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Departments of
*
Orthopaedic Surgery and
Cardiovascular Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan;
Department of Biochemistry, Tokyo University of Pharmacy and Life Science, Tokyo, Japan;
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan;
¶ Core Research for Evolutional Science and Technology, Research Development Corporation of Japan, Tokyo, Japan; and
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Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand (ODF/RANKL)
was identified as a key membrane-associated factor regulating
osteoclast differentiation. We demonstrate that B-lymphoid lineage
cells are a major source of endogenous ODF/RANKL in bone marrow and
support osteoclast differentiation in vitro. In addition, B-lymphoid
lineage cells in earlier developmental stages may hold a potential to
differentiate into osteoclasts when stimulated with M-CSF and soluble
ODF/RANKL in vitro. B-lymphoid lineage cells may participate in
osteoclastogenesis in two ways: they 1) express ODF/RANKL to support
osteoclast differentiation, and 2) serve themselves as osteoclast
progenitors. Consistent with these observations in vitro, a decrease in
osteoclasts is associated with a decrease in B-lymphoid cells in
klotho mutant mice (KL-/-),
a mouse model for human aging that exhibits reduced turnover during
bone metabolism, rather than a decrease in the differentiation
potential of osteoclast progenitors. Taken together, B-lymphoid lineage
cells may affect the pathophysiology of bone disorders through
regulating osteoclastogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteoclasts, multinucleated cells primarily responsible for bone
resorption, have been shown to differentiate from the
monocyte/macrophage lineage (1, 2, 3, 4). Mature osteoclasts can
be formed in vitro from bone marrow cells in the presence of two
cytokines, M-CSF and osteoclast differentiation factor/receptor
activator of NF-B ligand
(ODF/RANKL)3
(5, 6, 7, 8, 9). Osteoblastic cells and stromal cell lines such as
ST2 also support osteoclast formation from bone marrow cells in vitro.
In addition to the in vitro studies, recent experiments using knockout
mice demonstrated that disruption of either m-csf or
odf/rankl gene causes increased bone mass (osteopetrosis)
due to a complete lack of osteoclasts (10, 11). Thus,
M-CSF and ODF/RANKL are regarded as key regulators of
osteoclastogenesis. However, the osteoclast defect in m-csf
mutant mice can be restored by overexpression of anti-apoptotic
protein Bcl-2 in monocyte lineage cells (12), suggesting
that M-CSF is essential for the survival but not the differentiation of
osteoclast progenitors.
The monocyte/macrophage lineage cells that give rise to osteoclasts and lymphoid lineage cells are thought to be distantly related. However, evidence is accumulating in support of a close relationship between macrophage and B-lymphoid cell differentiation pathways. There are numerous reports on B cell lymphomas that acquired macrophage-like phenotypes, including characteristic morphological changes and expression of mature macrophage surface markers (see Ref. 13 for review). In addition, a subset of normal B cells (CD5+ B cells) is known to have properties of both B cells and macrophages (13, 14). CD5+ B cells are adherent and highly phagocytic, and they coexpress surface markers characteristic of macrophages (F4/80 and Mac-1) and B cells (B220, IgM, and IgD). Recently, it was reported that Pax5-deficient pro-B cells have the ability to take various differentiation pathways, including the monocyte/macrophage lineage, and can give rise to osteoclasts in vitro (15). These findings are challenging current ideas of lineage commitment in B-lymphoid lineage cells.
We recently established a mouse model for human aging termed klotho (16). The klotho mouse was serendipitously generated by insertional mutation of a transgene in a transgenic mouse, which disrupted the klotho gene encoding a novel single-pass membrane protein. A defect in klotho gene expression leads to a syndrome closely resembling human aging including a shortened lifespan, decreased spontaneous activity, infertility, skin atrophy, arteriosclerosis, premature thymic involution, pulmonary emphysema, and osteopenia, among others. Osteopenia observed in klotho mutant mice (KL-/- mice) is accompanied by low turnover during bone metabolism, in which the decrease in bone formation exceeds the decrease in bone resorption, resulting in a net bone loss (17). The number of osteoclasts was significantly decreased in KL-/- mice. In addition, osteoclast formation from bone marrow cells of KL-/- mice in vitro was significantly reduced (17). The pathophysiology of the bone in KL-/- mice recapitulates senile osteoporosis in humans rather than postmenopausal osteoporosis, which is primarily caused by increased bone resorption accompanied by an increase in the number of osteoclasts.
In an effort to find out the cellular mechanism by which KL-/- mice develop osteopenia resembling senile osteoporosis, we found two novel roles that B-lymphoid lineage cells appear to play in osteoclastogenesis. In the present study, we report that B-lymphoid lineage cells express abundant ODF/RANKL and have the ability to support osteoclast differentiation. In addition, we show that a subset of normal bone marrow cells that express markers of B-lymphoid lineage cells in early developmental stages can serve as osteoclast progenitors and are able to give rise to osteoclasts in vitro. We also show that KL-/- mice have significantly reduced numbers of B-lymphoid lineage cells in the bone marrow, which could cause a decrease in osteoclasts in KL-/- mice due to the simultaneous reduction in cells expressing ODF/RANKL and in cells serving as osteoclast progenitors. These findings provide new insights into current concepts of osteoclast differentiation and molecular mechanisms of bone disorders such as osteoporosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type (KL+/+) and KL-/- mice were generated by mating of heterozygous klotho mutant mice (KL+/-). The genetic background of the original klotho mouse was a mixture of C57BL/6J and C3H/J (16).
Histomorphometric analysis of the bone
All histological analyses were conducted using 8-wk-old KL+/+ and KL-/- littermates. Tibiae were excised, fixed with 100% ethanol, embedded in methyl methacrylate, and sectioned in 6-µm slices for Villanueva-Goldner staining and tartrate-resistant acid phosphatase (TRAP) staining. TRAP staining was conducted at pH 5.0 in the presence of L(+)-tartaric acid using naphthol AS-MX phosphate (Sigma, St. Louis, MO) in N,N-dimethyl formamide as a substrate. The specimens were subjected to histomorphometric analyses under a light microscope with a micrometer using an image analyzer (System Supply, Nagano, Japan). Parameters for bone resorption were measured in a trabecular bone area (1.2 mm in length from 0.1 mm below the growth plate) at the proximal metaphysis of the tibiae. The number and function of osteoclasts were determined by counting the number of TRAP-positive cells and by measuring the percentage of bone surface area eroded by osteoclasts with the image analyzer.
Cell preparation
Bone marrow cells were prepared from mouse tibiae by flushing out the bone marrow with Ca2+- and Mg2+-free PBS (PBS-). The bone marrow cells were resuspended in 2 ml of ammonium chloride-Tris buffer to lyse red blood cells. The cell suspension was washed with PBS- three times and resuspended in 1 ml of PBS- containing 1% BSA. Spleen cell suspensions were prepared in the same way. PBMC were separated using Ficoll-Conray solution for mouse lymphocytes.
Flow cytometric analysis
Cells (1 x 106) were incubated for
30 min on ice with various combinations of FITC-conjugated
anti-B220/CD45R (RA3-6B2; BD PharMingen, San Diego, CA), Gr-1
(RB6-8C5; BD PharMingen), CD3 (17A2; BD PharMingen), or F4/80 (MCA497F;
Serotec, Oxford, U.K.), and PE-conjugated TER-119 (TER-119; BD
PharMingen) or IgM-µ-chain Abs (Tago Scientific, Burlingame, CA),
washed twice with PBS-, and resuspended in
PBS- containing 1% BSA. The cells labeled with
fluorescent dyes were analyzed on a flow cytometer (FACSCalibur; BD
Biosciences, Mountain View, CA). For two-color analysis of ODF/RANKL
and B220, cells were incubated for 30 min on ice with FITC-conjugated
anti-B220 and an affinity-purified goat polyclonal
anti-ODF/RANKL Ab (C-20: sc-7627; Santa Cruz Biotechnology, Santa
Cruz, CA) raised against a peptide mapping at the carboxyl terminus of
human ODF/RANKL and shown to react with mouse, rat, and human
ODF/RANKL. After washing with PBS-, cells were
incubated with biotin-conjugated rabbit anti-goat IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA) and washed twice with
PBS-. The cells were then incubated for 10 min
at room temperature with streptavidin-PE conjugate (BD PharMingen), and
washed twice with PBS-. As negative controls,
goat IgG (Jackson ImmunoResearch Laboratories) and FITC-conjugated rat
IgG2a, (BD PharMingen) were used instead of anti-ODF/RANKL Ab
and FITC-conjugated anti-B220 (isotype: rat IgG2a, ),
respectively. Two-color FACS analysis with PE-labeled anti-ODF Ab
vs FITC-conjugated rat IgG2a and PE-labeled goat IgG vs
FITC-conjugated anti-B220 Ab was used to set the appropriate levels
of compensation.
Osteoclast formation in vitro
Bone marrow cells were collected from tibiae and femora of
6-wk-old KL+/+ or
KL-/- mice. The bone marrow cells
were cultured in 
MEM containing 10% FBS. Osteoclast differentiation
was induced for 8 days either with 1,25(OH)2
vitamin D3 (10-8 M) and
PGE2 (10-6 M) or with
ODF/RANKL (100 ng/ml; PeproTech, Rocky Hill, NJ) and M-CSF (recombinant
human M-CSF, 20 ng/ml; Genzyme, Cambridge, MA). TRAP-positive
multinucleated cells with three or more nuclei were counted as
osteoclastic cells. Genomic DNA from osteoclasts was prepared as
follows. First, the plates were washed five times with
PBS- to remove nonadherent cells that failed to
differentiate into osteoclasts. Then, the plates were treated with
0.25% trypsin to dislodge the nonosteoclast adherent cells.
Osteoclasts adhere to the culture plates so firmly that they are not
dislodged by trypsin treatment. After three times trypsin treatment,
every cell remaining on the culture plates was examined under the
microscopy to confirm that all were typical multinucleated osteoclasts.
The osteoclastic cells on the plates were then treated with proteinase
K (0.15 mg/ml) at 50°C for 3 h. The genomic DNA was precipitated
by adding isopropanol to the cell lysate.
Magnetic cell sorting of B-lymphoid lineage cells
B-lymphoid lineage cells were isolated with a magnetic cell-sorting system (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). After washing steel-wool columns with PBS, the matrix of the column was incubated with 1% BSA in PBS for 15 min to saturate nonspecific binding sites. After the column was flushed with ice-cold PBS/1% BSA, cells labeled with anti-B220, CD19, or CD43 Ab-conjugated magnetic beads were applied to the column. Unbound cells were then washed out with 2–4 column volumes of PBS/1% BSA or until no cells were collected at flow rates of 10 ml/h. The column was further washed with >3 volumes of PBS/1% BSA at an increased flow rate. After the steel-wool column was removed from the external magnetic field, bound cells were eluted and analyzed by flow cytometry. We repeated the sorting steps until >99% of eluted cells were positive for each marker.
Analysis of ODF/RANKL expression
Bone marrow cells were flushed out from femora and tibiae of 6-wk-old KL+/+ mice with PBS- and fractionated into B220+ and B220- cells as described above. Poly(A)+ RNA and total protein were extracted from B220+ cells, B220- cells, whole bone marrow cells, and residual bones (bones without the bone marrow cells). Poly(A)+ RNA was extracted using oligo(dT) columns (Pharmacia, Peapack, NJ). The poly(A)+ RNA (500 ng) was reverse-transcribed into single-strand cDNA with random hexamer and amplified with LA-Taq DNA polymerase with a commercially available kit (LA RT-PCR Kit; Takara, Kyoto, Japan), using primers specific for mouse ODF/RANKL (5'-CTCCGAGCTGGTGAAGAAA-3' and 5'-CAGGGGAATTACAAAGTGC-3') or G3PDH (5'-CATGTAGGCCATGAGGTCCACCAC-3' and 5'-TGAAGGTCGGTGTGAACGGATTTG GC-3'). Protein was extracted using TNE buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM aminoethyl-benzenesulfonyl fluoride, and 10 µg/ml aprotinin) and quantified with the BCA Protein Assay kit (Pierce, Rockford, IL). The same amounts of protein were separated by SDS-PAGE and then transferred to a nitrocellulose membrane. ODF/RANKL was detected using the anti-ODF/RANKL Ab, an HRP-conjugated goat anti-mouse IgG (1 µg/ml; Amersham, Arlington Heights, IL), and ECL Western blotting detection reagents (Amersham). The membrane was stained with Amido Black (Sigma) and reprobed with anti-p38 MAPK Ab (New England Biolabs, Beverly, MA) to confirm that equivalent amounts of protein were loaded in each lane.
Genotyping at the mouse klotho locus
Genotypes at the mouse klotho locus in the osteoclasts were determined by PCR using three primers (5'-CAAGGACCAGTCATCATCG-3', 5'-TTAAGGACTCCTGCATCTGC-3', and 5'-TGGAGATTGAAGTGGACG-3') specific to the mutated allele, wild-type allele, and both alleles, respectively. The mutated klotho allele (kl) and wild-type klotho allele gave PCR products of 920 and 458 bp, respectively.
Statistical analysis
Means of groups were compared by ANOVA, and significance of differences was determined by post hoc testing using the Bonferroni method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histomorphometric analysis of the bone of
KL-/- mice revealed a significant
reduction in the number of osteoclasts and bone resorption in vivo
(Fig. 1
, a and b)
(17). In addition, the number of osteoclastic cells
generated in vitro from whole bone marrow cells of
KL-/- mice was significantly lower
than that of wild-type (KL+/+) mice
when osteoclastogenesis was induced with
1,25(OH)2 vitamin D3 and
PGE2 (Fig. 1c). Three possible
mechanisms were considered to explain the impairment in osteoclast
formation in KL-/- mice. First, the
number of osteoclast progenitors may be reduced in the bone marrow.
Second, the cells and/or molecules that support the differentiation of
osteoclast progenitors may be defective. Third, the osteoclast
progenitors may have an intrinsic defect in differentiation. Although
distinct, these possibilities are not mutually exclusive.
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). Considering that the number of total
nucleated cells in KL-/- mice was
lower than that in KL+/+ mice (Table I), the decrease in the number of B220+ cells in
KL-/- mice was even larger. B220 is
a cell surface marker expressed on B-lymphoid lineage cells at all
stages of differentiation. Among the B220+ cells,
those expressing low levels of B220 (B220low)
were most significantly decreased in the bone marrow of
KL-/- mice (Fig. 2). The majority of
B220low cells were negative for IgM-µ-chain,
which is a marker of immature and mature B lymphocytes (Fig. 2). The
number of B220+ cells in the spleen and
peripheral blood of KL-/- mice was
also lower than in KL+/+ mice
(18). In addition, we previously reported that the number
of pro-B, pre-B, and mature B cells defined as
B220+CD43+IgM-,
B220+CD43-IgM-,
and B220+IgM+ cells,
respectively, was significantly lower in
KL-/- mice (18). These
data suggest that KL-/- mice have a
defect in early stages of B cell development.
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). The frequency
of T lymphoid lineage cells (CD3+ cells) was
slightly higher in KL-/- mice;
however, the slight increase did not translate into an increase in the
number of CD3+ cells because of the decrease in
the number of total nucleated cells in
KL-/- mice. Expression of ODF/RANKL on B-lymphoid lineage cells
To test whether KL-/- mice have
a defect in cells and/or molecules that support osteoclast
differentiation, we examined expression of ODF/RANKL in the bone
marrow. Dual-color flow cytometric analysis using anti-ODF/RANKL Ab
and Abs against several lineage-specific markers revealed that
ODF/RANKL is expressed primarily on the surface of B-lineage cells
expressing B220 (Fig. 3a).
Because the number of B220+ cells in the bone
marrow is significantly decreased in
KL-/- mice, the percentage of cells
expressing ODF/RANKL is also decreased from 13.3% in
KL+/+ to 6.2% in
KL-/- mice.
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a).
RT-PCR and immunoblot analysis confirmed that
B220+ cells are a major source of endogenous
ODF/RANKL in the bone (Fig. 3b). Expression of ODF/RANKL in
various tissues and cell lines has been described; however, there had
been no report on its expression in the bone marrow until quite
recently. These data indicate that ODF/RANKL is principally expressed
on B-lymphoid lineage cells in the bone. This finding is in agreement
with a recent study reporting that B lymphocytes and the pre-B cell
line 70Z/3 express ODF/RANKL more abundantly than bone marrow adherent
cells, which contain stromal cells (19). Taken together,
these observations have raised the possibility that ODF/RANKL expressed
on B-lymphoid lineage cells may be involved in osteoclastogenesis. B-lymphoid lineage cells support osteoclast differentiation
We tested whether ODF/RANKL expressed on
B220+ cells could support osteoclast formation in
vitro. Bone marrow cells from KL+/+
mice were separated into B220+ and
B220- cell fractions using magnetic cell
sorting. The sorted cells were cultured in medium containing
10-8 M 1,25(OH)2 vitamin
D3 and 10-6 M
PGE2 to induce osteoclast differentiation.
According to the current consensus on osteoclast differentiation,
osteoclast progenitors belong not to the lymphoid but to the
monocyte/macrophage lineage and should be enriched in the non-B
(B220-) cell fraction. Therefore, we expected
that robust osteoclast formation from B220-
cells would be observed. Contrary to our expectation, very few
osteoclasts were generated by B220- cells alone
(Fig. 4a). However, when
B220+ cells were added back to the fixed number
of B220- cells, osteoclast formation was
significantly augmented in proportion to the number of
B220+ cells added (Fig. 4c).
B220+ cells alone gave rise to very few
osteoclasts under these conditions (Fig. 4a). These results
clearly demonstrated that interactions between
B220+ and B220- cells
potentiated differentiation of osteoclasts. Osteoclast formation was
completely abolished by the addition of anti-ODF/RANKL Ab or
recombinant osteoprotegerin (100 ng/ml), a soluble decoy receptor of
ODF/RANKL, indicating that endogenous ODF/RANKL is essential for
osteoclastogenesis under these culture conditions.
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, c–f).
B220+ cells from
KL-/- mice supported osteoclast
formation to the same extent as those from
KL+/+ mice. In addition, the
B220- cells from
KL-/- mice generated almost the same
number of osteoclasts as those from
KL+/+ mice. Thus, the bone marrow
cells of KL-/- mice showed no
difference in osteoclastogenesis in vitro from those of
KL+/+ mice. Nevertheless, the number
of osteoclasts generated from whole bone marrow cells of
KL-/- mice was reduced to
approximately one-third that of KL+/+
mice (Fig. 1c). It should be noted that, in
KL-/- mice, the frequency of
B220+ cells is also reduced to almost one-third
(Table I). When the
B220+/B220- ratio was
reduced to that found in KL-/- mice
(1 x 105 B220+ cells
with 1 x 106 B220-
cells), the number of osteoclasts generated was almost the same as that
generated from the whole bone marrow mixture of
KL-/- mice. The results were
indifferent to the origin of the cells (Fig. 4, c–f).
Therefore, it is unlikely that the reduction of osteoclast formation
from whole bone marrow cells of
KL-/- mice in vitro is caused by an
inability of bone marrow cells to support osteoclast differentiation or
by an intrinsic defect in osteoclast progenitors to differentiate.
Rather, the reduction in osteoclastogenesis could simply be caused by
the reduction in the number of B220+ cells.
The most likely explanation for the results of the coculture experiment
is that ODF/RANKL expressed on the surface of
B220+ cells supported differentiation of
osteoclast progenitors in B220- cell fraction.
In fact, the effect of B220+ cells could be
replaced by soluble ODF/RANKL (Fig. 4b) and abolished with
anti-ODF/RANKL Ab or osteoprotegerin. If
B220+ cells serve exclusively as a source of
ODF/RANKL, osteoclasts generated in the coculture experiment should
have originated only from the B220- cell
fraction that contains osteoclast progenitors. To test this
possibility, we examined the origin of the osteoclasts. We generated
osteoclasts by coculture of B220+ cells from
KL-/- mice and
B220- cells from
KL+/+ mice (and vice versa) and
determined klotho locus genotypes of the osteoclasts.
Semiquantitative PCR demonstrated that 
75% of osteoclasts
originated from B220- cells and that 25% came
from B220+ cells (Fig. 5). The majority of osteoclasts
originated from the B220- cell fraction as
expected, verifying the idea that B220+ cells
support differentiation of osteoclast progenitors in the
B220- cell fraction. At the same time, however,
these observations imply that a significant portion of osteoclasts
(25%) came from B220+ cells. This high
percentage cannot be explained by contamination of
B220- cells in the B220+
cell fraction, because the contamination was determined to be <1% by
FACS analysis. Rather, it suggests that B220+
cells actually contributed to osteoclast formation. These observations
raised the possibility that the B-lymphoid lineage cells functioned not
only as a source of ODF/RANKL but also as osteoclast progenitors.
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To confirm that B220+ cells are able to give
rise to osteoclasts, we isolated cells from the bone marrow of
KL+/+ mice by cell sorting using
several different B-lineage markers (CD43, B220, CD19) and induced
osteoclast differentiation with soluble ODF/RANKL and M-CSF (Fig. 6). Under these conditions,
CD43+B220+ cells gave rise
to the highest number of osteoclasts. B220+ cells
and CD19+ cells gave rise to equivalent numbers
of osteoclasts as did B220- and
CD19- cells, respectively. However,
B220+ and CD19+ cells from
the spleen, most of which are positive for IgM-µ-chain, gave rise to
very few osteoclasts. These splenic B-lineage cells are regarded as
having already committed to the formation of mature B lymphocytes, and
lost the capability of differentiating into other cell lineages.
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). Therefore, we conclude
that B-lymphoid lineage cells in earlier developmental stages, before
the pre-BII stage, contain significant osteoclast progenitor
activity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies challenge the current concept of lineage commitment for B-lymphoid cells. Nutt et al. (15) showed that Pax5-deficient pro-B cells have multilineage potential. Bone marrow progenitors from Pax5-/- mice are unable to differentiate beyond the pre-BI stage of the B-lymphoid lineage. However, when induced with appropriate cytokines, these early pre-B cells can give rise to several different lineage cells including macrophages, osteoclasts, dendritic cells, granulocytes, and NK cells. The lineage switch of Pax5-/- pro-B cells to osteoclasts may not be an abnormal pathway due to the blockade in the normal B cell differentiation, because our present data indicate that normal B-lymphoid lineage cells may also have the potential to differentiate into osteoclasts when induced by soluble ODF/RANKL and M-CSF. We conclude that a subset of normal pro-B cells is uncommitted and may still hold a potential to give rise to osteoclasts.
The fact that the pro-B cell fraction contains osteoclast progenitors may have implications in pathophysiology of osteoporosis because it is known that the number of pro-B cells is positively correlated to the number of osteoclasts in vivo in various pathological conditions. Estrogen withdrawal causes a marked increase in the number of both B220low cells (containing pro-B cells) and osteoclasts, which leads to bone loss in humans (postmenopausal osteoporosis), and mice (osteopenia caused by ovariectomy) (1, 22, 23). In addition, IL-7, a cytokine that primarily stimulates B lymphopoiesis, also causes an increase in osteoclasts and bone loss when injected into mice (24). In contrast, a decrease in B220low cells is accompanied by a decrease in osteoclast numbers as observed in IL-7 receptor knock-out mice (24) and in KL-/- mice. Interestingly, the number of both osteoclasts and B220low cells decreases with age in mice (25) and humans (26). The positive correlation between the number of B220low cells and osteoclasts in various pathological conditions and the natural aging process may be partly explained by the possibility that pro-B cells actually serve not only as B cell progenitors but also as osteoclast progenitors in vivo.
ODF/RANKL expressed on B220+ cells may be another
factor that is involved in pathophysiology of osteoporosis. Although
our present study showed that B220low cells
express less ODF/RANKL than B220high cells (Fig. 3a), a significant increase in B220low
cells could result in a substantial increase in total amount of
ODF/RANKL in the bone marrow, which may contribute to acceleration of
osteoclastogenesis. A recent report indicated that the number of
B220low cells that express ODF/RANKL was higher
in bone marrow from ovariectomized mice than in bone marrow from
sham-operated mice (27). Because the expression level of
ODF/RANKL in each B220low cell was not
significantly different, the increase in the number of
B220low cells means a net increase in the amount
of ODF/RANKL expression. Conversely, the decrease in
B220low cells in
KL-/- mice results in a net decrease
in ODF/RANKL expression (Fig. 3a). Based on these
observations, the number of B220+ cells could
affect osteoclastogenesis and bone resorption in two ways: a change in
the amount of ODF/RANKL in the bone marrow and a change in the number
of osteoclast progenitors.
The mechanism by which B lymphopoiesis is impaired in KL-/- mice remains to be elucidated. A recent study indicated that the number of IL-7-responsive clonogenic progenitors (B-lymphoid progenitors) in bone marrow cells was markedly decreased in KL-/- mice (18). However, the number of hemopoietic stem cells and their capacity for B lymphopoiesis were normal (18), suggesting that a defect in hemopoietic environment might be responsible for poor B lymphopoiesis or survival in KL-/- mice. It remains to be determined whether the decrease in B220+ cells is the only explanation for the poor osteoclastogenesis in KL-/- mice. In addition to the decrease in B-lineage cells, it is possible that local and/or systemic factors affect osteoclast differentiation in KL-/- mice. In fact, we previously demonstrated that KL-/- mice had significantly higher blood osteoprotegerin levels than KL+/+ mice (17), which could also inhibit osteoclast formation in KL-/- mice in vivo. However, there was no significant difference in M-CSF mRNA levels between KL+/+ and KL-/- mice (18).
To our knowledge, there are no reports of osteoclast abnormality in B
cell-deficient mice. There are two major differences between
KL-/- mice and other B
cell-deficient mice. First, although many B cell-deficient mouse
strains, such as RAG2-/- and
5-/- mice (28, 29), lack
IgM+ cells, they have normal numbers of pro-B
cells, which contain the osteoclast progenitors. In contrast,
KL-/- mice show losses in both the
IgM+ and pro-B cells, which results in a
simultaneous reduction in both ODF/RANKL expression levels and the
number of osteoclast progenitors. Therefore,
KL-/- mice would be expected to have
a more severe defect in osteoclastogenesis than the other B
cell-deficient strains. Second, B-lymphoid lineage cells in the other B
cell-deficient strains have intrinsic defects in the ability to
differentiate into mature B lymphocytes. In contrast, the impairment of
B lymphopoiesis in KL-/- mice was
attributed to a defect in the "microenvironment" in the bone marrow
(18), which could also affect osteoclast
differentiation.
Our present study has indicated that B220+ cell
fraction contains both ODF/RANKL-expressing cells and osteoclast
progenitors. However, B220+ cells alone, when
stimulated with 1,25(OH)2 vitamin
D3 and PGE2, gave rise to
many fewer osteoclasts than those cocultured with
B220- cells (Fig. 4a). The poor
osteoclast formation was not completely restored by adding M-CSF (data
not shown), suggesting that endogenous ODF/RANKL was insufficient for
the effective osteoclast formation from B220+
cells and that B220- cells played an important
role in the osteoclast differentiation. Cell-cell interaction between
osteoclast progenitors and stromal cells is believed to be
indispensable for efficient osteoclast differentiation. Stromal cells
express several adhesion molecules including VCAM-1 that binds to very
late Ag-4 expressed on B-lymphoid lineage cells. Previous studies
showed that very late Ag-4/VCAM-1 interactions are important in the
adhesion of B cells to the bone marrow microenvironment and B cell
differentiation (30). It is possible that stromal cells,
which reside in the B220- cell fraction, act as
scaffolding to tether B220+ cells that express
ODF/RANKL to produce a microenvironment where ODF/RANKL is focally
concentrated. Such scaffolding may no longer be necessary when
excessive ODF/RANKL is present, as we observed robust osteoclast
formation from B220+ cells stimulated with M-CSF
and soluble ODF/RANKL (Fig. 6).
In conclusion, we propose two possible mechanisms by which B-lymphoid lineage cells regulate osteoclastogenesis: they act as the major source of ODF/RANKL and support differentiation of osteoclast progenitors into mature osteoclasts. In addition, normal pro-B cells may be able to serve as osteoclast progenitor cells. The close association between B-lymphoid cell and osteoclast differentiation pathways may also imply involvement of B-lymphoid cells in the pathophysiology of bone disorders such as osteoporosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Makoto Kuro-o, Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9072. E-mail address: kuroo.makoto{at}pathology.swmed.edu
3 Abbreviations used in this paper: ODF/RANKL, osteoclast differentiation factor/receptor activator of NF-B ligand; KL, klotho; TRAP, tartrate-resistant acid phosphatase; PBS-, Ca2+- and Mg2+-free PBS.
Received for publication September 21, 2000. Accepted for publication July 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand/osteoprotegrin ligand on pre-B cells: implications for accelerated osteoclastogenesis in estrogen deficiency. J. Bone Miner. Res. 15:1321.[Medline]
5 protein in B cell development. Cell 69:823.[Medline]
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  J. Lorenzo, M. Horowitz, and Y. Choi Osteoimmunology: Interactions of the Bone and Immune System Endocr. Rev., June 1, 2008; 29(4): 403 - 440. [Abstract] [Full Text] [PDF]
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 S. H. Windahl, M. K. Lagerquist, N. Andersson, C. Jochems, A. Kallkopf, C. Hakansson, J. Inzunza, J.-A. Gustafsson, P. T. van der Saag, H. Carlsten, et al. Identification of Target Cells for the Genomic Effects of Estrogens in Bone Endocrinology, December 1, 2007; 148(12): 5688 - 5695. [Abstract] [Full Text] [PDF]
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 Y. Li, G. Toraldo, A. Li, X. Yang, H. Zhang, W.-P. Qian, and M. N. Weitzmann B cells and T cells are critical for the preservation of bone homeostasis and attainment of peak bone mass in vivo Blood, May 1, 2007; 109(9): 3839 - 3848. [Abstract] [Full Text] [PDF]
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D. Min, A. Panoskaltsis-Mortari, M. Kuro-o, G. A. Hollander, B. R. Blazar, and K. I. Weinberg Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging Blood, March 15, 2007; 109(6): 2529 - 2537. [Abstract] [Full Text] [PDF]
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 T. Ichikawa, K. Horie-Inoue, K. Ikeda, B. Blumberg, and S. Inoue Steroid and Xenobiotic Receptor SXR Mediates Vitamin K2-activated Transcription of Extracellular Matrix-related Genes and Collagen Accumulation in Osteoblastic Cells J. Biol. Chem., June 23, 2006; 281(25): 16927 - 16934. [Abstract] [Full Text] [PDF]
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 H.-C. Chen, J. C. Byrd, and N. Muthusamy Differential Role for Cyclic AMP Response Element Binding Protein-1 in Multiple Stages of B Cell Development, Differentiation, and Survival J. Immunol., February 15, 2006; 176(4): 2208 - 2218. [Abstract] [Full Text] [PDF]
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 H. Shibata, M. Abe, K. Hiura, J. Wilde, K. Moriyama, T. Sano, K.-i. Kitazoe, T. Hashimoto, S. Ozaki, S. Wakatsuki, et al. Malignant B-Lymphoid Cells with Bone Lesions Express Receptor Activator of Nuclear Factor-{kappa}B Ligand and Vascular Endothelial Growth Factor to Enhance Osteoclastogenesis Clin. Cancer Res., September 1, 2005; 11(17): 6109 - 6115. [Abstract] [Full Text] [PDF]
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 P. Valverde, T. Kawai, and M.A. Taubman Potassium Channel-blockers as Therapeutic Agents to Interfere with Bone Resorption of Periodontal Disease Journal of Dental Research, June 1, 2005; 84(6): 488 - 499. [Abstract] [Full Text] [PDF]
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M. C. Horowitz, Y. Xi, D. L. Pflugh, D. G. T. Hesslein, D. G. Schatz, J. A. Lorenzo, and A. L. M. Bothwell Pax5-Deficient Mice Exhibit Early Onset Osteopenia with Increased Osteoclast Progenitors J. Immunol., December 1, 2004; 173(11): 6583 - 6591. [Abstract] [Full Text] [PDF]
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 W.-K. Suh, S. X. Wang, A. H. Jheon, L. Moreno, S. K. Yoshinaga, B. Ganss, J. Sodek, M. D. Grynpas, and T. W. Mak The immune regulatory protein B7-H3 promotes osteoblast differentiation and bone mineralization PNAS, August 31, 2004; 101(35): 12969 - 12973. [Abstract] [Full Text] [PDF]
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 U. H. Lerner NEW MOLECULES IN THE TUMOR NECROSIS FACTOR LIGAND AND RECEPTOR SUPERFAMILIES WITH IMPORTANCE FOR PHYSIOLOGICAL AND PATHOLOGICAL BONE RESORPTION Critical Reviews in Oral Biology & Medicine, March 1, 2004; 15(2): 64 - 81. [Abstract] [Full Text] [PDF]
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M. A. Karsdal, P. Hjorth, K. Henriksen, T. Kirkegaard, K. L. Nielsen, H. Lou, J.-M. Delaisse, and N. T. Foged Transforming Growth Factor-{beta} Controls Human Osteoclastogenesis through the p38 MAPK and Regulation of RANK Expression J. Biol. Chem., November 7, 2003; 278(45): 44975 - 44987. [Abstract] [Full Text] [PDF]
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S.-K. Lee, J. F. Kalinowski, S. L. Jastrzebski, L. Puddington, and J. A. Lorenzo Interleukin-7 Is a Direct Inhibitor of in Vitro Osteoclastogenesis Endocrinology, August 1, 2003; 144(8): 3524 - 3531. [Abstract] [Full Text] [PDF]
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D. Reynaud, N. Lefort, E. Manie, L. Coulombel, and Y. Levy In vitro identification of human pro-B cells that give rise to macrophages, natural killer cells, and T cells Blood, June 1, 2003; 101(11): 4313 - 4321. [Abstract] [Full Text] [PDF]
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S. V. Komarova, M. F. Pilkington, A. F. Weidema, S. J. Dixon, and S. M. Sims RANK Ligand-induced Elevation of Cytosolic Ca2+ Accelerates Nuclear Translocation of Nuclear Factor kappa B in Osteoclasts J. Biol. Chem., February 28, 2003; 278(10): 8286 - 8293. [Abstract] [Full Text] [PDF]
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G. Toraldo, C. Roggia, W.-P. Qian, R. Pacifici, and M. N. Weitzmann IL-7 induces bone loss in vivo by induction of receptor activator of nuclear factor kappa B ligand and tumor necrosis factor alpha from T cells PNAS, January 7, 2003; 100(1): 125 - 130. [Abstract] [Full Text] [PDF]
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T. Yamashita, S. Okada, K. Higashio, Y.-i. Nabeshima, and M. Noda Double Mutations in Klotho and Osteoprotegerin Gene Loci Rescued Osteopetrotic Phenotype Endocrinology, December 1, 2002; 143(12): 4711 - 4717. [Abstract] [Full Text] [PDF]
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 T N Crotti, M D Smith, H Weedon, M J Ahern, D M Findlay, M Kraan, P P Tak, and D R Haynes Receptor activator NF-{kappa}B ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis Ann Rheum Dis, December 1, 2002; 61(12): 1047 - 1054. [Abstract] [Full Text] [PDF]
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 I. Mikkola, B. Heavey, M. Horcher, and M. Busslinger Reversion of B Cell Commitment upon Loss of Pax5 Expression Science, July 5, 2002; 297(5578): 110 - 113. [Abstract] [Full Text] [PDF]
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