The Journal of Experimental Medicine
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 10 2381-2395
Hematopoiesis is associated with primitive stem
cell proliferation and diff erentiation leading to the
production of maturing cells in the BM, followed
by their continuous release to the circulation.
One of the basic characteristics of immature and
maturing hematopoietic cells is their unique abil-
ity to migrate between diff erent organs, particu-
larly in and out of the BM, as manifested during
both homeostasis and stress conditions ( 1 ).
Egress of progenitors and maturing cells from
the BM is accelerated during alarm situations that
are associated with urgent needs to rapidly cope
with physiological demands, such as host defense
and repair. This process is termed “ mobiliza-
tion, ” and it is induced by diff erent stimulations,
including cytokines and infl ammatory and chemo-
therapeutic agents. The cytokine G-CSF is clini-
cally used to induce stem cell mobilization as a
source harvested for BM transplantation protocols
( 2 – 5 ). The migration of circulating progenitor
cells back to their BM is termed “ homing, ” a
multistep process in which the immature cells
actively cross the endothelium barrier between the
circulation and the BM compartment. Homing
has physiological roles in adult BM homeostasis
and in the course of BM repopulation during
stem cell transplantations in patients ( 6 ).
Both mobilization and homing require ac-
tive navigation and use partially overlapping
Abbreviations used: CFC, col-
ony-forming cell; DC-STAMP,
DC-specifi c transmembrane
protein; Erk, extracellular sig-
nal-regulated kinase; FN, fi bro-
nectin; MMP, matrix
mononuclear cell; PB, periph-
eral blood; PYD, pyridinoline;
RANKL, receptor activator of
NF- ? B ligand; SCF, stem cell
factor; SDF-1, stromal-derived
factor 1; SKL, Sca-1 + /c-Kit + /
Lin ? ; Tb.N, trabecular num-
ber; TRAP, tartrate-resistant
acid phosphatase; WBC, white
CD45 regulates retention, motility,
and numbers of hematopoietic progenitors,
and aff ects osteoclast remodeling
of metaphyseal trabecules
Shoham Shivtiel , 1 Orit Kollet , 1 Kfi r Lapid , 1 Amir Schajnovitz , 1
Polina Goichberg , 1 Alexander Kalinkovich , 1 Elias Shezen , 1 Melania Tesio , 1
Neta Netzer , 1 Isabelle Petit , 1 Amnon Sharir , 2,3 and Tsvee Lapidot 1
1 Department of Immunology and 2 Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
3 The Laboratory of Musculoskeletal Biomechanics and Applied Anatomy, Koret School of Veterinary Medicine,
Hebrew University of Jerusalem, Rehovot 76100, Israel
The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating
hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression
on bone marrow (BM) leukocytes correlates with increased cell motility in response to
stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility,
including reduced homing (both steady state and in response to stromal-derived factor 1)
and reduced granulocyte colony-stimulating factor mobilization. These defects were associ-
ated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion
and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the recep-
tor activator of nuclear factor ? B ligand, and impaired modulation of the endosteal com-
ponents osteopontin and stem cell factor, suggested defective osteoclast function. Indeed,
CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which
we attributed to defective cell fusion and Src function. This led to irregular distribution
of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently,
CD45KO mice had less primitive cells in the BM and increased numbers of these cells
in the spleen, yet with reduced homing and repopulation potential. Uncoupling environ-
mental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates
progenitor movement and retention by infl uencing both the hematopoietic and nonhema-
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CD45 REGULATES PROGENITOR MOTILITY AND RETENTION | Shivtiel et al.
CD45 expression is dynamically modulated by stress signals
To evaluate the potential involvement of the panleukocyte
CD45 phosphatase in the mobilization and recruitment of im-
mature cells, we tested its expression levels in response to stress
signals. G-CSF stimulations signifi cantly increased expression
of CD45 by mononuclear cells (MNCs) in the BM ( Fig. 1,
A and B ). CD45 up-regulation in the BM was observed in ma-
ture leukocytes such as lymphoid CD4 + and myeloid CD11b +
cells and, moreover, in immature hematopoietic c-Kit + pro-
genitors ( Fig. 1 C ). Similar results were obtained when mice
were stimulated with LPS, mimicking bacterial-induced in-
fl ammation ( Fig. 1 D ). This increase in CD45 expression on
BM leukocytes correlated with their release to the circulation
during G-CSF mobilization ( Fig. 1 E ). CD45 expression was
elevated by 1.3-fold on day 1 and by 1.5-fold on day 3, reach-
ing 1.6-fold on day 5, when both cell mobilization and CD45
levels gained their peak values. Hence, G-CSF treatment led to
a gradual increase in the expression of CD45 on BM cells be-
fore their egress. Interestingly, we observed in the circulation a
reduction in CD45 expression on mobilized cells compared
with untreated blood cells ( Fig. 1, A and B ). These results re-
veal that CD45 expression is modulated during cell mobiliza-
tion, suggesting that dynamic CD45 expression participates in
egress from the BM in response to diff erent stress signals.
Impaired progenitor expansion and reduced mobilization
in mice lacking CD45
To further elucidate the role of CD45 in cell egress, the
model of CD45KO mice was used. We tested the potential
of CD45KO cells to egress from the BM under steady-state
conditions and in response to G-CSF stimulations. Impor-
tantly, diff erential BM cell count demonstrated no major dif-
ferences in the composition of mature cells obtained from
WT versus CD45KO mice (unpublished data). As shown
by others ( 23 ), we also did not detect substantial diff erences
in the total numbers of circulating white blood cells (WBCs)
in untreated CD45KO and WT mice ( Fig. 1 E ). However,
G-CSF stimulations in CD45KO mice resulted in a delayed
and reduced response. A suboptimal protocol of G-CSF, ad-
ministrated for 3 d only compared with the optimal protocol
of 5 d, was tested. After 3 d of injections, we documented a
signifi cant reduction ( ? 50%) in the number of mobilized
WBCs in the circulation of CD45KO mice compared with
their WT counterparts. Of note, in this suboptimal protocol
the numbers of mobilized WT cells were already very high,
near their peak levels, as documented after 5 d of stimulations
( Fig. 1 E ). Notably, mobilization of immature CD45KO
progenitor cells was also signifi cantly impaired, which is re-
fl ected by the frequency of circulating colony-forming cells
(CFCs) ( Fig. 1 F ). Moreover, we found in the peripheral
blood (PB) of CD45KO mice lower numbers of primitive
Sca-1 + /c-Kit + /Lin ? (SKL) cells (a rare population shown to
contain most of the stem cell activity) after 3 d of G-CSF in-
jections ( Fig. 1, G and I ). Progenitor cell proliferation within
the BM reservoir is a prerequisite process for cell egress and
mechanisms ( 7 ). These complex processes involve an interplay
between cytokines, chemokines, adhesion molecules, and pro-
teolytic enzymes. Adhesion molecules, including members
of the ? 1 and ? 2 integrins, are crucial for undiff erentiated
cell retention in their BM niches, maintaining the stem cell
pool and function. Breakdown of this anchorage is essential for
progenitor cell release ( 3, 8 ). Proliferation and migration of
primitive cells are regulated by various cytokines and chemo-
kines such as stromal-derived factor 1 (SDF 1; also termed
CXCL12), its receptor CXCR4, and the cytokine stem cell
factor (SCF) ( 7, 9 – 11 ). Proteolytic enzymes, especially metal-
loproteinases (MMPs), play central roles in various steps of
stem cell mobilization and homing. These enzymes cleave dif-
ferent adhesion interactions, extracellular matrix components,
and cytokines, which further facilitate cell egress through the
mechanical and endothelial barriers ( 9, 12 – 15 ). Recently, we
suggested a new regulator of progenitor cell mobilization by
demonstrating that bone-degrading osteoclasts play a major
role in homeostatic release and selective stress-induced pro-
genitor cell mobilization ( 16 ). These hematopoietic-derived
multinucleated, fused giant cells are involved in bone remodel-
ing processes. Diff erent players regulate osteoclast development,
recruitment, and function in their bone resorbing sites. The
seven-transmembrane-region receptor DC-specifi c transmem-
brane protein (DC-STAMP) was shown to mediate cell – cell
fusion of osteoclast precursors and the assembly of multi-
nucleated cells ( 17 ). In addition, Src kinases were shown to
be involved in sealing zone formation ( 18 ), and osteoclast
motility is regulated by MMP expression and secretion (for
review see reference 19 ). The role of osteoclasts in progenitor
cell mobilization involves cleavage of endosteal components
such as SDF-1, SCF, and osteopontin, which are regulators of
stem cell anchorage and retention ( 16 ). Previous studies have
introduced the linkage between bone remodeling, regulation
of hematopoiesis, and the dynamic nature of BM stem cell
niches ( 5 ). However, in spite of extensive experiments, the
exact mechanisms and regulators underlying the migration,
localization, and retention of hematopoietic progenitor cells
have not been fully elucidated.
All leukocytes, including hematopoietic stem and progeni-
tor cell populations, are characterized by unique cell surface
expression of CD45. CD45 is a transmembrane protein tyro-
sine phosphatase. It dephosphorylates diff erent sites on Src
family kinases, and can serve as both a positive and negative
regulator in a cell type – and context-dependent manner ( 20,
21 ). CD45 was shown to regulate diff erent stages of lympho-
cyte maturation ( 22 ), especially their activation and prolifera-
tion ( 23, 24 ). However, its potential role in the function of
earlier, undiff erentiated hematopoietic progenitor cells was
not identifi ed. The distinctive expression of CD45 led us to
postulate that this phosphatase may regulate fundamental pro-
cesses of immature hematopoietic cells. Our results show that
CD45 has multiple roles in regulating the cell autonomous
motility of progenitor cells and retention of these cells in the
BM, as well as osteoclast-mediated remodeling of the metaph-
yseal bone trabecules.
JEM VOL. 205, September 29, 2008
( Fig. 1 H ). However, in the CD45KO mice, we fi rst observed
that untreated mice have a priori lower numbers of CFCs in
the BM. Of note, CD45KO CFCs expanded to a lower ex-
tent in response to G-CSF stimulation for 3 d, a time frame
G-CSF – induced mobilization ( 25 ). We therefore examined
the ability of primitive BM cells to expand in vivo in re-
sponse to G-CSF stimulations. After 3 d of G-CSF treatment,
CFCs in the WT BM increased their numbers by 1.5-fold
Figure 1. CD45 expression is modulated and essential for cell mobilization. (A – D) CD45 expression. WT mice were untreated (untr.), or were treated
with G-CSF for 5 consecutive days (A – C) or with a single injection of LPS (D). MNCs from BM and PB samples were examined for their CD45 expression by
FACS. A representative histogram of CD45 levels is shown in A. Isotype control staining (dotted lines), untreated cells (black line), and cells after 5 d of
G-CSF stimulations (gray line) are shown. A summary of four independent experiments is shown for G-CSF (B and C) and LPS (D). CD45 expression (presented
as a geometric mean value) on different sorted subpopulations after G-CSF stimulations for 5 d is shown in C. (E) In vivo mobilization. WT and CD45KO
mice were injected with G-CSF for 3 and 5 d or were left untreated. Total numbers of viable circulating WBCs were evaluated. (F) Frequency of CFCs in the
PB of 3 d of G-CSF – treated mice. The data summarize three independent experiments ( ± SD). (G) Representative FACS analysis of MNCs derived from the
PB after 3 d of G-CSF stimulations. Indicated values represent the percentage of SKL primitive cells in PB MNCs. One representative fi gure out of fi ve inde-
pendent experiments is shown. (H) Kinetics of the increased CFC numbers in the BM of G-CSF – treated WT and CD45KO mice. Numbers of colonies were
evaluated in untreated mice or after G-CSF administration for 3 and 5 d. Bars represent at least three mice in each group, showing mean ± SD. *, P < 0.01;
**, P < 0.05. (I) Table summarizing the percentage of SKL cells out of BM and PB MNCs in WT or CD45KO mice after G-CSF treatment for 3 d.
CD45 REGULATES PROGENITOR MOTILITY AND RETENTION | Shivtiel et al.
a reduced potential to engraft the BM of WT recipients, also
showing defects in primitive repopulating cells ( Fig. 2 I ). These
results demonstrate systemic cell autonomous defects in
CD45KO cell motility.
Reduced MMP-9 secretion and hyperadhesion
of CD45KO cells
To elucidate possible mechanisms underlying CD45-regu-
lated cell motility, we assessed the secretion potential of MMPs,
in particular of MMP-9, which is involved in hematopoietic
cell invasion and is up-regulated upon stress signals ( 4, 9, 26 ).
BM and PB MNCs of CD45KO mice secreted lower levels of
pro – MMP-9 compared with their WT counterparts ( Fig. 3,
A and B ). In vivo 3-d G-CSF stimulations increased secre-
tion of this enzyme by both WT BM and PB MNCs ( Fig. 3,
A and B ). In contrast, MNCs obtained from G-CSF – treated
CD45KO mice still secreted signifi cantly lower levels of pro –
MMP-9 ( Fig. 3, A and B ).
Retention of leukocytes in the BM requires a concomi-
tant balance of adhesion and detachment. The role of CD45
in these processes was therefore studied. We found signifi -
cantly elevated expression levels of activated ? 1 integrin in
BM MNCs obtained from CD45KO mice (twofold higher
than their WT counterparts; Fig. 3 C ). This phenomenon was
associated with an increased adhesion capacity to FN ( Fig. 3 D ).
This hyperadhesion of CD45KO cells may further explain their
poor motility both in vitro and in vivo.
CD45 defi ciency leads to hyperactivation
of the Src signaling pathway
Several papers have demonstrated the importance of Src kinases,
the natural substrate of CD45, in integrin-mediated adhesion in
various mature lymphoid and myeloid cells ( 27, 28 ). We thus
examined the Src phosphorylation status and activity during
steady state and in response to G-CSF administration. G-CSF
stimulations of WT mice led to a reduction in Src phosphory-
lation and activity in BM MNCs ( Fig. 3, E and F ). Notably,
these modulations in Src were inversely correlated with CD45
expression ( Fig. 1, A and B ). Interestingly, MNCs derived from
the BM of untreated CD45KO mice displayed enhanced Src
phosphorylation and activity ( Fig. 3, E and F ), and G-CSF stim-
ulations only slightly reduced it in these CD45KO cells. Src
family kinases were shown to negatively regulate the mitogen-
activated protein kinase cascades, particularly extracellular sig-
nal-regulated kinase (Erk) activation ( 29 ). As Fig. 3 G shows,
G-CSF treatment activated Erk protein in WT-derived BM
cells, suggesting that Src kinase ac tivity is indeed reduced. Inter-
estingly, in the CD45KO BM, where Src kinases are hyper-
active, Erk proteins are inhibited, as demonstrated by the low
phosphorylation of Erk both in untreated and in G-CSF – treated
mice. Finally, inhibition of Src proteins in CD45KO BM-de-
rived cells using the PP2 inhibitor, which down-regulates the
hyperactivity of Src proteins, increased migration of these
CD45KO cells to a gradient of SDF-1 ( Fig. 3 H ). These results
ultimately demonstrate the link between CD45, Src kinase ac-
tivity, and the regulation of motility properties.
in which the number of immature progenitors in the WT BM
has already reached a plateau ( Fig. 1 H ). Importantly, reduced
numbers of primitive CD45KO SKL cells were also docu-
mented in the BM after G-CSF stimulation compared with
their WT counterparts ( Fig. 1 I ).
Reduced motility and response to SDF-1 are associated
with impaired homing of CD45KO progenitors
The lower numbers of progenitors in the CD45KO BM may
lead to their reduced appearance in the PB after G-CSF treat-
ment. To examine if CD45 is directly involved in progenitor
cell motility, we isolated BM subpopulations from CD45KO
mice and evaluated their spontaneous and SDF-1 – induced mi-
gration potential. The migration of CD45KO MNCs was sig-
nifi cantly reduced compared with WT cells ( Fig. 2 A ), including
both spontaneous as well as SDF-1 – directional migration. Of
note, no diff erences were observed in the expression of CXCR4,
the receptor for SDF-1 (unpublished data). MNCs isolated from
the BM of G-CSF – treated mice exhibited higher motility com-
pared with untreated cells ( Fig. 2 A ). This high motility of G-
CSF – treated WT cells correlated with their increased CD45
expression levels ( Fig. 1, A and B ). However, reduced mi-
gration was observed in cells obtained from CD45KO mice
despite G-CSF treatment. Importantly, although normal mobi-
lization levels were observed after 5 d of G-CSF administration,
CD45KO BM MNCs still demonstrated reduced spontaneous
and SDF-1 migration compared with their WT counterparts
( Fig. 2 A ). When CD45KO BM MNCs were allowed to mi-
grate through a fi bronectin (FN)-coated barrier, stronger re-
ductions in their migration were observed ( Fig. 2 B ). These
results suggest that together with their intrinsic defects in cell
motility, CD45KO cells predominantly have an impaired abil-
ity to cross extracellular matrix barriers. This notion was further
evident in vivo, as CD45KO MNCs showed reduced homing
compared with their WT counterparts ( Fig. 2 C ). Next, we ex-
amined the motility potential of sorted representative subpopu-
lations that strongly respond to G-CSF: CD11b + monocytes
and c-Kit + enriched progenitor cells. These cell fractions were
isolated from the BM of WT and CD45KO mice untreated or
treated with G-CSF for 5 d. As observed with the MNC popu-
lation, CD45KO CD11b + monocytes showed reduced sponta-
neous and SDF-1 – mediated migration compared with WT
CD11b + cells ( Fig. 2 D ). Similarly, CD45KO CD11b + cells
demonstrated reduced homing to the BM of the recipient mice
( Fig. 2 E ). Live cell images show that immature WT c-Kit + cells
responded to SDF-1 chemotactic signals by cell spreading and
the formation of elongated protrusions ( Fig. 2 F , left). In con-
trast, immature CD45KO c-Kit + cells remained mostly round,
forming only short cell protrusions in response to SDF-1
( Fig. 2 F , right). In vivo homing assays demonstrated the infe-
rior homing of CD45KO c-Kit + progenitors to the BM and
spleen, in comparison to their WT counterparts ( Fig. 2 G ). In-
terestingly, CD45KO spleen-derived colony-forming progeni-
tor cells also exhibited poor homing potential to the BM and
spleen of recipient mice ( Fig. 2 H ). Additionally, in a func-
tional repopulation assay, CD45KO spleen-derived cells showed
JEM VOL. 205, September 29, 2008
by injecting RANKL, which was shown to activate osteoclasts
leading to preferential expansion and mobilization of immature
cells ( 16 ). RANKL stimulation in WT mice increased the
Impaired receptor activator of NF- ? B ligand
(RANKL) – induced progenitor mobilization in CD45KO mice
Next, we chose to specifi cally stimulate CD45KO mice in vivo
Figure 2. Impaired migration and reduced response to SDF-1 by CD45KO progenitors. (A and B) In vitro migration of MNCs from the BM of
CD45KO or WT mice either untreated or treated with G-CSF for 3 or 5 d. Cells were allowed to migrate without (spont.) or with addition of SDF-1 to the
lower well. (B) Migration of WT and CD45KO BM MNCs of untreated mice through bare or FN-coated fi lters toward SDF-1. (C) Homing of BM MNCs de-
rived from untreated WT and CD45KO mice to the BM and spleens of recipient NOD/SCID mice 3 h after transplantation. (D and E). In vitro migration
(D) or homing assay (E) of BM CD11b + cells sorted from the BM of untreated or G-CSF – treated WT or CD45KO mice. (F) c-Kit + cells were sorted from the BM
of WT or CD45KO mice, untreated or treated with G-CSF for 5 d and stimulated in vitro with SDF-1. Representative images demonstrate cell polarization
(indicated by black arrows). Bar, 20 μ m. (G) Homing of c-Kit + cells sorted from the BM of WT or CD45KO mice to the BM of NOD/SCID recipient mice 3 h
after transplantation. (H) Homing of spleen progenitor cells isolated from WT or CD45KO mice to the BM and spleens of lethally irradiated NOD/SCID/ ? 2
recipients. (I) Percentages of engraftment in WT-recipient BM and PB transplanted with spleen-derived cells from WT and CD45KO mice. Data represent
the levels of specifi c lineages, as indicated. Shown are the means of four recipients in each group ± SE. *, P < 0.01; **, P < 0.05.
CD45 REGULATES PROGENITOR MOTILITY AND RETENTION | Shivtiel et al.
development of tartrate-resistant acid phosphatase – positive
(TRAP + ) osteoclasts along the endosteum of the trabecular bone,
as previously shown ( 16 ). However, no increase in TRAP + os-
teoclasts was observed in the CD45KO bones (unpublished
data). We next tested progenitor cell levels in response to
RANKL stimulations for 3 and 5 d. Immature WT progenitors
and primitive SKL cells ( Fig. 4, A and B , respectively) were ex-
panded in the BM in response to RANKL stimulation, which
was contrary to CD45KO cells. Levels of progenitors in the pe-
riphery of WT mice, such as the spleen ( Fig. 4 C ) or PB ( Fig.
4 D ), were increased, indicating progenitor mobilization. In
contrast, CD45KO progenitors were not mobilized by RANKL
stimulation for 3 d, and only at moderate levels after 5 d. Next,
deeper investigations concerning the degradation of niche com-
ponents were taken. We found only minor accumulation of
soluble SCF in the PB of CD45KO mice in contrast to their
WT counterparts ( Fig. 4 E ). Close examination of the trabecu-
lar endosteum, enriched with stem cell niches, demonstrated
that in comparison to the WT BM, the niche component os-
teopontin was poorly degraded in CD45KO bone-lining oste-
oblasts after RANKL administration for both 3 and 5 d ( Fig.
4 F ). A broader examination of the entire BM showed a clear
osteopontin-degraded product in the fl uids of the WT BM after
both 3 and 5 d of RANKL treatment ( Fig. 4 G ). However, deg-
radation products were below the detection levels in CD45KO
BM fl uids, suggesting impaired osteopontin degradation ( Fig.
4 G ). These results indicate possible defects in CD45KO osteo-
clasts, resulting in their impaired response to RANKL and poor
release of immature cells from the BM.
Abnormal development and maturation
of CD45KO osteoclasts
We continued by investigating the involvement of CD45
in osteoclast development and function. First, we confi rmed
that mature multinucleated osteoclasts derived from the BM
of WT mice express CD45 ( Fig. 5 A , top left), whereas, as
expected, CD45KO osteoclasts did not express this molecule
( Fig. 5 A , bottom left). Next, we observed that CD45KO oste-
oclasts cultured and developed in vitro demonstrated abnormal
morphology ( Fig. 5 B ). Appearance of the phosphatase TRAP
indicates the maturation status and functional stage of bone-
resorbing osteoclasts. TRAP staining of CD45KO osteoclasts
grown in vitro showed their inability to acquire the fl attened,
spread morphology and assemble the typical sealing zone that
can be seen as a purple ring in the perimeter of WT osteoclasts.
In addition, these CD45KO osteoclasts appeared smaller, with
a reduced ability to form multinucleated cells. Transcriptional
assessment of DC-STAMP mRNA expression revealed that
CD45KO BM cells exhib ited reduced expression of this re-
ceptor ( Fig. 5, C and D ), which may lead to defects in cell
fusion and maturation. We tested additional factors involved
in osteoclast development and function, and examined ex-
pression of MMP-9 and MT1-MMP expressed by CD45KO
osteoclasts. Notably, these cells secreted lower amounts of
MMP-9 compared with their WT counterparts ( Fig. 5 E ),
implying reduced osteoclast motility and activity. Moreover,
G-CSF, which was shown to activate osteoclast development
( 30 ), induced up-regulation of MT1-MMP in WT osteoclasts,
whereas expression of this enzyme in CD45KO osteoclasts
Figure 3. CD45 defi ciency reduces MMP-9 levels and increases cell
adhesion via activation of Src kinase. (A and B) BM or PB MNCs were
isolated from CD45KO or WT mice, either untreated or treated with G-CSF
for 3 d. Cells were cultured and the activity of MMP-9 in the conditioned
media was detected by the gelatin zymography assay. A summary of three
independent experiments and representative gel images of BM cells (A) and
PB cells (B) is shown ( ± SD). (C) FACS analyses of the ? 1-activated epitope
9EG7 in untreated WT or CD45KO BM MNCs. (C, i) Representative histogram
of background staining and 9EG7 expression in WT (black line) and CD45KO
(gray line). (C, ii) Summary of fi ve independent experiments ( ± SD). (D) Adhe-
sion assay of WT and CD45KO BM MNCs to FN-coated wells. Shown is a
summary of four independent experiments (mean ± SD). (E and F) Western
blot analysis of Src phosphorylation or Src activity levels (F) in whole-cell
lysates that were prepared from BM MNCs of WT or CD45KO mice, either
untreated or after G-CSF injections. Histogram represents a summary of four
independent experiments. Values represent the fold change of the control.
(G) Western blot analysis for Erk phosphorylation. Lysates were prepared as
described in E. (H) SDF-1 – induced migration of WT and CD45KO BM MNCs
pretreated with 1 μ M of the Src inhibitor PP2 or DMSO as a control. Data
represent mean ± SD values of three independent experiments and are
shown as the fold increase from DMSO-treated cells. *, P < 0.01; **, P < 0.05.
JEM VOL. 205, September 29, 2008
sembly of CD45KO osteoclasts ( Fig. 5 G , bottom). Src activity
assay confi rmed that treating CD45KO osteoclast precursors
with PP2 reduced their hyperactive Src to levels equivalent to
WT cells ( Fig. 5 H ), enabling the formation of CD45KO os-
teoclasts exhibiting a normal phenotype. These results suggest
that the expression and function of CD45 and its downstream
target Src in monocyte-derived osteoclasts are required for the
regulation of normal osteoclast development.
remained low, as in the steady state ( Fig. 5 F ). CD45 defi -
ciency is associated with hyperphosphorylation and activation
of its substrate, Src, leading to impaired cell movement ( Fig. 3 ).
We therefore assessed osteoclast development in the presence
of the Src inhibitor PP2. Src inhibition impaired the develop-
ment and organization of WT osteoclasts ( Fig. 5 G , top),
resembling the Src ? / ? phenotype ( 31 ). Conversely, PP2 treat-
ment restored the normal morphology and sealing zone as-
Figure 4. CD45KO mice show impaired progenitor mobilization and endosteal modulation induced by RANKL. (A) Colony-forming progenitor
cells in the BM of WT and CD45KO mice after RANKL treatment for 3 d. Values indicate fold changes compared with control mice ± SE (*, P < 0.01).
(B) The percentage of SKL primitive cells in the BM of WT versus CD45KO mice after treatment with RANKL for 3 or 5 d. Values indicate the fold changes
compared with control mice ± SE (*, P < 0.02; **, P < 0.05). (C) Colony-forming progenitor cells in the spleens of WT and CD45KO mice after RANKL treat-
ment. Values indicate fold changes compared with control mice ± SE (*, P < 0.01). (D) Numbers of colony-forming progenitor cells in the PB of WT and
CD45KO mice after RANKL treatment for 5 d ( ± SE). (E) Soluble SCF levels in the plasma of WT and CD45KO, control, or RANKL-treated mice. Values indi-
cate the fold change in plasma SCF compared with control mice ± SE (*, P < 0.05). (F) Immunoreactivity (brown) of osteopontin in femoral metaphyseal
trabecules of WT and CD45KO, control, and RANKL-stimulated mice for 3 and 5 d. Black arrowheads indicate endosteal bone-lining osteoblasts. Bars,
20 μ m. (G) Western blot analysis of an osteopontin-degraded product (32 kD) in BM superannuates of WT and CD45KO mice after RANKL treatments
for 3 and 5 d.
CD45 REGULATES PROGENITOR MOTILITY AND RETENTION | Shivtiel et al.
levels of PYD in the plasma of CD45KO mice, indicating
impaired osteoclast resorbing activity ( Fig. 6 A ). In addition,
we tested the levels of plasma osteocalcin as a marker for bone
turnover. CD45KO mice demonstrated reduced amounts of
osteocalcin in the plasma in comparison to their WT controls
( Fig. 6 B ). Hence, these fi ndings suggested that bone-re-
modeling processes are impaired in CD45KO mice, resem-
bling mild osteopetrosis. A three-dimensional μ CT scanning
Abnormal bone microenvironment and defective
hematopoiesis caused by reduced osteoclast function
in CD45KO mice
The reduced response of CD45KO mice to RANKL stimu-
lations further suggested a defective function of CD45KO os-
teoclasts after their activation. Accumulation of pyridinoline
(PYD) in the mouse plasma refl ects an ongoing bone resorp-
tion process as a result of osteoclast function. We found lower
Figure 5. Defective maturation of CD45KO osteoclasts in vitro involving impaired expression of MMPs, DC-STAMP, and Src kinase. (A) BM-
derived osteoclasts from WT (top) and CD45KO (bottom) mice immunolabeled for CD45 expression (green), and stained for polymerized actin (red) and
nuclear DNA (blue). Bars, 20 μ m. (B) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts. Bars, 200 μ m. (C and D) Semiquan-
titative PCR analysis for DC-STAMP mRNA levels in WT and CD45KO BM-derived cells. (C) Representative PCR image. (D) Summary of four independent
experiments showing the ratio between DC-STAMP and GAPDH mRNA expression ( ± SE; *, P < 0.01). (E) Conditioned medium of WT versus CD45KO BM-
derived osteoclasts was tested for the activity of secreted MMP-9 in a gelatin zymography assay. (F) WT and CD45KO BM-derived osteoclasts cultured
with or without G-CSF were immunolabeled for expression of MT1-MMP (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bar,
10 μ m. (G) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts cultured in vitro in the presence of DMSO vehicle (left) or the
Src inhibitor PP2 (right). Bars, 200 μ m. (H) Src activity assay for osteoclast precursors incubated with 1 μ M PP2 or DMSO (ctrl) for 5 d. Values indicate the
fold changes of WT control mice ± SE (indicated by a horizontal line), showing a representative experiment.
JEM VOL. 205, September 29, 2008
distribution was observed in the femurs of CD45KO mice,
demonstrating a substantially higher number of trabecules in
region 2 ( Fig. 6 C , yellow frame). Accordingly, stereological
analyses of CD45KO bones showed lower Tb.Ns at region 1
(red frame, 75 ± 1.5%) and higher numbers at region 2 (yel-
low frame, 122 ± 11%) compared with the femurs of WT
mice. Interestingly, in vivo TRAP staining of bone sections
demonstrated higher numbers of CD45KO TRAP + cells along
the trabecular endosteum, in comparison to their WT coun-
terparts ( Fig. 6 D ). Similarly, a signifi cantly larger portion of
the CD45KO bone surface was covered with TRAP + cells
( Fig. 6 E ), apparently compensating for their impaired function,
as previously suggested in other osteoclast-defective models
( 18, 33 ). Of note, previous studies showed that osteopetrosis
of WT and CD45KO mice femurs was applied to analyze the
trabecular and cortical microarchitecture. Based on a previous
paper showing preferred localization of stem cells close to the
bone edge ( 32 ), two regions of interest were determined: the
fi rst included 2 mm from the distal metaphysis (region 1), and
the second was 1 mm ahead toward the diaphysis (region 2).
Various bone morphometric and steorologic parameters, in-
cluding bone volume and bone mineral mass; trabecular vol-
ume, trabecular number (Tb.N), and trabecular thickness;
and the gaps between the trabecules were measured. The most
prominent diff erence was the distribution of femoral trabe-
cules. In WT mice, most of the femoral trabecules were found
near the growing plates of the bone edge, in region 1 ( Fig.
6 C , red frame). However, an abnormal pattern of trabecular
Figure 6. Reduced osteoclast function in CD45KO mice is associated with elongated trabecular zone and irregular localization of progenitors.
(A) Plasma levels of PYD indicating bone resorption in WT and CD45KO. (B) Plasma levels of osteocalcin in WT and CD45KO mice. Values represent the mean ±
SE (*, P < 0.05). (C) Three-dimensional constructions of the distal femurs of WT and CD45KO mice. The red frame indicates the metaphyseal region, near the
bone growing plates, and the yellow frame indicates a region 1 mm ahead toward the diaphysis (see Materials and methods). Bars, 2 mm. (D) TRAP staining of
femoral bone sections. Osteoclasts stained in red (arrowheads) are shown along the metaphyseal trabecules (Tb). (E) The ratio between osteoclast surface and
bone surface (Ocl.S/B.S). Data are presented as the percentage of WT cells ± SE (*, P < 0.05). (F – H) Flow cytometry analyses of BM immature Lin ? /c-Kit + pro-
genitors (F), presented as the percentage of WT (mean ± SE), and the more primitive SKL stem cells. A representative FACS plot (G) and summary (H) indicate
lower levels of these primitive cells in the BM of CD45KO mice. *, P < 0.01. (I) Flow cytometry analyses of spleen-derived immature Lin ? /c-Kit + progenitors,
presented as the percentage of WT (mean ± SE). Numbers indicate higher levels of these primitive cells in the spleen of CD45KO mice, *, P < 0.01.
CD45 REGULATES PROGENITOR MOTILITY AND RETENTION | Shivtiel et al.
First, we revealed autonomous defects in CD45KO cell mo-
tility into and out of the BM compartment, including reduced
mobilization and homing of mature leukocytes and immature
progenitor cell populations derived from diff erent organs. We
suggest that CD45 regulates two cellular processes that have
key roles in the migration and retention of leukocytes in general,
and other defects associated with changes in the architecture
of the bone are accompanied by alterations in the hematopoi-
etic stem cell pool size and location ( 33, 34 ). We therefore
anticipated that the abnormal bone microenvironment in
CD45KO mice is related to altered hematopoietic stem and
progenitor cell retention. Despite the regular cellularity of
mature BM leukocytes and normal diff erential counts of cir-
culating mature cells ( Fig. 1 E ), we identifi ed lower numbers
of immature Lin ? /c-Kit + cells ( Fig. 6 F ) and the primitive
SKL subset ( Fig. 6, G and H ) in the BM of CD45KO mice.
Notably, signifi cantly higher numbers of these undiff eren-
tiated cell populations were documented in the spleens of
CD45KO mice in comparison to their WT counterparts ( Fig.
6 I ). Yet, despite their increased levels, CD45KO spleen-
derived progenitors showed inferior homing and repopula-
tion potentials ( Fig. 2 I ).
Mutual defects in the CD45KO BM environment
and hematopoietic cells in response to rapid mobilization
Our results suggested that both environmental and cell-intrin-
sic defects can account for the impaired progenitor mobiliza-
tion in CD45KO mice. To directly dissect these options, we
established two sets of chimera models in which CD45KO
mice were tested either as recipients or donors compared with
WT mice. In the fi rst set of experiments, WT and CD45KO
mice were used as recipients for WT donor cells. In this setting,
we observed lower engraftment levels of WT donor cells in
CD45KO recipients compared with WT recipients ( Fig. 7 A ).
This suggested an impaired ability of the CD45KO BM en-
vironment to support stem cell maintenance. Next, we stud-
ied rapid mobilization by injecting the CXCR4 antagonist
AMD3100, which was shown to induce mobilization without
mediating cell expansion in the BM. Our results showed that
after AMD3100 administration, WT (normal) donor cells have
impaired mobilization in CD45KO compared with WT hosts
( Fig. 7 B ). These fi ndings demonstrated that the CD45KO BM
environment failed to facilitate normal mobilization. In the
second set of experiments, high cell doses of WT and CD45KO
cells were used as donor cells to repopulate WT recipients. In
this set the mobilization potential of CD45KO cells in a normal
environment was further assessed. In highly engrafted mice,
AMD3100 stimulation resulted in only a minor cell mobiliza-
tion when CD45KO cells served as donors, compared with
their WT counterparts, although their BM environment was
normal ( Fig. 7 C ). These experiments suggested that CD45
defi ciency results in multiple defects, combining both cell
autonomous and environmental mechanisms.
In this study, we identifi ed important roles for the panleuko-
cyte CD45 in key processes of immature hematopoietic cell
function: its BM retention and release to the periphery, pro-
cesses that are dramatically aff ected by stress conditions. By
using the CD45KO mouse model, we investigated two major
parameters involved in progenitor motility and location, in-
cluding intrinsic properties and environmental regulation.
Figure 7. Defective engraftment and reduced rapid mobilization
in CD45KO chimeras. (A) Percentage of donor-derived WT cells in the
BM of WT and CD45KO recipients. Shown is a summary of seven mice in
each group ( ± SE; *, P < 0.01). Values demonstrate reduced engraftment
levels in CD45KO hosts. (B) AMD3100-induced mobilization of donor
WBCs in the PB of WT and CD45KO hosts. Values indicate fold change
of mobilization index compared with PBS control mice ( ± SE). **, P <
0.05. (C) Rapid mobilization of WT and CD45KO donor cells. WT chimeric
hosts were injected with PBS or AMD3100 and were tested for the pres-
ence of donor-derived cells in the PB. The mobilization index of specifi c
lineages was tested, as indicated in the fi gure legend. Values indicate
the fold change compared with PBS control mice, which are marked as
a dashed line (mean ± SE). *, P < 0.01; ***, P = 0.07.
JEM VOL. 205, September 29, 2008
Osteoclasts derived from hematopoietic precursors in the
BM of CD45KO mice show abnormal morphology and func-
tion both in vitro and in vivo, refl ecting mild osteopetrosis. Of
note, osteoclasts derived from precursors in the spleen exhib-
ited the same defective phenotype (unpublished data), although
the numbers of hematopoietic progenitors were higher in the
CD45KO spleens. This demonstrates that the decreased os-
teoclast numbers are not caused by the lack of progenitor
cells but rather an intrinsic defect in osteoclast diff erentia-
tion. Our investigations further showed that CD45 regulates
osteoclast formation via controlling Src kinase activity and DC-
STAMP expression. In support of our fi ndings, previous re-
ports showed that osteoclasts derived from DC-STAMP ? / ?
mice were TRAP + MNCs exhibiting a reduced bone-resorb-
ing activity ( 17 ). Interestingly, DC-STAMP ? / ? osteoclasts dem-
onstrated enhanced Src expression, suggesting a link between
these two regulators ( 17 ). Thus, our data propose a role for the
CD45 – Src axis in osteoclast fusion and maturation. In addition,
low expression of MMPs in CD45KO osteoclasts showed that
by regulating MMP-9 and MT1-MMP expression, CD45 is
eventually involved in osteoclast motility and bone degrada-
tion activity ( 42, 43 ). These defects in osteoclasts may thus
explain the poor mobilization observed in CD45KO mice.
However, distinguishing between the environmental versus
hematopoietic eff ects using chimera models revealed a parallel
and perhaps additive impact of both compartments on pro-
genitor retention and mobilization potentials.
The abnormal phenotype and activity of CD45KO osteo-
clasts are associated with lower numbers of trabecules in the fem-
oral metaphysis, a region known to harbor stem cells ( 32 ). Mouse
models of severe osteopetrosis exhibit extramedular hematopoi-
esis, demonstrating lower levels of stem and progenitors cells
in the BM caused by several bone structure defects, and higher
levels of progenitor cells in the spleen ( 33, 44 ). CD45KO mice
demonstrated a similar phenotype of the primitive SKL pool size
and location driven by multiple defects of both the CD45KO
primitive cells and their osteoclast progeny. The reduction in
CD45KO primitive cells in the BM is complementary to previ-
ous fi ndings showing that Lyn ? / ? mice (members of the Src ki-
nase family) display higher numbers of primitive SKL cells in the
BM ( 45 ), demonstrating the central role of the CD45 – Src cas-
cade in stem cell retention. Our fi ndings indicate that stem and
progenitor cells can modulate their CD45 expression and signal-
ing via Src kinase, infl uencing their retention, survival, and mo-
tility. Moreover, CD45KO spleen progenitors, which are not
directly infl uenced by osteoclasts, exhibited poor mobility and
repopulation potentials, and an unusual distribution of these pro-
genitors was observed between the spleen and the PB. However,
previous studies showed that in normal settings, spleen progeni-
tors reside in equilibrium with the blood, suggesting no barrier
between these organs ( 46 ), as opposed to the BM ( 6, 46 ). Hence,
progenitor accumulation in the spleen may also be aff ected by
the impaired intrinsic ability of CD45KO spleen progenitors to
traffi c to the circulation. Additional factors may also be involved,
including increased survival and/or proliferation of these pro-
genitor cells in extramedullary locations such as the spleen.
and progenitors in particular: proteolytic enzyme secretion and
adhesion interactions. Lower secretion of MMP-9 by CD45KO
BM MNCs after G-CSF stimulation implies that CD45 regu-
lates MMP activation, and may thus further explain the reduced
egress of CD45KO leukocytes. Corroborating our fi ndings,
mouse multiple myeloma CD45KO cells also secrete lower
levels of MMP-9, correlating with their reduced invasive capac-
ities compared with CD45 + cells ( 35 ).
BM-derived cells lacking CD45 have increased activation
of ? 1 integrins and hyperinduction of adhesion properties,
demonstrating that CD45 is a negative regulator of signaling
cascades, inducing cell detachment and release. We found that
Src kinase, the CD45 substrate, is a potential target by which
CD45 regulates the migration of hematopoietic cells. Indeed,
Src kinase inhibition enhanced CD45KO cell motility, dem-
onstrating that Src activity is unbalanced in these cells. Several
studies support the involvement of Src kinases in adhesion and
motility properties. Src kinases were shown to regulate ? 1 and
? 2 integrins in diff erent cells and cell lines ( 28, 36 ). Moreover,
in mice defi cient in members of the Src family, immature, he-
matopoietic Sca-1 + cells demonstrated increased homing ( 37 ),
and primitive, BM-derived SKL cells showed enhanced G-
CSF – induced mobilization that was associated with elevated
MMP-9 and accelerated breakdown of vascular cell adhesion
molecule 1 ( 38 ). In line with these studies, our results demon-
strate the opposite eff ects when Src is hyperactive because of
CD45 defi ciency. Still, such fundamental defects in the motil-
ity of both CD45KO progenitors and maturing leukocytes
strongly suggest that additional pathways are also imbalanced
by the lack of CD45 function. This is especially apparent in the
defective cell polarization in response to chemotactic signals of
CD45KO c-Kit + progenitors. Moreover, SDF-1 is considered
as a survival factor for stem and progenitor cells ( 39 ). Thus, the
impaired ability of immature CD45KO c-Kit + cells to nor-
mally respond to SDF-1 stimulation may further explain their
inferior retention in the BM.
Previously, we suggested that interactions between hema-
topoietic stem and progenitor cells with their BM microenvi-
ronment are mutual ( 5, 11 ). We further reveal that CD45 also
plays a role in progenitor mobilization by regulating com-
ponents of the BM microenvironment. Reduced progenitor
expansion and release in response to RANKL activation in
CD45KO mice was associated with impaired modulation of
the stem cell niche regulating components osteopontin and
SCF. Osteopontin was shown to negatively regulate and limit
the number of endosteal stem cells ( 40, 41 ). The impaired deg-
radation of osteopontin in the endosteum of RANKL-treated
CD45KO mice may explain the low numbers and reduced ex-
pansion of progenitors and stem cells in their BM. In addition,
it was previously shown that shedding of membrane-bound
SCF by MMP-9 shifts stem cells from a quiescent to a prolifer-
ative state, enabling their release from the BM ( 9 ). The im-
paired resorption activity and the low secretion of MMP-9 by
CD45KO osteoclasts may also contribute to the reduced pro-
genitor expansion in the CD45KO BM, a prerequisite step for
immature cell mobilization.
CD45 REGULATES PROGENITOR MOTILITY AND RETENTION | Shivtiel et al.
CD45 ? cells. Mobilization index refers to the ratio between BM engraft-
ment level and the amount of donor-derived cells in the circulation.
Colony-forming assay. PB samples were subjected to Ficoll separation.
Total BM and spleen cells or PB MNCs were seeded (1.5 × 10 4 , 5 × 10 5 ,
and 2 × 10 5 cells/ml, respectively) in semisolid cultures, as previously de-
scribed ( 15 ). Colonies were scored 7 d later under an inverted microscope
(CK2; Olympus), applying morphological criteria.
Flow cytometry analysis. Membrane expression of diff erent molecules
on mouse BM and PB MNCs was detected by fl ow cytometry, using one-
or two-step staining procedures. CD45 expression was assessed with FITC
anti – mouse CD45.2 (BD Biosciences). CD45 expression on lineage-spe-
cifi c populations was determined by double staining using anti – CD45-PE
(BD Biosciences) and antibodies for lineage markers (CD4- and CD11b-
FITC, and c-Kit – allophycocyanin; eBioscience). The percentage of SKL
cells in the BM and PB was tested by staining MNCs, as previously de-
scribed ( 16 ). Activated mouse ? 1 was detected by using anti-CD29 (clone
9EG7; BD Biosciences) and secondary PE – donkey anti – rat (Jackson Immuno-
Research Laboratories). After staining, cells were washed and analyzed on a
FACSCalibur (Becton Dickinson) using CellQuest software.
Sorting for CD11b + and c-Kit + cells. Total BM cells from untreated
mice or mice treated with G-CSF for 5 d were stained using anti – CD11b-
FITC and anti – c-Kit – APC. Cells were sorted to these two populations si-
multaneously using a FACSAria (Becton Dickinson). Cells were washed and
tested applying in vitro and in vivo assays.
Migration assay. Chemotaxis assays were performed in Costar transwells
(6.5-mm diameter, 5- μ m pore size; Corning). Upper fi lters were untreated
(bare) or precoated overnight with 25 μ g/ml FN at 4 ° C (Millipore). 10 5
mouse BM MNCs were added to the upper fi lters and were allowed to mi-
grate toward 50 ng/ml SDF-1 ? (PeproTech) for 2 h. Migrating cells were
counted using a FACSCalibur. CD45KO BM MNCs were pretreated with
1 μ M of the Src inhibitor PP2 (EMD), or as a control with the PP2 solvent
DMSO for 30 min at 37 ° C. The cells were then washed and submitted to
migration toward 50 ng/ml SDF-1 and analyzed as described.
Cell polarization microscopy images. Response to 200 ng/ml SDF-1 of
c-Kit + – sorted cells was observed using a 40 × objective (NA = 1.35; Olym-
pus) on uncoated ? slides (Integrated BioDiagnostics). Phase-contrast images
were acquired using scientifi c-grade charge-coupled device (CCD) camera
(LIS-700; Applitech) and processed by the DeltaVisionRT system using
SoftWoRx software (Applied Precision).
Adhesion assay. 96-well plates were coated by overnight incubation with
25 μ g/ml FN at 4 ° C, washed with PBS, and blocked with 0.1% BSA. 2.5 ×
10 5 WT or CD45KO BM MNCs per well were allowed to adhere to the
plates for 16 h at 37 ° C in serum-free RPMI 1640. Nonadherent cells were
washed twice in PBS. Adherent cells were collected in 200 μ l PBS buff er
plus 0.5 mM EDTA. The number of adherent cells was determined by FACS
analysis using a FACSCalibur.
MMP-9 zymography. Zymography assay was performed as previously
described ( 4 ), with the following modifi cations. BM and PB MNCs were
incubated in vitro at 37 ° C (10 5 cells per 100 μ l of serum-free RPMI 1640)
for 40 h. For measurement of osteoclast-secreted MMP-9, total BM cells
were cultured with M-CSF and RANKL, as previously described ( 16 ).
The resulting conditioned medium was collected and loaded (10 μ l) on
10% SDS-PAGE gels containing 1 mg/ml gelatin.
Immunoblotting. Whole-cell lysates were prepared from BM MNCs of
WT or CD45KO mice, intact or after G-CSF injections for 3 – 5 d. Lysates
were obtained by a 15-min incubation with modifi ed RIPA buff er (20 mM
Hepes [pH 7.3], 150 mM NaCl, 10% glycerol, 0.1% SDS, 1% Triton X-100,
Our results suggest that hematopoietic stem cells and their
leukocyte progeny have dual CD45-mediated self-regulation
modes: their motility, proliferation, and adhesion are autono-
mously and dynamically regulated. In addition to stem cell
regulation by the niche, functional CD45 is needed for osteo-
clast development and activity, which indirectly aff ect hemato-
poiesis and the progenitor pool size via interactions with the
bone and BM stromal cells. This notion of a dynamic cross talk
between all components of the system is also supported by a
recent study, which shows that primitive signaling lymphocyte
activation molecule stem and progenitor cells can directly reg-
ulate osteoblast development ( 47 ). Collectively, our results re-
veal that hematopoietic stem and progenitor cells are involved
in regulating their own levels and the dynamic BM microen-
vironment via their osteoclast progeny, which require modu-
lated CD45 activity.
MATERIALS AND METHODS
Mice. All experiments were approved by the animal care committee of the
Weizmann Institute. The experiments were performed on CD45 exon 6 –
defi cient mice (provided by T. Mak, University of Toronto, Toronto, Can-
ada), which were bred and maintained under defi ned fl ora conditions at the
Weizmann Institute. Age- and sex-matched C57BL/6 (CD45.2) mice (Har-
lan) were used as a WT control. All mice used were 6 – 8 wk of age at the
onset of the experiments.
Stress-induced mobilization. Mice received a daily s.c. injection of 300
μ g/kg G-CSF (Filgrastim; Roche) for 3 or 5 consecutive days and were killed
4 – 6 h after the last injection. Single injections of 12.5 mg/kg LPS ( Escherichia
coli serotype O111:B4; Sigma-Aldrich) were administrated i.p. Mice were
killed 16 h after injection. 2 μ g of mouse RANKL (R & D Systems) was in-
jected into 5 – 6-wk-old WT and CD45KO mice, s.c. over the femur, twice
a day for the fi rst 3 d followed by 2 d of rest, or for 5 consecutive days.
Homing assay. 5 × 10 6 mouse BM MNCs per mouse, or 2.5 × 10 6 CD11b +
or c-Kit + sorted cells per mouse, were prelabeled with CFSE dye (5 μ M/10 7
cells; Invitrogen) and i.v. injected into NOD/SCID mice. Recipient mice
were killed after 3 h, and the number of CFSE + cells that reached the BM
and spleens of recipient mice was determined by FACS. The homing of pro-
genitor cells was examined as previously described ( 48 ) and modifi ed using
spleen cells by injecting 20 × 10 6 total spleen cells into lethally irradiated (600
cGy from a cesium source) NOD/SCID/ ? 2 ? / ? mice. A fraction of the in-
jected cells was plated in colony assays to quantify the number of injected
CFU-Cs. Recipients were killed 18 h after injections, and fractions of BM
and spleen (1 – 2 × 10 6 cells) were plated in methylcellulose medium to fi nd
the numbers of functional CFU-Cs (progenitors) that lodged to these organs.
Homing effi ciency was calculated as the percentage of homed progenitors
out of the number of injected CFU-Cs in total spleen or four bones.
Repopulation and rapid mobilization in chimera models. Recipient
C57BL/6 (CD45.2) or CD45KO (CD45.2) mice were irradiated (600 cGy
from a cesium source) and injected 4 h later with 10 6 total BM cells derived
from B6.SJL donors (CD45.1). Alternatively, C57BL/6 recipients were
transplanted with 1 – 10 × 10 6 CD45KO BM or 10 6 spleen-derived cells
from B6.SJL or CD45KO mice. PBS (as a control) or 5 mg/kg AMD3100
(Sigma-Aldrich) was injected s.c into chimeric mice 5 wk after transplanta-
tion. Engraftment levels and mobilization of donor WBCs in the PB were
evaluated 1 h after AMD3100 injections using diff erent combinations of cell
staining and were analyzed by FACS. B6.SJL cells in diff erent hosts were
detected by tracing CD45.1 + cells, followed by staining with anti – CD45.1-
PE/CD45.2-FITC (eBioscience). Detection of CD45KO cells in C57BL/6
hosts was performed by staining with Lineage + /CD45.2 antibodies (eBio-
science), evaluating donor cells as CD19 + , CD11b + , and Gr1 + , which are
JEM VOL. 205, September 29, 2008
For osteoclast formation in vitro, BM cells were seeded in 96-well plates
(10 5 cells/0.2 ml) and cultured with M-CSF and RANKL, as previously de-
scribed ( 16 ). In some experiments, the culture medium was supplemented
with 1 μ M of the Src inhibitor PP2 or its vehicle DMSO in the respec-
Immunohistochemistry of osteopontin and SDF-1. Bone sections
were prepared and stained as previously described ( 16 ).
ELISA for mouse SCF. Blood plasma samples were obtained from con-
trol and RANKL-treated mice and tested for SCF by ELISA, as previously
described ( 16 ).
ELISA for PYD and osteocalcin. We tested plasma PYD (Metra; Quidel
Corp.) and osteocalcin (Biomedical Technologies, Inc.) on frozen plasma
samples according to the manufacturers ’ instructions.
Semiquantitative RT-PCR for DC-STAMP. We prepared cDNA
from mouse BM cells using standard protocols. We performed semiquan-
titative PCR analysis for DC-STAMP expression for 35 cycles: 95 ° C for
1 min, 60 ° C for 1 min, and 72 ° C for 1 min. We used the following primer
sequences: 5 ? -GGGTCTCAACACCACGAACT-3 ? and 5 ? -GACTCT-
GTTTGCCCAGCTTC-3 ? (251 bp).
Statistical analysis. Signifi cance levels of the data were determined by the
Student ’ s t test using Microsoft Excel.
The authors would like to thank Prof. Tak Mak for providing the CD45 KO mice;
Dr. E. Zelzer for the usage of the μ CT machine; and Prof. G. Wagemaker, Prof.
A. Globerson, Prof. D. Zipori, Prof. R. Alon, Dr. S. Feigelson, and Prof. S. Berrih-Aknin
for critical remarks and fruitful discussions.
This work was partially supported by grants from the Israel Science Foundation
(796/04), the European Union FP6 Magselectofection, the Charles and David
Wolfson Charitable Trust, and the Helen and Martin Kimmel Institute for Stem Cell
Research at the Weizmann Institute of Science.
The authors have no confl icting fi nancial interests.
Submitted: 10 January 2008
Accepted: 14 August 2008
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2 mM EDTA, 2 mM EGTA, 0.5% deoxycholate, 50 mM ? GP, and 50 mM
NaF) freshly supplemented with 1% protease inhibitor cocktail (Sigma-
Aldrich) and 0.2 mM pervanadate (Sigma-Aldrich). 50 μ g of total protein was
separated on 10% SDS-PAGE and transferred to nitrocellulose membranes.
The membranes were blocked with TBST (5 mM Tris, 154 mM NaCl, 0.1%
Tween-20 [pH 7.6]) containing 5% milk and probed with rabbit anti – human/
mouse phospho-Src (Invitrogen), rabbit anti – human/mouse ERK1 (pThr 202 /
pTyr 204 ) and ERK2 (pThr 185 /pTyr 187 ; Sigma-Aldrich), or rabbit anti – total
ERK1/2 (Sigma-Aldrich), as a control for total protein. Osteopontin ex-
pression was evaluated in BM superannuates, separated on 10% SDS-PAGE
(20 μ g). Polyclonal anti – mouse osteopontin antibodies (R & D Systems) were
used to detect the 32-kD degraded product, as previously described ( 49 ).
Src activity assay. Whole-cell lysates were prepared from BM MNCs and
sorted CD11b + cells. Alternatively, lysates were prepared from osteoclast
precursors grown for 5 d in the presence of RANKL and 20 ng/ml M-CSF
supplemented with 1 μ M PP2 or DMSO vehicle. Lysis was performed using
HNTG lysis buff er (20 mM Hepes [pH 7.5], 150 mM NaCl, 1% Triton X-
100, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 50 mM NaF) freshly sup-
plemented with 1% protease inhibitor cocktail, 0.2 mM pervanadate, and
0.5 mM okadaic acid (A.G. Scientifi c). Src kinases were immunoprecipitated
by incubating cell lysates with 1 μ g of anti – v-Src antibodies (EMD) for 2 h
at 4 ° C. Protein G plus agarose beads (Santa Cruz Biotechnology, Inc.) were
added to the mixture and incubated for an additional 12 h at 4 ° C. Immuno-
complexes were precipitated after three washes with HNTG wash buff er
(20 mM Hepes [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol,
1 mM EDTA, 1 mM EGTA, 50 mM ? GP, 50 mM NaF, 1 mM sodium or-
thovanadate) and a fi nal wash with Src kinase buff er (20 mM MOPS, 5 mM
MgCl 2 ). Src kinase activity was tested using a tyrosine kinase activity assay
kit (Millipore) according to the manufacturer ’ s instructions. Src activity in
BM samples was calculated in correlation to the total amount of Src that was
precipitated in each sample stated by immunoblot assay.
μ CT imaging and trabecular morphometry. Femurs from CD45KO
and WT control mice were removed, disarticulated from the pelvic bone
and tibia, cleaned of soft tissues, and stored at ? 20 ° C. After thawing at
room temperature, bones were scanned using a μ CT device (eXplore Lo-
cus SP; General Electric) with custom software (version 5.2.2; MicroView).
Scanning was performed with 80-kV x-ray voltage, 80- ? A current, 400-
ms integration time, and 8- μ m pixel size. Based on preliminary work, two
volumes of interest (VOI) were defi ned ( Fig. 6 C ). The fi rst VOI consisted
of the distal metaphyseal region, defi ned as starting at a distance of 32 im-
age slices (250 μ m) from the growth plate in the direction of the diaphysis,
and extending a further 220 slices (1.75 mm) in the same direction. The
second proximal metaphyseal VOI started from the end of the previous
VOI and extended a further 125 slices (1 mm) in the same direction. The
trabecular volume was separated from the surrounding cortical shell by
manual segmentation, and a direct three-dimensional model ( 50 ) was used
to evaluate the Tb.N.
Osteoclast immunocytochemical staining. Total BM cells were seeded
on glass cover-slips (10 6 cells/1 ml) and cultured for 6 d in ? -MEM supple-
mented with 20 ng/ml M-CSF (PeproTech) and RANKL that were changed
every other day. Where indicated in the fi gures, culture medium was supple-
mented with 200 ng/ml G-CSF (Filgrastim; Roche). Samples were fi xed with
3% paraformaldehyde (Merck), permeabilized with 0.5% Triton X-100 (Sigma-
Aldrich), and immunolabeled at room temperature in a humidifi ed chamber
with FITC-conjugated anti-CD45.2 (eBioscience) or rabbit anti – mouse/hu-
man MT1-MMP polyclonal antibody (Millipore), followed by secondary goat
anti – rabbit – Alexa Fluor 488. TRITC-phalloidin and DAPI (Sigma-Aldrich)
were added. Images were acquired using scientifi c-grade CCD camera and
processed by the DeltaVisionRT system using SoftWoRx software.
TRAP staining of bone sections and osteoclasts. TRAP staining of
bone sections and osteoclasts was performed as previously described ( 16 ).
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