expression of GPI-anchored proteins. Therefore, GPI and
GPI+ cells had exactly the same genetic background, the
sole phenotypic difference being in the expression of GPI-anchored proteins.
Using these mice, we tested whether GPI
hematopoietic stem cells dominate GPI+ counterparts.
Because proliferation of the hematopoietic stem cells may be regulated
by interactions with bone marrow stromal cells through the cell surface
proteins on the stem cells, we reasoned that GPI
hematopoietic stem cells might be more resistant to a negative regulation, leading to dysregulation and expansion. The results showed
that this was not true because proportions of GPI
cells in various hematopoietic lineages were stable from 6 to 42 weeks
after transplantation. Therefore, other events must occur to cause
dominance of GPI hematopoietic stem cells seen in
PNH. It should be noted, however, that in this mouse model the hosts
were irradiated before transplantation of a mixture of
GPI and GPI+ stem cells. If irradiation
eliminates an expression of a molecule on bone marrow stromal cells
that acts as a ligand of a relevant GPI-anchored protein on stem cells,
then a critical regulatory interaction between the stem cells and the
stromal cells might have been eliminated, preventing a possible
expansion of GPI stem cells. But it seems unlikely
that irradiation causes such effects.
Although expansion of GPI hematopoietic stem cells
did not occur in the model mice when a large number of fetal liver
cells (3 million) were transplanted, percentages of
GPI cells varied when a smaller number of fetal
liver cells were transplanted. Under these conditions, the number of
transplanted hematopoietic stem cells was small, a few or several,
because percentages of GPI cells were variable among
mice. In one mouse, the proportion of GPI monocytes
was 80% at 12 weeks after transplantation and decreased to 20% at 30 weeks (Fig 6). We think that when hematopoiesis is maintained by a
small number of stem cells, fluctuations can readily occur as the
fractional contribution of any one stem cell is relatively great. This
indicates that if the number of hematopoietic stem cells is decreased
for some reason, the GPI hematopoietic stem cells
would contribute more to and account for most of the hematopoiesis. It
is, therefore, implied that expansion of GPI
hematopoietic stem cells could occur as a consequence of a decrease in
the number of hematopoietic stem cells.
Our results indicated that GPI-anchored proteins act early in
T-lymphocyte development. Percentages of GPI cells
in peripheral blood CD4 and CD8 T lymphocytes were much higher than in
other blood lineage cells. Percentages of GPI cells
were similarly higher in T lymphocytes in lymph nodes and spleen,
eliminating the possibility that GPI T lymphocytes
are defective in the ability to home into lymphoid tissues from the
blood. Therefore, GPI cells dominated the entire
fraction of mature CD4 and CD8 T lymphocytes. This indicated that
GPI cells became dominant during T-lymphocyte
development. Within the thymus, the percentage of
GPI cells was already high in the most immature CD4
and CD8 double negative cells, and similar values were obtained for the
CD4 and CD8 double positive cells, and both CD4 single and CD8 single positive cells. The GPI immature T cells, therefore,
developed into mature T lymphocytes at a similar efficiency to
GPI+ cells. Further dissection and analysis of CD4 and CD8
double negative cells based on expressions of CD44 and CD25 showed that CD44-positive and CD25-negative cells, the most primitive thymic T
lineage cells, contained a high percentage of GPI
cells. This indicated that GPI cells were dominant
at a very early stage in T-lymphocyte development. This domination may
have occurred within the thymus, outside the thymus, or while entering
the thymus. We think it is most likely that the domination occurred
after the cells had entered the thymus. It is possible that some
GPI-anchored proteins are involved in the negative regulation of cell
proliferation, and their lacks result in the dysregulation of
proliferation and expansion of GPI cells. It was
reported that CD44-positive and CD25-negative cells slowly proliferate
and that active proliferation occurs in the subsequent CD44 and CD25
double positive stage before rearrangement of T-cell receptor
(TCR) genes.28 If proliferation is not
well-regulated at the CD44-positive and CD25-negative stage because of
a lack of GPI-anchor, the percentage of GPI cells
would increase at this stage. While entering the thymus, T precursor
cells would interact with other cells. Some GPI-anchored proteins might
be involved in a specific interaction that facilitates the entrance of
cells into the thymus. Therefore, GPI cells would be
less, rather than more, efficient at entering the thymus if
GPI-anchored proteins play such a role. It is also unlikely that the
domination exists in the prethymic common lymphocyte precursor
cells because the domination was not seen in B lymphocytes. It is still
possible, however, that the domination occurred after further
commitment to the T-cell lineage, although such a prethymic stage has
not been well characterized.
Four genes of GPI-anchored proteins expressed in T lineage cells,
namely Thy1, Ly6A, CD24, and CD48, have been knocked out. None of them
showed apparent abnormality in early T-cell
development.29-32 It is possible that other GPI-anchored
proteins are responsible or more than one of those four proteins are
redundantly responsible.
In contrast with the mouse system, the percentage of
GPI cells in T lymphocytes from patients with PNH is
no higher than in other cell types. This can be accounted for by the
different role of thymus in the development of GPI T
lymphocytes in the model mice and patients with PNH. In patients with
PNH, the thymus has usually already lost its function in T-lymphopoiesis when the somatic mutation of PIG-A occurred,
whereas in the model mice, the thymus was actively involved in
T-lymphopoiesis after transplantation.
The percentage of GPI cells in the peripheral blood
B lymphocytes was much lower than that in the T lymphocytes and similar to or lower than those in other blood cells, indicating that
GPI-anchored proteins do not play a significant role in regulation
during B-lymphocyte development. In contrast, the percentage of
GPI cells in lymph node and in spleen B lymphocytes
was much lower than in the peripheral blood B lymphocytes. This
indicates that some GPI-anchored proteins would be involved in either
the entering of B lymphocytes into or keeping of them in the peripheral
lymphoid organs. Therefore, GPI-anchors have different roles in T and B lymphocytes; they are important to the development of T lymphocytes but
not B lymphocytes and to the homing of B lymphocytes but not T
lymphocytes into lymphoid organs.
The important role GPI-anchored proteins play in the homing from blood
to organs is unique to B lymphocytes. They do not seem to play a
significant role in the homing of granulocytes and monocytes into lymph
nodes and spleen, nor in the homing of hematopoietic stem cells into
bone marrow. It is likely, therefore, that the roles of GPI-anchored
proteins vary in cells of different lineages.
Studies with the model mice described here provided strong evidence
that a second factor is involved in the expansion of
GPI hematopoietic stem cells in PNH. These mice
should be useful to test candidates of the second factor for their
ability to cause expansion of GPI hematopoietic stem
cells. Studies with these mice also provided evidence that GPI-anchored
proteins play a role in the negative regulation of cell proliferation
at an early stage of T-lymphopoiesis and are important for efficient
homing of B lymphocytes into lymph nodes and spleen. Further studies
are necessary to identify specific GPI-anchored proteins involved in
these processes.
| |
ACKNOWLEDGMENT |
The authors thank Dr A. Nagy (Samuel Lunenfeld Research Institute,
Toronto, Canada) for transgenic mice and Dr T. Iwabuchi (Hokkaido
University, Sapporo, Japan) for discussions.
| |
FOOTNOTES |
Submitted March 5, 1999; accepted July 2, 1999.
Supported by grants from the Ministry of Education, Science, Sports and
Culture and the Ministry of Health and Welfare of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to T. Kinoshita, PhD, Department
of Immuno- regulation, Research Institute for Microbial
Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: tkinoshi{at}biken.osaka-u.ac.jp.
| |
REFERENCES |
1.
Miyata T, Takeda J, Iida Y, Yamada N, Inoue N, Takahashi M, Maeda K, Kitani T, Kinoshita T:
Cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.
Science
259:1318, 1993[Abstract/Free Full Text]
2.
Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T:
Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria.
Cell
73:703, 1993[Medline]
[Order article via Infotrieve]
3.
Bessler M, Mason PJ, Hillmen P, Miyata T, Yamada N, Takeda J, Luzzatto L, Kinoshita T:
Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene.
EMBO J
13:110, 1994[Medline]
[Order article via Infotrieve]
4.
Luzzatto L, Bessler M, Rotoli B:
Somatic mutations in paroxysmal nocturnal hemoglobinuria: A blessing in disguise?
Cell
88:1, 1997[Medline]
[Order article via Infotrieve]
5.
Young NS:
The problem of clonality in aplastic anemia: Dr Dameshek's riddle, restated.
Blood
79:1385, 1992[Free Full Text]
6.
Rotoli B, Luzzatto L:
Paroxysmal nocturnal hemoglobinuria.
Semin Hematol
26:201, 1989[Medline]
[Order article via Infotrieve]
7.
Rosse WF:
Paroxysmal nocturnal hemoglobinuria.
Curr Top Microbiol Immunol
178:163, 1992[Medline]
[Order article via Infotrieve]
8.
Kinoshita T, Inoue N, Takeda J:
Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria.
Adv Immunol
60:57, 1995[Medline]
[Order article via Infotrieve]
9.
Rosse WF, Ware RE:
The molecular basis of paroxysmal nocturnal hemoglobinuria.
Blood
86:3277, 1995[Free Full Text]
10.
Miyata T, Yamada N, Iida Y, Nishimura J, Takeda J, Kitani T, Kinoshita T:
Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.
N Engl J Med
330:249, 1994[Abstract/Free Full Text]
11.
Yamada N, Miyata T, Maeda K, Kitani T, Takeda J, Kinoshita T:
Somatic mutations of the PIG-A gene found in Japanese patients with paroxysmal nocturnal hemoglobinuria.
Blood
85:885, 1995[Abstract/Free Full Text]
12.
Inoue N, Watanabe R, Takeda J, Kinoshita T:
PIG-C, one of the three human genes involved in the first step of glycosylphosphatidylinositol biosynthesis is a homologue of Saccharomyces cerevisiae GPI2.
Biochem Biophys Res Commun
226:193, 1996[Medline]
[Order article via Infotrieve]
13.
Takahashi M, Inoue N, Ohishi K, Maeda Y, Nakamura N, Endo Y, Fujita T, Takeda J, Kinoshita T:
PIG-B, a membrane protein of the endoplasmic reticulum with a large lumenal domain, is involved in transferring the third mannose of the GPI anchor.
EMBO J
15:4254, 1996[Medline]
[Order article via Infotrieve]
14.
Ware RE, Howard TA, Kamitani T, Chang HM, Yeh ETH, Seldin MF:
Chromosomal assignment of genes involved in glycosylphosphatidylinositol anchor biosynthesis: Implications for the pathogenesis of paroxysmal nocturnal hemoglobinuria.
Blood
83:3753, 1994[Abstract/Free Full Text]
15.
Ohishi K, Inoue N, Endo Y, Fujita T, Takeda J, Kinoshita T:
Structure and chromosomal localization of the GPI-anchor synthesis gene PIG-F and its pseudogene YPIG-F.
Genomics
29:804, 1995[Medline]
[Order article via Infotrieve]
16.
Parker CJ:
Molecular basis of paroxysmal nocturnal hemoglobinuria.
Stem Cells
14:396, 1996[Abstract]
17.
Kawagoe K, Kitamura D, Okabe M, Taniuchi I, Ikawa M, Watanabe T, Kinoshita T, Takeda J:
GPI-anchor deficient mice: Implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria.
Blood
87:3600, 1996[Abstract/Free Full Text]
18.
Rosti V, Tremmi G, Soares V, Pandolfi PP, Luzzatto L, Bessler M:
Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion.
J Clin Invest
100:1028, 1997[Medline]
[Order article via Infotrieve]
19.
Nagy A, Moens C, Ivanyi E, Pawling J, Gertsenstein M, Hadjantonakis A, Pirity M, Rossant J:
Multipurpose gene alterations from a single targeting vector: Dissecting the role of N-myc in development.
Curr Biol
8:661, 1998[Medline]
[Order article via Infotrieve]
20.
Tarutani M, Itami S, Okabe M, Ikawa M, Tezuka T, Yoshikawa K, Kinoshita T, Takeda J:
Tissue specific knock-out of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development.
Proc Natl Acad Sci USA
94:7400, 1997[Abstract/Free Full Text]
21.
Nozaki M, Ohishi K, Yamada N, Kinoshita T, Nagy A, Takeda J:
Developmental abnormalities of glycosylphosphatidylinositol-anchor deficient embryos revealed by Cre/loxP system.
Lab Invest
79:293, 1999[Medline]
[Order article via Infotrieve]
22.
Sheid MP, Triglia D:
Further description of the Ly-5 system.
Immunogenetics
9:423, 1979
23.
Heyworth CM, Spooncer E:
In Vitro Clonal Assays for Murine Multipotential and Lineage Restricted Myeloid Progenitor Cells. Oxford, UK, IRL Press, 1993
24.
Pangburn MK, Schreiber RD, Trombold JS, Muller Eberhard HJ:
Paroxysmal nocturnal hemoglobinuria: Deficiency in factor H-like functions of the abnormal erythrocytes.
J Exp Med
157:1971, 1983[Abstract/Free Full Text]
25.
Molina H, Wong W, Kinoshita T, Brenner C, Foley S, Holers VM:
Distinct receptor and regulatory properties of recombinant mouse complement receptor 1 (CR1) and crry, the two genetic homologues of human CR1.
J Exp Med
175:121, 1992[Abstract/Free Full Text]
26.
Spicer AP, Seldin MF, Gendler SJ:
Molecular cloning and chromosomal localization of the mouse decay-accelerating factor genes.
J Immunol
155:3079, 1995[Abstract]
27.
Rosse WF, Adams JP, Thorpe AM:
The population of cells in paroxysmal nocturnal haemoglobinuria of intermediate sensitivity to complement lysis: Significance and mechanism of increased immune lysis.
Br J Haematol
28:181, 1974[Medline]
[Order article via Infotrieve]
28.
Rothenberg E:
The development of functionally responsive T cells.
Adv Immunol
51:85, 1992[Medline]
[Order article via Infotrieve]
29.
Hueber AO, Bernard AM, Battari CL, Marguet D, Massol P, Foa C, Brun N, Garcia S, Stewart C, Pierres M, He HT:
Thymocytes in Thy-1/ mice show augmented TCR signaling and impaired differentiation.
Curr Biol
7:705, 1997[Medline]
[Order article via Infotrieve]
30.
Stanford WL, Haque S, Alexander R, Liu X, Latour AM, Snodgrass HR, Koller BH, Flood PM:
Altered proliferative response by lymphocytes of Ly-6A (Sca-1) null mice.
J Exp Med
186:705, 1997[Abstract/Free Full Text]
31.
Nielsen PJ, Lorenz B, Muller AM, Wenger RH, Brombacher F, Simon M, von der Weid T, Langhorne J, Mossmann H, Kohler G:
Altered erythrocytes and a leaky block in B cell development in CD24/HSA-deficient mice.
Blood
89:1058, 1997[Abstract/Free Full Text]
32.
Cabrero JG, Wise CJ, Latchman Y, Freeman GJ, Sharpe AH, Reiser H:
CD48-deficient mice have a pronounced defect in CD4+ T cell activation.
Proc Natl Acad Sci USA
96:1019, 1999[Abstract/Free Full Text] |
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