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Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3363-3370
HEMATOPOIESIS
Intramedullary and extramedullary B lymphopoiesis in osteopetrotic
mice
Hisashi Tagaya,
Takahiro Kunisada,
Hidetoshi Yamazaki,
Toshiyuki Yamane,
Takeshi Tokuhisa,
Erwin F. Wagner,
Tetsuo Sudo,
Leonard D. Shultz, and
Shin-Ichi Hayashi
From the Department of Immunology, School of Life Science, Faculty
of Medicine, Tottori University, Yonago, Tottori, Japan; Division of
Developmental Genetics, Chiba University Graduate School of Medicine,
Chiba, Japan; Research Institute of Molecular Pathology, Vienna,
Austria; Basic Research Laboratories, Toray Industries, Inc, Kamakura,
Kanagawa, Japan; and Jackson Laboratory, Bar Harbor, ME.
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Abstract |
Adult bone marrow is a major site for hematopoiesis, and reduction
of the bone marrow cavity induces hematopoiesis in extramarrow tissues.
To investigate the rudimentary intramarrow and the compensatory extramarrow hematopoiesis, particularly B lymphopoiesis, we used 3 osteopetrotic mouse strains [op/op, mi/mi, and Fos
( / )], which are severely deficient in functional osteoclasts and
therefore form inadequate bone marrow cavities. We found that bone
marrow in these osteopetrotic mice supports
myelopoiesis but not B lymphopoiesis, although cells that
have the potential to differentiate into B lineage cells are present in
the bone marrow. Although B lymphopoiesis normally occurs both in the
spleen and liver of newborn mice, compensatory B lymphopoiesis in adult
op/op and mi/mi mice is observed only in the liver,
while myelopoiesis is enhanced in both organs. Interestingly, mice
lacking the Fos proto-oncogene exhibit B lymphopoiesis in the
spleen as well as liver. The amounts of expression of steel factor,
Flt3/Flk-2 ligand, and interleukin-7 in the bone marrow, spleen, or
liver were not significantly affected in these osteopetrotic mutants.
These findings suggest that the volume of the bone marrow cavity
regulates B lymphopoiesis without affecting the production of certain
hematopoietic growth factors. The splenic microenvironments that
support both myelopoiesis and B lymphopoiesis in the neonatal stage are
lost in adults and are not reactivated even in the osteopetrotic adults
unless the Fos gene is disrupted.
(Blood. 2000;95:3363-3370)
© 2000 by The American Society of Hematology.
 |
Introduction |
Hematopoiesis is initiated in the yolk sac and
subsequently occurs in the paraaortic splanchnopleural mesoderm and
aorta-gonad-mesonephros regions.1-3 Hematopoietic stem
cells (HSCs) in the splanchnopleural mesoderm or
aorta-gonad-mesonephros migrate to the fetal liver and spleen and
finally localize in the bone marrow. Blood formation in extramarrow
tissues gradually decreases, and adult hematopoiesis mainly occurs in
the bone marrow.2-5
In adult life, extramedullary hematopoiesis is induced by low-dose
X-irradiation6,7 and by osteopetrosis, fibrosis, or malignant blood diseases in which the bone marrow cavity is
incapacitated for hematopoiesis.8-11 Usually,
extramedullary hematopoiesis occurs in the spleen and liver, which are
the sites for fetal and neonatal hematopoiesis in the normal
state.12 It is speculated that adult spleen and liver
maintain the potential to support hematopoiesis and that stimuli such
as X-irradiation or reduction of bone cavity induce molecules essential
for the support of extramedullary hematopoiesis.
Colonization from HSCs in spleen (CFU-s, or colony-forming
units-spleen) depends on the presence of steel factor (hereafter abbreviated as SLF, also named stem cell factor, mast cell growth factor, and a ligand for c-Kit receptor protein tyrosine kinase and the
encoding gene symbolized as Mgf). SLF-deficient steel mice and
the c-Kit-deficient dominant spotting
(KitW) mice lack CFU-s and have severely
reduced definitive-type hematopoiesis.13-20 Another
receptor protein tyrosine kinase, Flt3/Flk-2 (the encoding gene
symbolized as Flt3), is also expressed in immature
hematopoietic cells,21-23 and disruption of the Flt3
gene results in deficiencies both in HSCs and in early B progenitor
cells, while mice with combined Flt3/Flk-2 and c-Kit deficiencies
exhibit more severe phenotypes.24 Thus, SLF and Flt3/Flk-2
ligand (Flt3L) are candidate critical molecules for extramedullary hematopoiesis.
For B lymphopoiesis, interleukin (IL)-7, which is produced by stromal
cells that support hematopoiesis, is known to be
essential.25-28 Disruption of the Il7 or its
receptor (Il7r) genes results in severe
lymphopenia.29-31 Overexpression of the Il7 gene
induces B cell hyperplasia in the bone marrow and extramedullary organs in transgenic mice.32,33 These findings indicate that SLF, Flt3L, and IL-7 production might regulate lymphohematopoiesis.
In the current study, we used 2 different spontaneous osteopetrotic
mutations, osteopetrosis (Csfmop),
abbreviated as op, and microphthalmia
(Mitfmi), abbreviated as mi,
and the induced mutation Fos ( / ), which result in deficiencies of osteoclasts and a reduced hematopoietic "niche" in the bone marrow.34-38 Hematopoiesis in the
bone marrow and in the extramedullary organs was analyzed in these
osteopetrotic mice. We found that the regulation of B lymphopoiesis is
different from that of hematopoiesis represented by colony-forming
cells responsive to IL-3 (CFU-IL-3). Rudimentary bone marrow in the osteopetrotic mice contained CFU-IL-3 at a normal frequency, but the
frequencies of B lineage cells, which are characterized by stromal
cell- and IL-7-dependent B precursor cells and
CFU-IL-7,39,40 were significantly diminished. However,
when the bone marrow cells of these osteopetrotic mice were cultured
with stromal cells in the presence of IL-7, they differentiated into B
lineage cells, indicating that defective B lymphopoiesis in
osteopetrotic bone marrow may result from defects in the
microenvironment rather than from defective B progenitor cells.
Moreover, the finding that aged op/op mice with spontaneously
cured osteopetrosis had restored levels of B precursor cells in their
bone marrow confirms the critical role of the hematopoietic niche on
medullary B lymphopoiesis.41,42 Because numbers of
peripheral blood cells, including mature B cells in fetal, newborn, and
aged op/op mice are relatively normal8,43,44 and
production of immunoglobulin (Ig)M and IgG antibodies is also indistinguishable from normal mice,45,46 we reasoned that
the osteopetrotic mice use alternative sites for hematopoiesis. While myelopoiesis was enhanced in both the spleen and liver of adult osteopetrotic mice, extramedullary B lymphopoiesis in op/op and mi/mi mice was observed only in the liver. In contrast, Fos
( / ) spleen contained substantial numbers of
CFU-IL-7.
Expression of the critical hematopoietic growth factors SLF,
Flt3L, and IL-7 in the bone marrow, spleen, and liver of osteopetrotic mice was relatively indistinguishable in osteopetrotic mice and normal
littermates. This suggests the possible importance of the reduction of the hematopoietic niche in the bone marrow in inducing the
arrest of B lymphopoiesis and implies that compensatory extramedullary hematopoiesis is induced by as yet unknown mechanisms without an
increase of production of SLF, Flt3L, or IL-7.
 |
Materials and methods |
Mice
C57BL/6 (B6) mice were purchased from Clea Japan (Yokohama, Japan);
B6C3Fe-a/a- Csfmop/+ (op/+)
and B6C3Fe-a/a- Mitfmi/+
(mi/+) heterozygous mice were obtained from the Jackson
Laboratory (Bar Harbor, ME); and 129/Sv-Fos (+/ )
[Fos (+/ )] gene-targeted mice37 were
obtained from Chiba University, Japan, and were originally derived from
the European Molecular Biology Laboratory, Heidelberg, Germany. All
mice were maintained in the Research Animal Center, Faculty of
Medicine, Tottori University, Yonago, Japan. Genomic DNA from the tails
of op/+ and mi/+ mice was screened to detect the
mutated sequences, and that from Fos (+/ ) heterozygous mice carrying the neoR gene was screened by
using appropriate primers and the polymerase chain reaction
(PCR).34,36 Homozygous op/op, mi/mi, and
Fos ( / ) mice were obtained by heterozygous
sibling mating of each strain and were identified by the lack of incisors.
Cell preparation
Bone marrow cells from normal mice were collected by flushing
femoral shafts using a 26G sterile needle, and those from osteopetrotic mice were obtained by crushing the femora to small pieces and using
vigorous pipetting. Spleens and livers were minced and then homogenized
by disruption between frosted glass slides in alpha-minimum essential
medium ( -MEM; Gibco-BRL, Grand Island, NY). The cells were passed
through nylon mesh and washed once. To prepare hematopoietic cells from
the liver, the cells were washed 3 more times and then suspended in
40% Percoll (Gibco-BRL) and layered onto 70% Percoll. The tubes were
centrifuged at 600g at 20°C for 25 minutes, and the
interface between the 40% layer and the 70% layer containing hematopoietic cells was collected.47
Assays of colony-forming cells
Cells were incubated in 1 mL of -MEM containing 1.2%
methylcellulose (Muromachi Kagaku Kogyo, Tokyo, Japan), 30% fetal
bovine serum (Bio-Whittaker, Walkersville, MD), 1% deionized bovine
serum albumin (Sigma Chemical, St Louis, MO), 50-µM
2-mercaptoethanol, 50-U/mL streptomycin, and 50-µg/mL penicillin
(Meiji Chemical, Tokyo, Japan) in the presence of 100 U/mL mouse
recombinant IL-3, 100 U/mL granulocyte-macrophage colony-stimulating
factor (GM-CSF), or 20 U/mL IL-7. Numbers of colonies were counted on
the seventh day after the inoculation39 and expressed as
the mean ± SD of triplicate cultures.
Clonal expansion of B progenitor cells on stromal cell layers
The limiting dilution assay on PA648 stromal cell layers
was performed as previously described.39 Briefly, stromal
cell monolayers were allowed to form in 96-well culture plates (Falcon Labware, Oxnard, CA). Bone marrow cells were diluted to various concentrations, inoculated into the wells, and cultivated for 7 or 8 days in the presence of 20-U/mL mouse recombinant IL-7. Outgrowth of B
lineage cells was assessed with the aid of a microscope.
Flow cytometry
Cells were incubated on ice with heat-inactivated normal rabbit
serum (Gibco-BRL) and then stained with biotinylated anti-Mac-1 (M1/70,
PharMingen, San Diego, CA), biotinylated goat antimouse IgM (ICN
Pharmaceuticals Inc, Costa Mesa, CA), and fluorescein isothiocyanate
(FITC)-conjugated anti-B220 (6B2, PharMingen). The stained cells were
further incubated with phycoerythrin (PE)-labeled streptavidin (Becton
Dickinson Immunocytometry Systems, San Jose, CA) and analyzed using an
EPICS-XL flow cytometer (Coulter Electronics Inc, Haleah, FL).
Quantitative reverse transcription-polymerase chain reaction and
Southern blotting
Total RNA was extracted from bone marrow, spleen, and liver using
ISOGEN (Nippon Gene, Toyama, Japan). RNA (5 µg) was
reverse-transcribed using SuperScript RNase H
reverse transcriptase (Gibco-BRL) and oligo(dT) primer according to the
protocol provided by the manufacturer. PCR assays were performed in a
reaction mixture containing 1 × rTaq buffer (Toyobo, Osaka,
Japan), 0.16-mM dNTP, 1.5-mM MgCl2, 30-U/mL rTaq
DNA polymerase (Toyobo), and each primer at 0.5 µmol/L. The forward
primers and reverse primers, respectively, were as follows:
Mgf, 5'-GTGGCAAATCTTCCAAATGA-3' and
5'-CTCGGGACCTAATGTTGAAG-3'; Il7,
5'-ACATCATCTGAGTGCCACA-3' and
5'-CTCTCAGTAGTCTCTTTAG-3'; Flt3l,
5'-GGACGAGAAGCACTGCAAGG-3' and
5'-GTGAGAGGCAGCAGCAGCAG-3'; glyceraldehyde-3-phosphate
dehydrogenase (GAPD), 5'-CCATGGAGAAGGCTGGGG-3' and
5'-CAAAGTCATGGATGACC-3'. PCR amplification of complementary
DNAs (cDNAs) was performed under the following conditions: first cycle
consisting of 94°C for 3 minutes, annealing for 3 minutes, and
72°C for 3 minutes, followed by 24 cycles of 94°C for 1 minute,
annealing for 1 minute, and 72°C for 1.5 minutes; the temperatures
for annealing were 55°C (Mgf), 57°C (Il7),
55°C (Flt3l), and 60°C (GAPD). To compare relative levels of gene expression, serially diluted cDNA (1:1, 1:8,
1:64) was amplified. The PCR products were subjected to electrophoresis on 2% agarose gels and Southern blot hybridization with appropriate [32P]-radiolabeled gene probes.
 |
Results |
B lymphopoiesis is observed in neonatal spleen but not in adult
spleen of wild-type mice
The spleen is one of the main sites for perinatal hematopoiesis in
normal mice.2,3 We assessed hematopoiesis in the spleens of
2-day-old neonatal and 8-week-old adult C57BL/6 mice by enumerating the
cells that had the potential of colony formation in the presence of
IL-3 (CFU-IL-3) and IL-7 (CFU-IL-7). Numbers of cells recovered from
2-day-old neonatal spleen and femora were approximately 20- and 10-fold
lower than those from 8-week-old adult mice, respectively. Both
neonatal and adult bone marrow contained CFU-IL-3 and CFU-IL-7 at
approximately comparable frequencies (Table
1). In the spleen, the frequency of
CFU-IL-3 in adult mice was reduced to less than 1/36 of that in
neonatal mice, and the total number of CFU-IL-3 in the spleens of
8-week-old mice was half of that in the spleens of 2-day-old mice.
While CFU-IL-7 existed in the neonatal spleen, we did not detect
CFU-IL-7 in the adult spleen (Table 1). These results indicated that
adult hematopoiesis, particularly B lymphopoiesis, mainly occurs in
bone marrow rather than in the spleen.
Lack of B lymphopoiesis in the bone marrow from osteopetrotic mice
Osteopetrotic mice are known to have reduced bone marrow cavities
available for hematopoiesis and to have elevated extramedullary hematopoiesis.8 We first analyzed the residual
intramedullary hematopoiesis in op/op mice. Hematopoietic cells
in the op/op rudimentary bone marrow cavity were obtained by
crushing the femora into small pieces and using vigorous pipetting, and
recovery of cells from 8-week-old op/op bone marrow was 10 to
20 times lower than that from the bone marrow of normal littermates.
However, the frequencies of CFU-IL-3 and CFU-GM in op/op mice
were comparable with those of the normal littermates (Table
2), although in 1 experiment the frequency
of CFU-IL-3 in op/op mice was only half of that in the normal
littermates (Table 2, experiment no. 3). On the other hand, the
frequency of CFU-IL-7 in op/op bone marrow was diminished to
1/8 of that in the bone marrow of normal littermates, and in 1 experiment (Table 2, experiment no. 3) we could not detect any
colony-forming cells per 105 op/op bone marrow
cells.
The defect of op/op mice is attributed to hematopoietic
microenvironments that lack functional M-CSF produced by stromal cells, resulting in reduced osteoclast development and therefore in
osteopetrosis.34,35 To determine whether the abnormality in
B lymphopoiesis associated with osteopetrosis is an
op/op-specific characteristic or a common phenomenon in
osteopetrotic mice, we used osteopetrotic mi/mi mice mutated in
the Mitf gene, which encodes a basic helix-loop-helix, leucine
zipper-type transcriptional factor and affects hematopoietic cells such
as osteoclasts and mast cells rather than affecting supportive
microenvironments.36 The total number of CFU-IL-3 (mi/mi: 1306 ± 24 cells/femur, mi/+:
55 692 ± 7224 cells/femur) was significantly reduced in
mi/mi mice, whereas the frequency of CFU-IL-3 was comparable
with that of mi/+ littermates (Figure 1). No CFU-IL-7 was detected
(< 1/105), which is similar to the finding
in op/op mice. We also studied Fos ( / )
mice, which lack osteoclasts and thus have osteopetrosis, and obtained
similar results (Figure 1).

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| Fig 1.
Extramedullary hematopoiesis in 8-week-old mi/mi
and Fos ( / ) mice.
The numbers of colony-forming cells elicited by 100 U/mL IL-3 or 20 U/mL IL-7 per 105 cells from bone marrow, spleen, liver,
thymus, peripheral lymph nodes (cervical, axillary, and inguinal),
mesenteric lymph nodes, Peyer patches, and peritoneal cavity were
measured. Cells from tissues other than bone marrow, spleen, and liver
did not form any colonies (< 1/105). Data
are expressed as the mean ± SD of triplicate cultures. *Values of
osteopetrotic mice that are significantly different from those of the
corresponding normal littermates (P < .05).
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Next, we examined at what differentiation stage the
B-cell lineage in osteopetrotic bone marrow was affected.
The culture of bone marrow cells with PA6 stromal cells in the
presence of IL-7 (PA6 + IL-7) allows growth and differentiation of B
lineage cells more immature than CFU-IL-7.39 Using
this culture system, the frequency of clonal expansion of PA6 + IL-7-dependent B lineage cells was examined. Freshly prepared bone
marrow cells from op/op mice showed a significantly
reduced frequency of PA6 + IL-7-dependent B lineage cells (Table
3), and in some experiments we observed more than 90-fold reduction compared with normal littermates
(op/op: < 1/9500, +/?: 1/103.9). These results
indicate that B lymphopoiesis in op/op bone marrow is affected
prior to the PA6 + IL-7-dependent stage.
However, after culturing op/op bone marrow cells with PA6 and
IL-7 for 2 weeks, the frequency of PA6 + IL-7-dependent B lineage cells was restored and that of CFU-IL-7 was increased (Table 3). These
results suggest that the precursor cells of the B lineage were present
in op/op bone marrow, but the microenvironment for B-cell
differentiation was defective.
Selective reduction of B precursor cells in the bone marrow of
osteopetrotic mice
Because numbers of CFU-IL-7 and PA6 + IL-7-dependent B
lineage cells in osteopetrotic bone marrow were reduced, we
examined whether mature B cells exist in the osteopetrotic mice by flow cytometric analysis. B220+ cells in op/op bone
marrow were selectively reduced (24.3% B220+ cells
in +/? bone marrow cells and 7.0% in op/op bone marrow cells), and almost all B220+ cells also
expressed surface IgM (8.7% B220+ IgM+ cells
in op/op bone marrow cells). In contrast,
two-thirds of the B lineage cells in normal bone marrow were immature
B220+ IgM cells (8.0% B220+
IgM+ cells in +/? bone marrow
cells). There were few immature B lineage cells in
osteopetrotic bone marrow, indicating that B lymphopoiesis may
not occur in the bone marrow of osteopetrotic mice and mature B cells
are generated in extramarrow tissues.
B precursor cells appear in the spontaneously cured osteopetrotic
marrow of op/op mice
It has been reported that age-related progressive formation of bone
marrow cavity by an unknown mechanism cures osteopetrosis in
op/op mice.41,42 We have observed that some adult
op/op mice (30 weeks old) had a relatively normal number of
hematopoietic cells in the bone marrow (op/op:
4.23 × 106/femur, op/+ littermate:
15.6 × 106/femur). The partially cured
op/op bone marrow cells showed nearly normal relative
frequencies of CFU-IL-3 and CFU-IL-7 (Figure
2). This result confirmed the close
relationship between the amount of marrow cavity available for
hematopoiesis and maintenance of B lymphopoiesis in the bone marrow,
and it ruled out the possibility that M-CSF is essential to support
B-cell development in the bone marrow.

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| Fig 2.
Bone marrow from aged op/op mice in which
osteopetrosis was spontaneously cured produced CFU-IL-7.
The numbers of CFU-IL-3 and CFU-IL-7 in 30-week-old op/op
homozygous and heterozygous littermates were examined. Data are
expressed as the mean ± SD of triplicate cultures.
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Because op/op mice are spontaneously cured, we could not
precisely determine the state of osteopetrosis of each op/op
mouse; thus, the following experiments were mainly carried out using mi/mi mice unless otherwise indicated.
Extramedullary B lymphopoiesis in osteopetrotic mice
If bone marrow is the only site for adult B lymphopoiesis,
osteopetrotic mice should lack mature B lymphocytes. We analyzed whether B cells expressing both B220 and IgM were present in 8-week-old mi/mi and op/op spleens and detected numbers of mature
B cells comparable with those of normal littermates (Figure
3), indicating that sites other than bone
marrow for B lymphopoiesis may exist in these mice. To determine the
sites for extramedullary B lymphopoiesis, we assessed the presence of
the B precursor cells indicated by CFU-IL-7 in mi/mi spleens.
However, we could not detect any CFU-IL-7 (< 1/105) either in mi/mi or mi/+
adult spleens (Figure 1). Because our experiments could detect 1 CFU-IL-7 in 105 cells, there were fewer than 1000 CFU-IL-7 per spleen. In contrast, the frequency of CFU-IL-3 was
significantly increased in mi/mi spleens (Figure 1), indicating
that extramedullary hematopoiesis was induced in the spleens of
mi/mi mice whereas B lymphopoiesis was not. We also examined the
numbers of CFU-IL-7 in the thymus; peripheral lymph nodes, including
cervical, axillary, and inguinal nodes; mesenteric lymph nodes; Peyer
patches; and peritoneal cavity; however, no colony-forming cells were
detected (< 1/105 cells) from mi/mi
or mi/+ mice.

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| Fig 3.
Presence of mature B cells in the mi/mi spleen.
Spleen cells (106) from 8-week-old mi/mi
homozygotes or mi/+ normal littermates were stained with
FITC-6B2 (anti-B220), biotinylated-IgM or -M1/70 (anti-Mac-1), and
PE-streptavidin, and analyzed using a flow cytometer.
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Further examination was done by using fractionated liver cells
separated by Percoll density gradients (40%-70%). The fractionated cells, containing 64% to 84% viable hematopoietic cells, were used
for further analysis. The fractionated cells from both mi/mi and mi/+ livers contained CFU-IL-7, and the frequencies of
both CFU-IL-3 and CFU-IL-7 in mi/mi liver cells were
significantly increased (Figure 1). When op/op mice were used,
we obtained results similar to those with mi/mi mice (data not
shown). These results indicated that osteopetrotic mice have
up-regulated hematopoiesis in both spleen and liver, but B
lymphopoiesis was only up-regulated in the liver.
It was previously reported that Fos ( / ) mice have
reduced numbers of mature B cells.49 This implies a lack of
extramedullary B lymphopoiesis and resultant B lymphopenia in Fos
( / ) mice. However, we found that some Fos
( / ) mice have normal numbers of B cells in their spleens,
although other Fos ( / ) mice have significantly
reduced numbers of B cells, as reported.49 We selected the
Fos ( / ) mice without B lymphopenia and analyzed the presence of CFU-IL-7. Interestingly, we detected CFU-IL-7 in both
spleen and liver of Fos ( / ) mice (Figure 1).
Expression of Mgf, Flt3l, and Il7 genes in the
osteopetrotic mice
To investigate the enhanced myelopoiesis in the spleen and liver and
B lymphopoiesis in the liver of osteopetrotic mice, we examined the
expression of genes encoding critical hematopoietic factors, SLF,
Flt3L, and IL-7, of mi/mi mice by using quantitative reverse
transcription-PCR. The Mgf, Flt3l, and Il7 genes in
mi/mi mice were expressed at levels relatively
indistinguishable from those of normal mi/+ mice (Figure
4). Myelopoiesis in spleen and liver of
mi/mi mice was enhanced, but the levels of Mgf and
Flt3l gene messenger RNAs (mRNAs) did not significantly increase.
In contrast, B lymphopoiesis in mi/mi bone marrow was reduced,
while almost equal amounts of Il7 mRNA were detected in the
bone marrow of mi/mi mice and normal littermates. Not only
liver where B lymphopoiesis occurred in osteopetrotic mice, but also
spleen where no B lymphopoiesis occurred, revealed a high level
expression of Il7 mRNA both in mi/mi and mi/+
mice. Although all osteopetrotic mouse strains showed enhanced
myelopoiesis in the spleens, op/op but not mi/mi spleens demonstrated increased Mgf mRNA (Figure 4). B
lymphopoiesis was observed in Fos ( / ) spleen;
however, significant differences between Fos (+/ ) and
Fos ( / ) spleens in the expression of Il7 gene were not detected (Figure 4). Furthermore, we did not detect any
differences in Flt3l gene expression between mutants and their controls. These results suggest that although SLF, Flt3L, and IL-7 play
critical roles in hematopoiesis, including B lymphopoiesis, induction
or initiation of extramedullary hematopoiesis might be regulated by
factors other than those we have tested here.

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| Fig 4.
Expression of mRNA of Mgf, Flt3l, and Il7
in the osteopetrotic mice.
Total RNAs were prepared from bone marrow, spleens, or livers of
8-week-old osteopetrotic mice. Different dilutions (1:1, 1:8, 1:64) of
cDNA were subjected to PCR amplification specific for Mgf,
Flt3l, Il7, and GAPD transcripts. The
products were electrophoresed, blotted, and probed to detect messages
for each corresponding gene.
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 |
Discussion |
Osteopetrotic mice have remarkably diminished total hematopoietic
cell numbers in the bone marrow.8,50-52 In this study, we
showed selective reduction of B lymphopoiesis in the rudimentary bone
marrow of osteopetrotic mice regardless of whether the mutated gene was
Csfm, Mitf, or
Fos.34-38 All these mutant mice had
enhanced extramedullary hematopoiesis in their spleens and livers, but B lymphopoiesis in op/op and mi/mi mice was enhanced
only in the liver. Despite the fact that early stages of
lympho-hematopoiesis are dependent on SLF, Flt3L, and IL-7, no
significant differences of the expression of these genes were observed
in the bone marrow, spleen, or liver of osteopetrotic versus normal mice.
The frequencies of stromal cell + IL-7-dependent B precursors and
CFU-IL-7 in osteopetrotic bone marrow were all reduced; however, the
progenitor cells that give rise to stromal cell + IL-7-dependent B
precursors and CFU-IL-7 were maintained in the osteopetrotic marrow
(Table 3). These results suggest that the progenitor cells of the B
lineage are present in the osteopetrotic bone marrow, but the
microenvironment may be defective for support of further development of
B cells.
The relationship between the volume of the bone marrow cavity and
medullary B lymphopoiesis is obvious in vertebrates. Intramarrow hematopoiesis first appears in amphibians,53 and the bone
marrow in land Rana is an active site for hematopoiesis
involving myelopoiesis and lymphopoiesis, whereas the bone marrow of
aquatic amphibians such as Xenopus is rudimentary and is not a
lymphopoietic tissue.54 Moreover, aquatic mammals such as
Trichechiformes, with significantly diminished bone
cavity,55 have been observed to carry out erythropoiesis and granulopoiesis, but not lymphopoiesis, in their
vertebrae.56 Furthermore, the fact that bone marrow cells
from the op/op mice spontaneously cured of osteopetrosis showed
a normal frequency of CFU-IL-7 also supports the existence of such a
relationship.41,42
Each lineage of hematopoietic cells in the bone marrow develops in a
specialized location, indicating that local requirements necessary for
each cell lineage are not identical.26 Jacobsen and
Osmond5 proposed specially organized intramarrow B
lymphopoiesis in the bone marrow. According to their proposal, B
lymphopoiesis is centrally directed from the peripheral subendosteal
area, near the bone cortex, in which the earliest B progenitor cells
are located in extravascular spaces of marrow, in accordance with progression of maturation.5 Hematopoiesis in the bone
marrow may be initiated first by erythropoiesis or myelopoiesis and
then followed by B lymphopoiesis. Therefore, it is probable that the subendosteal microenvironment, which supports early B lymphopoiesis, is
preserved, but the microenvironment required for subsequent differentiation is not retained properly in the osteopetrotic bone marrow.
We have previously reported that even in scid/scid mice, which
completely lack lymphocytes and thus might have room available for
hematopoiesis in all lymphoid organs, hematopoiesis is not enhanced in
the spleen, indicating that the spleen cannot support adult
hematopoiesis because of a microenvironmental defect, and the defect is
not simply caused by a defective space or niche for
hematopoiesis.7 When scid/scid mice are treated
with low-dose X-irradiation (150 rad), hematopoiesis in the spleen is
induced and Mgf gene expression in the spleen is
up-regulated,7 indicating that SLF is one of the candidates
of essential factors for the construction of hematopoietic
microenvironments.57-59
It has been reported that injection of high doses of soluble SLF does
not significantly increase the number of HSCs but promotes the
migration of HSCs from the bone marrow to the spleen and peripheral blood.60 Recently, we prepared transgenic mice expressing
Mgf transgenes in the spleen, but Mgf expression was
not necessarily accompanied by splenic hematopoiesis (T. Kunisada, H. Tagaya, and H. Yamazaki, unpublished observation).61 In the
present study, although myelopoiesis was promoted in the spleens of
osteopetrotic mice, Mgf up-regulation was not observed. All of
these findings suggest that Mgf gene expression is not
necessarily enhanced during extramedullary hematopoiesis.
Major histocompatibility complex class II promoter-driven
Il7-transgenic mice show extremely elevated B lymphopoiesis in
the bone marrow, spleen, liver, and lymph nodes.32,33 This
may suggest that insufficiency of IL-7 production in the spleen is a
reason for the lack of B lymphopoiesis in the spleens of osteopetrotic mice, because hematopoiesis other than B lymphopoiesis is enhanced in
osteopetrotic mice. However, Il7 mRNA is expressed in the
spleen as well as liver both in mi/mi and mi/+ mice,
indicating that induction of extramedullary hematopoiesis might be
regulated by factors other than those tested here.
An alternative explanation for the extramedullary hematopoiesis is that
not only bone marrow but also spleen and liver constitutively produce
these factors sufficiently to support adult hematopoiesis, and
distribution of HSCs is a crucial requirement in this process. Because
HSCs in osteopetrotic mice rarely migrate into and lodge in the bone
marrow, mainly due to the lack of adequate space, HSCs could only be
retained in the extramedullary organs.62-68
Recently, analyses of knock-out mice targeting the genes for the
chemokine SDF-1 or its receptor, CXCR4, have shown that HSCs or
immature cells, especially B lineage cells, require signaling via CXCR4
for migration into and retention in the bone marrow69,70; TEL/ETV6 transcription factor null mice show virtually the same phenotypes.71 The possibility remains that SDF-1 production may be insufficient in the osteopetrotic marrow, and therefore immature
B lineage cells selectively leave the bone marrow.62 However, because our experiments did not detect the differences of
Sdf-1 messages between mi/mi and mi/+ mice
(data not shown), other explanations will have to be found for the
defect of B lymphopoiesis in the osteopetrotic marrow.
Il7-transgenic mice have also been reported to display
accelerated bone resorption, resulting in an increase of the bone
marrow cavity.33 Because B lineage cells are known to
produce osteoclast differentiation factor (also named osteoprotegerin
ligand, TRANCE, and RANKL), which induces osteoclastogenesis in the
presence of M-CSF,72-75 it is possible that the B lineage
cells themselves control osteoclast development and regulate the volume
of the bone marrow cavity.51,76,77
Lastly, it is still unclear why only Fos ( / )
spleen is able to support CFU-IL-7 generation. Unknown critical
molecules for B lymphopoiesis might be activated when c-Fos
transcription factor is deleted. In summary, osteopetrotic mice should
provide us opportunities to elucidate molecular and structural
requirements for intramedullary and extramedullary hematopoiesis.
 |
Acknowledgments |
We thank Dr Paul W. Kincade (Oklahoma Medical Research Foundation), Dr
Shin-Ichi Nishikawa, Dr Minetaro Ogawa, Dr Koichi Ikuta (Kyoto
University), Dr Seiji Okada (Chiba University), Dr Toru Naskano (Osaka University), and Dr Shumpei Niida (Hiroshima University) for helpful suggestions and generous gifts of reagents. We also thank
Dr Toshiyuki Shibahara and Dr Takashi Iwaki for maintenance of the mice
and Ms Toshie Shinohara for secretarial assistance.
 |
Footnotes |
Submitted October 19, 1999; accepted January 31, 2000.
Supported by grants from the Ministry of Education, Science, Sports and
Culture in Japan; the Ministry of Science and Technology of Japan; the
Cellular Technology Institute; Otsuka Pharmaceutical Co, Ltd; Osaka
Foundation for Promotion of Clinical Immunology; and NIH grant
CA20 408 (L.D.S.). T. Yamane is a research fellow of the Japan Society
for the Promotion of Science.
Reprints: Shin-Ichi Hayashi, Department of Immunology, School
of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-machi, Yonago, Tottori 683-8503, Japan; e-mail:
shayashi{at}grape.med.tottori-u.ac.jp.
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.
 |
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