ArticlePDF Available

B-lymphopoiesis is stopped by mobilizing doses of G-CSF and is rescued by overexpression of the anti-apoptotic protein Bcl2

Authors:

Abstract and Figures

Background Osteoblasts are necessary to B-lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However the effect of these mobilizing agents on B-lymphopoiesis has not been reported. Methods Mice (wild-type, knocked-out for TNF-α and TRAIL, or overexpressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. Results G-CSF stopped medullar B-lymphopoiesis with concomitant loss of B cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B cell colony-forming units and pre-pro-B cells in the marrow and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B-lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B-lymphopoiesis but caused instead robust B cell mobilization. Conclusions G-CSF, cyclophosphamide and AMD3100 have distinct effects on B-lymphopoiesis and B cell mobilization with 1) G-CSF inhibiting medullar B-lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B-lymphopoiesis observed during inflammation or viral infections, 2) CYP mobilizing B cells but blocking their maturation, and 3) AMD3100 mobilizing B cells without affecting B-lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.
Content may be subject to copyright.
325
haematologica | 2013; 98(3)
ARTICLES
Introduction
The interface between the compact bone and the bone
marrow (BM), the endosteum, is a privileged site where
bone formation and turnover take place. In the past decade,
it has emerged that this endosteal region of the BM, particu-
larly the metaphyseal spongiosa rich in trabecular bone, har-
bors the most primitive hematopoietic stem cells (HSC) able
to reconstitute long-term multi-lineage hematopoiesis upon
serial transplantation into lethally irradiated mice.1-5 Hence,
it was concluded that HSC niches are not distributed ran-
domly in the BM tissue but preferentially locate within 2-3
cell diameters from endosteal bone surfaces.3-4 These conclu-
sions were further supported by the observation that HSC
express calcium receptors sensing the calcium gradient
formed by osteoclast-mediated bone degradation and help-
ing HSC to lodge in these endosteal niches.6This drew the
attention to the potential role of osteoblasts, osteoprogeni-
tors and their mesenchymal precursors in regulating most
primitive HSC. Conditional gene deletion in, and specific
ablation of osteoblasts,7osteoprogenitors8or mesenchymal
stem cells9have shown that osteoblast-lineage and mes-
enchymal progenitor cells are critical to maintain normal
HSC within the BM. It has also recently emerged that in
addition to regulating HSC, osteoblasts and their progenitors
critically regulate medullar B lymphopoiesis. Indeed, abla-
tion of osteoblasts or conditional deletion of the Gs
α
gene
specifically in osteoblasts impairs primitive B lymphopoiesis
in the BM.10-11 Therefore, osteoblastic lineage cells at the
endosteum control the maintenance of two different arms of
hematopoiesis: 1) primitive hematopoiesis via HSC; and 2)
B-lymphopoiesis.
We and others have previously reported that specific popu-
lations of BM macrophages are critical to maintain HSC
within their BM niches. Indeed, ablation of these
macrophages12 and/or their stimulation by granulocyte
colony-stimulating factor (G-CSF)13 results in inhibition of
bone formation, disappearance of endosteal osteoblasts, and
impairment of HSC niche function as measured by expres-
sion of HSC-supportive factors such as CXCL12, Kit ligand,
angiopoietin-1, and VCAM-1, leading to robust mobilization
of HSC into the peripheral blood.12-14 We identified two
macrophage subsets that potentially exert this regulatory
role: 1) osteomacs, a specific population of BM macrophages
that form a canopy over active osteoblasts at the endosteum
and are necessary to maintain osteoblast function; and 2)
CD11b+F4/80+Ly6-G+macrophages.15 It is still unclear as to
whether osteomacs are a subset of the CD11b+F4/80+Ly6-G+
©2013 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2012.069260
The online version of this article has a Supplementary Appendix.
Manuscript received May 2, 2012. Manuscript accepted August 2, 2012.
Correspondence: jplevesque@mmri.mater.org.au
Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit
osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lym-
phopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-
α
and TRAIL, or over-expressing Bcl-2)
were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node con-
tent in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-
forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism.
Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-
pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous
soluble TNF-αwith Etanercept, or combined deletion of the TNF-
α
and TRAIL genes did not prevent B lym-
phopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not
suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and
AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B
lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lym-
phopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their matura-
tion; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood
mobilized with these three agents may have distinct immune properties.
ABSTRACT
B-lymphopoiesis is stopped by mobilizing doses of G-CSF and
is rescued by overexpression of the anti-apoptotic protein Bcl2
Ingrid G. Winkler,1Linda J. Bendall,2Catherine E. Forristal,1 Falak Helwani,1Bianca Nowlan,1 Valerie Barbier,1
Yi Shen,1Adam Cisterne,2Lisa M. Sedger,3,4 and Jean-Pierre Levesque1,5
1Mater Research at the Translational Research Institute, Woolloongabba, Queensland; 2Westmead Institute for Cancer Research,
Westmead Millennium Institute, University of Sydney, Westmead, New South Wales; 3Institute for Immunology and Allergy,
Westmead Millennium Institute, The University of Technology, Sydney; 4School of Medical and Molecular Biosciences, The
University of Sydney; and 5University of Queensland, School of Medicine, Brisbane, Queensland, Australia
Hematopoiesis & Hematopoietic Stem Cells
macrophages that specifically support osteoblasts or
whether these are separate populations. Nevertheless, we
and others have found that continuous treatment with
the cytokine G-CSF causes HSC mobilization by deplet-
ing these niche-supportive macrophages, causing deple-
tion of endosteal osteoblasts, and reducing HSC niche
function leading to HSC mobilization into the peripheral
blood.13,15 We have also found that the alkylating agent
cyclophosphamide (CYP) also depletes osteomacs and
osteoblasts from endosteal surfaces leading to impair-
ment of HSC niches and HSC mobilization.16 In contrast,
the CXCR4 antagonist AMD3100 (Plerixafor), which
mobilizes HSC by binding directly to CXCR4 and block-
ing the chemotactic signaling elicited by the binding of
the chemokine CXCL12,17has no effect on osteoblasts or
niche-supportive macrophages.16
Considering that both G-CSF and CYP inhibit
osteoblasts and HSC niches, whereas AMD3100 does
not,16and that endosteal osteoblasts are critical to main-
tain medullar B lymphopoiesis,10-11 we have evaluated the
effect of these three mobilizing agents on B lymphopoiesis
in the mouse.
Design and Methods
All experiments were approved by the Animal Experimentation
Ethics Committees of the University of Queensland and
University of Sydney, Australia.
Mouse mobilization and tissue harvesting
All experiments were performed on 8-12 week-old male
C57BL/6 mice. vavBcl2 transgenic mice18 were produced by breed-
ing transgenic males with wild-type C57BL/6 females. B6.TNFa–/–
mice19were crossed with B6.TRAIL–/– mice20to generate mice het-
erozygous for both alleles. These were interbred to yield
B6.TNFa–/–.TRAIL–/– (B6.TT) mice defective in both alleles. Pups
were identified by PCR on ear punch biopsy using primers specific
to the Bcl2 transgenene as described.18 Polymerase chain reaction
(PCR) detection of TNF-
α
and TRAIL wild-type and targeted alle-
les was performed essentially as described previously20 but using
TRAIL gene specific neomycin-target allele primers.
Recombinant human G-CSF (Filgrastim, Amgen, Thousand
Oaks, CA, USA) was injected twice daily subcutaneously at 125
mg/kg per injection for up to six consecutive days. Control mice
were injected with an equivalent volume of saline. In some exper-
iments, 20 mg/kg/day etanercept was injected once daily intraperi-
toneally for four days to block endogenous soluble tumor-necrosis
factor (TNF)-α21 and G-CSF administered for the last three days of
the experiment, as described above.
Cyclophosphamide (CYP)-treated mice were injected intraperi-
toneally with a single dose of 200 mg/kg CYP diluted in saline.
AMD3100 octohydrochloride (Tocris Bioscience, Bristol, UK)
was injected intraperitoneally as a single 16 mg/kg dose correspon-
ding to 10 mg/kg of AMD3100 base. Tissues were harvested 1 h
after AMD3100 administration.
At specified time points, mice were anesthetized with isofluo-
rane and approximately 1 mL of blood collected into heparinized
tubes by cardiac puncture before cervical dislocation. Femoral BM
was flushed and spleens dissociated in phosphate-buffered saline
(PBS) containing 2% fetal calf serum (FCS) for further analyses. For
flow cytometry analyses, red cells were lysed from blood samples
as previously described.22 Spleens were harvested, weighed, and
dissociated in PBS with 2% FCS. Cells and RNA from the central
region of the BM, and RNA from the endosteum were isolated
from femurs as previously described.23 Inguinal and popliteal
lymph nodes draining hind legs24 were harvested and dissociated
in PBS with 2% FCS similar to spleens.
Cell counts and colony assays
Leukocytes were counted on a Sysmex KX-21 automated cell
counter. For colony-forming unit B cell (CFU-B) assays, 105cells
were plated in 1 mL of Methocult CFU-B medium containing 10
ng/mL human interleukin (IL)-7 following manufacturer’s instruc-
tions (Stem Cell Technologies, Vancouver, Canada). Colonies of
small lymphocytes were counted after seven days culture.
CFU-C assays to test the mobilization of myeloid progenitors
were performed in methylcellulose medium containing recombi-
nant mouse granulocyte-monocyte colony-stimulating factor
(GM-CSF), Kit ligand and IL-6, as previously described.12
Quantitative real-time RT-PCR (RT-qPCR)
RNA was precipitated, DNase was treated and reverse tran-
scribed using random hexamers. qRT-PCR with CXCL12 and IL-7
were performed using Taqman probes (labeled 5’with 6-carboxy-
fluorescein (FAM) and 3’ with blackhole quencher-1 (BHQ-1)).
Oligonucleotide sequences are shown in Online Supplementary
Table S1 except for mouse G-CSF receptor (Csf3r) primers. Primers
and probe set were purchased from Applied Biosciences (set
Mm00432735_m1). All primers crossed intron-exon boundaries
and did not amplify genomic DNA in the absence of reverse tran-
scription. RNA levels were standardized by parallel RT-qPCR
using primers to the housekeeping gene b2-microglobulin.
TNF-
α
measurement in bone marrow fluids
At sacrifice, femurs were flushed with 1 mL ice-cold PBS, BM
cells dissociated by serial pipetting and then pelleted at 400 x g for
5 min at 4°C. BM fluids in supernatants were collected, aliquoted
and stored at -70°C until use. TNF-αprotein concentration was
measured using the BD Cytometric Bead Array for mouse inflam-
matory cytokines following the manufacturer’s instructions (BD
Biosciences, Sydney, Australia) and analyzed on an LSRII flow
cytometer (BD Biosciences).
Flow cytometry
Bone marrow, blood and spleen leukocytes were pelleted at 370
x g for 5 min at 4°C and resuspended in 2.4G2 anti-CD16/CD32
hybridoma culture supernatant to block IgGFc receptors.
Measurement of B-progenitor cells were performed by staining
cell suspensions with CD11b-PECy7, NK1.1-PE, biotinylated don-
key F(ab)’2 anti-mouse-IgM with streptavidin-Pacific Blue, B220-
APCCy7, CD19-PercPCy5.5 or CD19-APC and CD43-FITC (BD
Biosciences) monoclonal antibodies. For sorting, cells were labeled
in an identical manner and sorted in medium containing 50% FCS
on an Aria cell sorter (BD Biosciences).
Mobilization of HSPC was measured by flow cytometry using
a cocktail of anti-lineage (Lin) antibodies (CD3e, CD5, B220,
CD11b, Gr-1, CD41, Ter119), anti-Sca-1, anti-Kit, CD48 and
CD150 antibodies, as previously described.25
For apoptosis assays, BM cells were labeled as above, pelleted,
resuspended in 100 mL of annexin V binding buffer (BD
Biosciences) and incubated with APC-conjugated annexin V at
room temperature for 15 min. Cells were then analyzed using an
LSRII flow cytometer following addition of 2 mg/mL 7-actino-
mycin D (7-AAD).
Statistical analysis
Calculations for whole body content in B-cell progenitors were
based on the assumption that one femur represents 5.6% of total
mouse BM and total volume of blood is 0.08 mL per g of body
I.G. Winkler et al.
326 haematologica | 2013; 98(3)
weight.26 Significance levels were calculated using the Student’s t-
test for colony assays, flow cytometry analyses, and RT-qPCR.
Results
G-CSF inhibits medullar B-lymphopoiesis
Adult mice were mobilized by bi-daily injections of G-
CSF for up to six days (Figure 1A) and expression of IL-7
and CXCL12 was measured in the endosteal region of the
BM by RT-qPCR (Figure 1B). TNF-αprotein concentration
was directly measured in BM fluids (Figure 1C). IL-7 and
CXCL12 mRNA expression were both significantly inhib-
ited by G-CSF treatment whereas TNF-αprotein concen-
tration in BM fluids increased during the first four days of
G-CSF treatment. As CXCL12 and IL-7 are B-lymphopoi-
etic cytokines27-29 and TNF-αhas been reported to inhibit
B-lymphopoiesis,30 these results suggested that G-CSF
may impair B lymphopoiesis in the BM.
We next measured the number of B220+CD11bNK1.1
B cells in BM, blood and spleen by flow cytometry. The
frequency of these B cells in the BM declined 28-fold at
Day 6 of G-CSF treatment and progressively returned to
normal levels eight days following cessation of G-CSF
administration (Figure 2A). This decrease in medullar B
cells was not accompanied by any significant increase in
the number of B cells in blood or spleen during G-CSF
treatment (Days 0-6) (Figure 2A) although a significant
increase in blood B cells was observed at Day 10 when the
number of B cells in the BM began to recover, returning to
normal values by Day 14. Assuming that a femur repre-
sents 5.6% of total BM, and that total blood volume is pro-
portional to mouse body weight (0.08 mL/g),26 we found
that the total number of B cells in whole BM, blood and
spleen per mouse declined progressively by 60% at Day 6
of G-CSF administration and returned to baseline values
by Day 14. To further clarify whether this decline in BM B
cells was due to a halt in medullar B-lymphopoiesis or
involved relocation of some B progenitors into the spleen,
B progenitors and B cells were enumerated by flow
cytometry. B-lineage cells were gated within the side-
scatterlowCD11b-NK1.1-population as surface IgM (sIgM)
B220+CD19-CD43+pre-pro-B cells,
sIgMB220lowCD19+CD43-pro-B cells, sIgM-B220+CD19+
pre-B cells, and sIgM+B220+mature B cells, as previously
reported by Zhu et al.,10 and Hardy et al. for the specific
expression of CD43 in pre-pro-B cells31 (Figure 3A). These
analyses revealed a rapid and pronounced decline of all B-
Effect of mobilizing agents on B-lymphopoiesis
haematologica | 2013; 98(3) 327
Figure 1. Effect of G-CSF, CYP and
AMD3100 on CFU-C mobilization and
expression of IL-7, CXCL12 and TNF-α.
(A) Mobilization of CFU-C in peripheral
blood was measured at indicated time
points. Data are means±SD, 4 mice per
time point. (B) IL-7 and CXCL12 mRNA
were measured in the endosteal region
of the BM by RT-qPCR. Data are relative
to b2-microglobulin mRNA. Each dot is
the result from an individual mouse.
Bars are the average of each group. (C)
Concentration of TNF-αprotein in BM flu-
ids. Each dot is the result from an indi-
vidual mouse. Bars are the average of
each group. ***P<0.001, **P<0.01, and
*P<0.05. Gray boxes on kinetics show
time period during which HSPC were
mobilized into the blood.
A
B
C
G-CSF
CFC x 10-3/mL blood
mRNA relativve to b2m
TNF-α, pg/femur
G-CSF
CYP
CYP
AMD
0246810
Days
0246810
Days
0246810
Days
0246810
Days
0369
Days after CYP
03 6 9
Days after CYP
03 6 9
Days after CYP
03 6 9
Days after CYP
Saline AMD
Saline AMD
Saline AMD
CXCL12
CXCL12
TNF-αTNF-α
CXCL12
IL-7
IL-7 IL-7
n.s.
8
6
4
2
0
12
10
8
6
4
2
0
12
10
8
6
4
2
0
0.1
0.01
1
0.1
1
0.1
0.01
1
0.1
0.01
10
1
0.1
1
0.1
16
12
8
4
0
0.5
0.4
0.3
0.2
0.1
0
cell populations in the BM, with the exception of pro-B
cells whose decline was delayed, occurring only at Day 4
of G-CSF administration (Figure 3B). Importantly,
although some pre-pro/pro/pre-B cells relocated to blood
and spleen, this did not compensate for the loss of BM B
cells and their progenitors, resulting in a net decrease in
pre-pro-B (3.5-fold), pro-B (1.7-fold), pre-B (4.6-fold), and
mature B (1.9-fold) cells on a per mouse basis for the sum
of the BM, blood and splenic compartments (Figure 3B).
These results were confirmed at the level of more primi-
tive CFU-B in functional B-cell colony assays, with a rapid
and severe decline in medullar CFU-B with only marginal
mobilization into blood and spleen (Figure 3B).
Importantly, while mature sIgM+B220+B cells were abun-
dant in inguinal and popliteal lymph nodes, all sIgMB220+
B-progenitor subsets were very rare (less than 1/100 of
mature B cells). Furthermore, their number did not
increase after four days of G-CSF (Online Supplementary
Figure S1), excluding the possibility that B-cell progenitors
home to lymph nodes in response to G-CSF.
To determine whether this loss in medullar B cells was
mediated by apoptosis, BM cells were stained with 7-
AAD and annexin-V (Figure 3C). Apoptosis of immature
sIgMand mature sIgM+B cells was in part responsible for
this collapse in medullar B-lymphopoiesis with a signifi-
cant increase in the proportion of 7-AAD+and/or annexin-
V+apoptotic cells in these two B-cell populations after four
days of G-CSF. Conversely, the proportion of apoptotic
BM myeloid cells was significantly decreased by G-CSF
treatment.
Blockage of TNF-
α
 does not prevent HSPC mobilization
or arrest in B-lymphopoiesis
To determine whether the effect of G-CSF on B cells
was direct, sIgMB220+CD19CD43+pre-pro-B cells,
sIgM-B220+CD19+CD43pre-B and pro-B cells, and
sIgM+B220+mature B cells were sorted from the BM, RNA
extracted and G-CSF receptor mRNA quantified by RT-
qPCR. G-CSF receptor mRNA were undetectable in all B-
cell populations but abundantly expressed in BM CD11b+
myeloid cells and LinSca1-Kit+myeloid progenitors
(Online Supplementary Figure S2). Therefore, in the absence
of receptor, the effect of G-CSF on B cells must be indi-
rect.
As TNF-αinhibits B-lymphopoiesis30 and is increased in
the BM during G-CSF treatment (Figure 1), we treated
mice for four days with etanercept, a human TNFR2-IgG1
Fc chimera that cross-reacts with mouse TNF-αand
inhibits endogenous soluble mouse TNF-α.21 Mice were
then mobilized with G-CSF for the last three days.
Etanercept treatment did not alter mobilization of CFU-
C, Lin-Sca1+Kit+HSPC or LinSca1+Kit+CD48CD150+HSC
into the blood or spleen (Online Supplementary Figure S3).
Etanercept did not prevent the loss of pre-pro-B cells or
more mature B-cell precursors in the BM following G-CSF
treatment, although it did increase the number of mature
B cells in steady-state (Online Supplementary Figure S3).
These results were confirmed in mice knocked-out for
both TNF-
α
and TRAIL genes. A 4-day treatment with G-
CSF also strongly reduced medullar B lymphopoiesis in
these mice (Online Supplementary Figure S4). Therefore,
I.G. Winkler et al.
328 haematologica | 2013; 98(3)
Figure 2. Comparative effect of G-CSF, CYP and AMD3100 on B cells in BM, blood and spleen. At indicated time points, SSClow CD11bB220+
B cells were measured by flow cytometry in BM, blood and spleen. The charts on the right column show the number of B cells in total BM,
blood, spleen per mouse calculated after summation of content in BM, blood and spleen. Data are average±SD of 4 mice per time point per
treatment group. ***P<0.001, **P0.01, and *P<0.05.
A
B
C
0246810 12 14 02 46810 11 14 02 4 6 810 12 14 024 6810 12 14
Days Days Days Days
0246810 12 14 02 46810 11 14 02 4 6 810 12 14 024 6810 12 14
Days Days Days Days
BM
BM
BM
G-CSF
G-CSF G-CSF G-CSF
Blood
Blood
Blood
CYP CYP
CYP
CYP
Spleen
Spleen
Spleen
Mouse
Mouse
Mouse
B cells, 106/femur
B cells, 106/mL blood
B cells, 106/mL blood
B cells, 106/mL blood
B cells, 106/mL spleen
B cells, 106/mL spleen
B cells, 106/mouse
B cells, 106/mouse
B cells, 106/mouse
B cells, 106/femur
B cells, 106/femur
6
4
2
0
6
4
2
0
4
3
2
1
0
80
60
40
20
0
120
90
60
30
0
60
40
20
0
200
150
100
50
0
200
150
100
50
0
150
100
50
0
6
4
2
0
3
2
1
0
5
4
3
2
1
0
B cells, 106/mL spleen
Saline AMD3100 Saline AMD3100 Saline AMD3100 Saline AMD3100
TNF-α, and TNF-αrelated TRAIL are dispensable to the
B-lymphopoiesis inhibition in response to G-CSF.
Overexpression of Bcl-2 rescues CFU-B and
pre-pro-B cells in response to G-CSF
To further investigate the contribution of apoptosis in
the inhibition of B-lymphopoiesis in response to G-CSF,
we next mobilized vavBcl2 transgenic mice which over-
express the Bcl2 gene under the vav gene promoter in all
hematopoietic cells. Bcl2 is an anti-apoptotic protein nec-
essary to early B-cell development32 and mutations in Bcl2
gene are frequent in B-cell neoplasms.33-34 Overexpression
of Bcl2 in hematopoietic cells causes a pan-leukophilia, B-
cell accumulation in the spleen, evolving to follicular lym-
phoma after 40-week latency.18,35 We conducted experi-
ments in 8-week old mice that did not shown any sign of
malignancy despite a pronounced leukophilia in steady-
state (data not shown). Colony assays in the presence of
GM-CSF, Kit ligand and IL-6 showed that the number of
myeloid progenitors CFU-C in blood and spleen was more
than a degree of magnitude higher in vavBcl2 mice com-
pared to wild-type, both in steady-state and after a 4-day
course of G-CSF (Figure 4A). There was no significant dif-
ference in CFU-C content in the BM between the two
strains in steady-state or during mobilization.
B-cell subset analysis revealed that, unlike the wild-
type, there was no significant loss of pre-pro-B cells and
CFU-B in mobilized BM from vavBcl2 transgenic mice
(Figure 4B). Mobilization of pre-pro-B cells and CFU-B
into the blood was also more pronounced (Figure 4B).
Therefore, overexpression of Bcl2 rescued the loss of most
primitive B-cell progenitors in G-CSF mobilized BM. CFU-
Effect of mobilizing agents on B-lymphopoiesis
haematologica | 2013; 98(3) 329
Figure 3. G-CSF induces loss of all B-
cell subsets in the BM. (A) Gating
strategy to identify B-cell subsets
during mobilization. B-cell subsets
were defined within the SSClow
CD11b-NK1.1- B220+population as
shown in the top two dot-plots.
Mature B cells were defined as
sIgM+whereas immature B cells
were sIgM-. Immature B cells were
then subdivided in B220+CD19
CD43+pre-pro-B cells, B220low CD19+
pro-B cells and B220+CD19+pre-B
cells. Typical B-cell profiles are
shown for BM from control saline
treated mice (top row) and G-CSF-
treated mice (bottom row). Note the
strong reduction of all B-cell subsets
in G-CSF-mobilized BM. (B) The
number of cells in each B-cell sub-
set (pre-pro-B, Pro-B, pre-B and
mature B cells) was measured by
flow cytometry in BM, blood and
spleen at indicated time points of G-
CSF treatment. Results per mouse
were calculated after summation of
content in BM, blood and spleen.
The number of CFU-B was measured
in B-cell colony assays. (C)
Apoptosis in BM cells was meas-
ured by flow cytometry. Cells were
considered apoptotic when they
were positive for annexin-V and/or
7-AAD. Data are average±SD of 4
mice per time-point per treatment
group. ***P<0.001, **P<0.01, and
*P<0.05.
A
B
C
Saline
CD11b
CD11b
cells x10-6 femur
cells x10-6 /mL blood
cells x10-6 /spleen
cells x10-6 /mouse
B220
B220
SSC
SSC
B220
B220
B220
B220
B220
NK1.1
NK1.1
B220
sIgM
sIgM
CD19
Mature Pre-pro-B
CD19
CD43
CD43
Pre-pro-B Pro-B Pre-B Mature B CFU-B
G-CSF
BM
BM+blood+spleen
Total BM CD11b+B220+IgMB220+IgM+
% apoptotic cells
Saline 4 days
G-CSF 4 days
Blood
Spleen
60
50
40
30
20
10
0
0246 0246 0246 0246
0246 0246 0246 0246
0246 0246 0246 0246
0246 0 246 0246 0246
Days of G-CSF
0246
Cells/femur
Cells/mL blood
Cells/spleen
Cells/mouse
0246
02 46
024 68
5,000
4,000
3,000
2,000
1,000
1,000
800
600
400
200
0
3,500
3,000
2,500
2,000
1,500
1,000
500
0
80000
60000
40000
20000
0.6
0.5
0.4
0.3
0.2
0.1
0
0.16
0.12
0.08
0.04
0
0.25
0.20
0.15
0.10
0.5
0
0.20
0.15
0.10
0.5
0
10
8
6
4
2
0
12
10
8
6
4
2
0
2.5
2.0
1.5
1.0
0.5
0
1.2
0.8
0.4
0
0.06
0.04
0.02
0
4
3
2
1
0
4
3
2
1
0
60
40
20
0
80
60
40
20
0
120
100
80
60
40
20
0
3
2
1
0
4
3
2
1
0
B were also dramatically increased in the spleen of mobi-
lized vavBcl2 mice (Figure 4B).
Analysis of more mature B cells revealed that, although
the number of pro-B, pre-B and mature B cells was dramat-
ically decreased in the BM of mobilized vavBcl2 mice, this
loss in the BM was fully compensated by a robust mobi-
lization of these B-cell subsets into the blood.
Consequently, the total number of CFU-B, pre-pro-B, pro-
B, pre-B and mature B cells did not decrease on a per mouse
basis that included the summation of BM, blood and
spleen compartments (Figure 4B). This is in sharp contrast
to wild-type mice in which the loss of B cells in the BM
was not compensated by their number in the blood, spleen
or lymph nodes, resulting in an overall decrease of all B-cell
types on a per mouse basis (Figure 3B). Taken together,
these data show that Bcl2 overexpression rescues the arrest
in medullar B lymphopoiesis during G-CSF administration
with maintenance of pre-pro-B cell and CFU-B pools in the
BM, and redistribution of all B-cell stages from the BM into
the blood and spleen without loss in B cells.
Cyclophosphamide (CYP) and AMD3100 induce
B-cell mobilization without impairing B-lymphopoiesis
We next examined the effect of two other mobilizing
agents on medullar B-lymphopoiesis. A single dose of the
alkylating agent CYP causes rapid myelosuppression in
BM during the acute cytotoxic phase in the first three days
of administration in mice. This is followed by a robust
rebound of hematopoiesis between Days 6-8 that coin-
cides with HSPC mobilization into the blood.16,36 Total
B220+B cells were profoundly reduced in BM, blood and
spleen at Day 3 and slowly recovered in BM and spleen
between Days 6 and 14 (Figure 2). To better understand
the effect of CYP on lymphopoiesis, B-cell precursors
were further analyzed in these three tissues following the
same gating strategy as that used in G-CSF-mobilized
mice. At Day 3, during the acute cytotoxic phase, pro-B,
pre-B and mature B cells were almost undetectable in the
BM (Online Supplementary Figure S5). Although significant-
ly reduced, detectable numbers of pre-pro-B cells
remained in the BM. At Days 6 and 8, when HSPC are
I.G. Winkler et al.
330 haematologica | 2013; 98(3)
Figure 4. Overexpression of Bcl-2 in
vavBcl2 mice rescues medullar B
lymphopoiesis during G-CSF treat-
ment. (A) CFU-C in blood, BM and
spleen were measured in colony
assays. (B) The number of cells in
each B-cell subset (pre-pro-B, Pro-B,
pre-B and mature B cells) was
measured by flow cytometry in BM,
blood and spleen at Day 6 of G-CSF
or saline treatments. Results per
mouse were calculated after sum-
mation of content in BM, blood and
spleen. The number of CFU-B was
measured in B-cell colony assays.
Data are average±SD of 4 mice per
time-point per treatment group.
***P<0.001, **P<0.01, and
*P<0.05.
A
B
Blood
Blood
Spleen
Spleen
BM
CFU-C/ml blood
CFU-C/spleen
CFU-C/femur
BM
BM+blood+spleen
WT Bcl2 WT Bcl2 WT Bcl2
IgMB220+CD19CD43+IgMB220lowCD19+IgMB220+CD19+IgMB220+
WT Bcl2 WT Bcl2 WT Bcl2 WT Bcl2
WT Bcl2 WT Bcl2 WT Bcl2 WT Bcl2
WT Bcl2 WT Bcl2 WT Bcl2 WT Bcl2
WT Bcl2 WT Bcl2 WT Bcl2 WT Bcl2
105
104
103
102
101
1
1.0
0.8
0.6
0.4
0.2
0
0.4
0.3
0.2
0.1
0
2.0
1.6
1.2
0.8
0.4
0
40
30
20
10
0
4
3
2
1
0
6
4
2
0
20
16
12
8
4
0
25
20
15
10
5
0
200
150
100
50
0
200
150
100
50
0
300
200
100
0
8
6
4
2
0
120
100
80
60
40
20
0
100,000
80,000
60,000
40,000
20, 000
160,000
120,000
80,000
40,000
0
1,000
800
600
400
200
0
15,000
10,000
5,000
0
0.3
0.2
0.1
0
5
4
3
2
1
0
15
10
5
0
105
104
103
102
101
1
105
104
103
102
101
1
Pre-pro-B Pro-B Pre-B Mature B CFU-B
Saline
G-CSF
WT Bcl2
WT Bcl2
WT Bcl2
WT Bcl2
n.s.
n.s.
n.s.
cells x 10-6/femur
cells/femur
cells/blood
cells/spleen
cells/mouse
cells x 10-6/spleen
cells x 10-6/mouse cells x 10-6/mL blood
mobilized into the blood (Figure 1A),36 medullar B-lym-
phopoiesis was re-initiated with increased pre-pro-B, pro-
B and pre-B cells in the BM. These B cell precursors were
also robustly mobilized into the blood and spleen (Online
Supplementary Figure S5). As a result, all these B-cell precur-
sor populations rebounded from the Day 3 nadir on a per
mouse basis based on the summation of the BM, blood
and spleen compartments (Online Supplementary Figure S5).
Therefore, unlike G-CSF, HSPC mobilization in response
to CYP is not concomitant with an arrest in medullar
B-lymphopoiesis. Importantly, we noted that B-cell matu-
ration to sIgM+was delayed with the numbers of mature
B cells remaining low in BM, spleen and BM even ten days
after CYP injection (Online Supplementary Figure S5). As
mature B cells represent a large proportion of B cells in the
blood, this low number of mature B cells explains the per-
sistent low number of total B cells in the blood even 14
days after CYP treatment (Figure 2B).
Finally, to determine the effects of CXCR4, mice were
treated with AMD3100, a CXCR4 antagonist that blocks
the chemotactic effect of the chemokine CXCL12,37 and
tissues harvested 1 h later at the peak of HSPC mobiliza-
tion17 (Figure 1A). AMD3100 caused a slight decrease in
medullar B cells and significant B-cell mobilization into
blood and spleen (Figure 2C). A more detailed analysis of
B-cell subsets showed very robust mobilization of all B-
cell types into the blood (Online Supplementary Figure S6).
A significant proportion of pro-B, pre-B and mature B cells
had already homed to the spleen after 1 h of AMD3100
injection, demonstrating that these B cells can very rapidly
home to the spleen. There was no significant reduction in
pre-pro-B cells and pro-B cells in the BM following
AMD3100 treatment. However, the number of more dif-
ferentiated pre-B and mature sIgM+B cells was significant-
ly reduced in the BM (Online Supplementary Figure S6)
explaining the overall reduction of B cells in the BM
(Figure 2C). Finally, AMD3100 treatment did not alter the
numbers of pre-pro-B, pro-B and mature B cells on a per
mouse basis (in BM, blood and spleen) (Online
Supplementary Figure S6). Together, these results suggest
that AMD3100 does not compromise medullar B lym-
phopoiesis and B-cell survival despite a robust mobilizing
effect on all differentiation stages of the B-cell lineage.
Discussion
G-CSF causes a rapid loss of osteoblasts on endosteal
surfaces12,16 with reduction in the transcription of both
CXCL12 and IL-7 mRNA in the endosteal region, and
increased TNF-αprotein in BM fluids. This is accompa-
nied by a very pronounced reduction in the numbers of
pre-pro-B, pro-B, pre-B and sIgM+mature B cells in the
BM. The loss of B-cell precursors in the BM is not compen-
sated by relocation to the blood, spleen or lymph nodes.
Furthermore, the proportion of apoptotic B cells increased
in the BM in response to G-CSF, a loss that was rescued by
the overexpression of the anti-apoptotic protein Bcl2. This
suggests that G-CSF blocks medullar B-lymphopoiesis by
increasing B-progenitor cell apoptosis via the mitochondr-
ial-triggered pathway. This is consistent with the observa-
tion that Bcl2 overexpression also rescues B-cell numbers
in the BM and spleen of mice deficient in BOB.1/OBF.1, a
B-cell specific transcriptional co-activator.32
This effect of G-CSF was indirect as B cells and their
progenitors do not express G-CSF receptor. A possible
candidate for mediating this indirect effect was TNF-αas:
1) TNF-αadministration directly inhibits B lymphopoiesis;
and 2) inhibition of B-lymphopoiesis by LPS- or adjuvant-
induced acute inflammation are in part TNF-αdepen-
dant.30 However, we have found that neither blockade of
endogenous TNF-αwith etanercept nor targeted deletion
of the TNF-
α
or TRAIL genes prevents the arrest in B-lym-
phopoiesis caused by G-CSF. Therefore, although G-CSF
and acute inflammation38-39 both enhance myelopoiesis
and suppress B-lymphopoiesis, the mechanisms are some-
what distinct in respect to the role of TNF-α. Clearly,
cytokines other than TNF-αand TRAIL play a role in
inhibiting B-lymphopoiesis in response to G-CSF. One
possibility is lymphotoxin-α, since this cytokine is known
to alter B-cell maturation in vivo19,40 but this remains to be
investigated in relation to G-CSF. A possible mechanism
leading to increased B-cell apoptosis in the BM in response
to G-CSF could be the downregulation of both CXCL12
and IL-7 mRNA in the endosteal region. Indeed, both
CXCL1227-28 and IL-729 are indispensable to B-lym-
phopoiesis. CXCL12 and IL-7 have been reported to be
produced by separate BM stromal cells and from distinct
niches for pre-pro-B cells and pro-B cells respectively.41 An
important role of osteoblasts in regulating B-lym-
phopoiesis is suggested by expression of both IL-7 and
CXCL1211 in osteoblasts. Furthermore, deletion of the
protein Gs
α
gene specifically in primitive osteoprogeni-
tors and osteoblasts using Cre recombinase under the con-
trol of the osterix gene promoter (GsαosxKO mice) also
reduces the number of osteoblasts on endosteal surfaces,
and down-regulates IL-7 expression leading to impaired
medullar B lymphopoiesis with reduced numbers of pro-
B- and pre-B cells, whereas pre-pro-B and sIgM+B cells are
unaffected in the BM of these mice.11This phenotype is
somewhat similar to our observation in wild-type mice
mobilized with G-CSF except that G-CSF strongly
reduced pre-pro-B cells and sIgM+B cells in addition to
pro-B and pre-B cells (Figure 3). In these GsαosxKO mice,
administration of exogenous IL-7 was also able to partially
rescue medullar B-lymphopoiesis.11 Therefore, it is excit-
ing to speculate that: 1) the attenuation of IL-7 expression
in pro-B and pre-B cell niches observed in GsαosxKO mice
mice or wild-type mice treated with G-CSF, induces apop-
tosis of IL-7-dependent pro-B and pre-B cells; and 2) that
the subsequent B-cell apoptosis can be rescued either com-
pletely by overexpression of the Bcl2 anti-apoptotic pro-
tein, or partially by administering IL-7. The main differ-
ence between GsαosxKO mice mice and G-CSF-treated wild-
type mice is that CXCL12 was down-regulated in the lat-
ter, not in the former. This may explain the pronounced
decrease in pre-pro-B cells and mature sIgM+B cells that
was not observed in GsαosxKO mice mice.
The lesser effect of CYP on medullar B-lymphopoiesis
was unexpected as CYP also ablates osteoblasts, although
this occurs during the rebound phase between Days 6-10
following CYP administration when HSPC are mobilized
into the blood.16 The persistent absence of endosteal
osteoblasts at Days 6-10 of CYP16 did not prevent the re-
expression of IL-7 at Day 8, or the rebound in medullar B-
lymphopoiesis with the number of pro-B and pre-B cells
increasing in the BM from Day 6 and more primitive pre-
pro-B cells from Day 8 after CYP administration. This
was highly unexpected as osteoblasts have been reported
to be necessary to maintain medullar B-lymphopoiesis in
Effect of mobilizing agents on B-lymphopoiesis
haematologica | 2013; 98(3) 331
vivo.10 Of note, less primitive pro-B and pre-B cells
rebounded in the BM two days prior to pre-pro-B cells
which rebounded in the BM at Day 8 only. As pre-pro-B
cells rebounded in the spleen earlier than in the BM (Day
6 instead of Day 8), this suggests that B-lymphopoiesis re-
started from the spleen sometime between Day 3 and
Day 6 post-CYP and seeded the BM despite persistent
absence of osteoblasts. Clearly, expression of IL-7 and
CXCL12 resumed in other BM stromal cells in the
absence of osteoblasts. This is consistent with previous
observations showing that IL-7 and CXCL12 are also
expressed by distinct stromal cells away from the endos-
teum.41-42 However, it is important to note that despite the
re-expression of both IL-7 and CXCL12 and re-initiation
of medullar B-lymphopoiesis, the maturation of pre-B
cells into sIgM+cells was still blocked at Day 10 after CYP
when osteoblasts are still absent,16 with very few sIgM+B
cells in BM, blood and spleen.
Finally, we found that all B-cell subsets were robustly
mobilized 1 h after AMD3100 administration without inhi-
bition of CXCL12 or IL-7 expression or medullar
B-lymphopoiesis. This is consistent with our previous obser-
vations that AMD3100 has no effect on osteoblast numbers
and expression of the osteoblast marker osteocalcin.16
Therefore, AMD3100 mobilizes B cells by directly antago-
nizing CXCR4 which is expressed by B cells,43-44 without
interfering with B-cell niches or medullar lymphopoiesis.
In conclusion, the three HSC mobilizing agents G-CSF,
CYP and AMD3100 have very distinct effects on B-lym-
phopoiesis and B-cell mobilization with: 1) G-CSF inhibit-
ing medullar B-lymphopoiesis without mobilizing B cells
in a mechanism distinct from the loss of B-lymphopoiesis
observed during inflammation or viral infections;30,38 2)
CYP mobilizing B cells but blocking their maturation into
sIgM+B cells; and 3) AMD3100 mobilizing B cells without
affecting B lymphopoiesis. These results suggest that
blood mobilized with these three agents may have distinct
immune properties.
Finally, as G-CSF is a potent inhibitor of B-lym-
phopoiesis, it could be a useful agent to treat B-cell neo-
plasms. However, as Bcl2 overexpression is common in
and drives B-cell neoplasms, combinations of G-CSF with
Bcl2 inhibitors such as ABT-73745-47 or obatoclax48 could
efficiently target B-cell malignancies or lymphoprolifera-
tive disorders associated with Bcl2 overexpression.
Funding
During the course of this study, JPL was supported by a Senior
Research Fellowship from the Cancer Council Queensland, IGW
and LJB by Career Development Fellowships, and FH by a
Biomedical Fellowship from the National Health and Medical
Research Council (NHMRC #488817, #511965 and #488821,
respectively). This work was supported by NHMRC Project
Grants ns. 434515 to JPL and IGW, and an Anthony Rothe
Grant to LJB.
Authorship and Disclosures
Information on authorship, contributions, and financial & other
disclosures was provided by the authors and is available with the
online version of this article at www.haematologica.org.
I.G. Winkler et al.
332 haematologica | 2013; 98(3)
References
1. Nilsson SK, Johnston HM, Coverdale JA.
Spatial localization of transplanted hemo-
poietic stem cells: inferences for the local-
ization of stem cell niches. Blood.
2001;97(8):2293-9.
2. Arai F, Hirao A, Ohmura M, Sato H,
Matsuoka S, Takubo K, et al. Tie2/angiopoi-
etin-1 signaling regulates hematopoietic
stem cell quiescence in the bone marrow
niche. Cell. 2004;118(2):149-61.
3. Lo Celso C, Fleming HE, Wu JW, Zhao CX,
Miake-Lye S, Fujisaki J, et al. Live-animal
tracking of individual haematopoietic
stem/progenitor cells in their niche. Nature.
2009;457(7225):92-7.
4. Xie Y, Yin T, Wiegraebe W, He XC, Miller
D, Stark D, et al. Detection of functional
haematopoietic stem cell niche using real-
time imaging. Nature. 2009;457(7225):97-
101.
5. Grassinger J, Haylock DN, Williams B,
Olsen GH, Nilsson SK. Phenotypically
identical hemopoietic stem cells isolated
from different regions of bone marrow
have different biologic potential. Blood.
2010;116(17):3185-96.
6. Adams GB, Chabner KT, Alley IR, Olson
DP, Szczepiorkowski ZM, Poznansky MC,
et al. Stem cell engraftment at the endosteal
niche is specified by the calcium-sensing
receptor. Nature. 2006;439(7076):599-603.
7. Visnjic D, Kalajzic Z, Rowe DW, Katavic V,
Lorenzo J, Aguila HL. Hematopoiesis is
severely altered in mice with an induced
osteoblast deficiency. Blood. 2004;103(9):
3258-64.
8. Raaijmakers MHGP, Mukherjee S, Guo S,
Zhang S, Kobayashi T, Schoonmaker JA, et
al. Bone progenitor dysfunction induces
myelodysplasia and secondary leukaemia.
Nature. 2010;464(7290):852-7.
9. Mendez-Ferrer S, Michurina TV, Ferraro F,
Mazloom AR, MacArthur BD, Lira SA, et
al. Mesenchymal and haematopoietic stem
cells form a unique bone marrow niche.
Nature. 2010;466(7308):829-34.
10. Zhu J, Garrett R, Jung Y, Zhang Y, Kim N,
Wang J, et al. Osteoblasts support B-lym-
phocyte commitment and differentiation
from hematopoietic stem cells. Blood.
2007;109(9):3706-12.
11. Wu JY, Purton LE, Rodda SJ, Chen M,
Weinstein LS, McMahon AP, et al.
Osteoblastic regulation of B lymphopoiesis
is mediated by Gsa-dependent signaling
pathways. Proc Natl Acad Sci USA. 2008;
105(44):16976-81.
12. Winkler IG, Sims NA, Pettit AR, Barbier V,
Nowlan B, Helwani F, et al. Bone marrow
macrophages maintain hematopoietic stem
cell (HSC) niches and their depletion mobi-
lizes HSCs. Blood. 2010;116(23):4815-28.
13. Christopher MJ, Rao M, Liu F, Woloszynek
JR, Link DC. Expression of the G-CSF
receptor in monocytic cells is sufficient to
mediate hematopoietic progenitor mobi-
lization by G-CSF in mice. J Exp Med.
2011;208(2):251-60.
14. Chow A, Lucas D, Hidalgo A, Méndez-
Ferrer S, Hashimoto D, Scheiermann C, et
al. Bone marrow CD169+ macrophages
promote the retention of hematopoietic
stem and progenitor cells in the mesenchy-
mal stem cell niche. J Exp Med. 2011;
208(2):261-71.
15. Winkler IG, Barbier V, Wadley R,
Zannettino ACW, Williams S, Levesque J-P.
Positioning of bone marrow hematopoietic
and stromal cells relative to blood flow in
vivo: serially reconstituting hematopoietic
stem cells reside in distinct nonperfused
niches. Blood. 2010;116(3):375-85.
16. Winkler IG, Pettit AR, Raggatt LJ, Jacobsen
R, Forristal CE, Barbier V, et al.
Hematopoietic stem cell mobilizing agents
G-CSF, cyclophosphamide or AMD3100
have distinct mechanisms of action on
bone marrow HSC niches and bone forma-
tion. Leukemia. 2012;26(7):1594-601.
17. Broxmeyer HE, Orschell CM, Clapp DW,
Hangoc G, Cooper S, Plett PA, et al. Rapid
mobilization of murine and human
hematopoietic stem and progenitor cells
with AMD3100, a CXCR4 antagonist. J
Exp Med. 2005;201(8):1307-18.
18. Ogilvy S, Metcalf D, Print CG, Bath ML,
Harris AW, Adams JM. Constitutive Bcl-2
expression throughout the hematopoietic
compartment affects multiple lineages and
enhances progenitor cell survival. Proc Natl
Acad Sci USA. 1999;96(26):14943-8.
19. Korner H, Cook M, Riminton DS,
Lemckert FA, Hoek RM, Ledermann B, et
al. Distinct roles for lymphotoxin-alpha
and tumor necrosis factor in organogenesis
and spatial organization of lymphoid tis-
sue. Eur J Immunol. 1997;27(10):2600-9.
20. Sedger LM, Glaccum MB, Schuh JC, Kanaly
ST, Williamson E, Kayagaki N, et al.
Characterization of the in vivo function of
TNF-alpha-related apoptosis-inducing lig-
and, TRAIL/Apo2L, using TRAIL/Apo2L
gene-deficient mice. Eur J Immunol.
2002;32(8):2246-54.
21. Zalevsky J, Secher T, Ezhevsky SA, Janot L,
Steed PM, O’Brien C, et al. Dominant-neg-
ative inhibitors of soluble TNF attenuate
experimental arthritis without suppressing
innate immunity to infection. J Immunol.
2007;179(3):1872-83.
22. Levesque JP, Hendy J, Winkler IG,
Takamatsu Y, Simmons PJ. Granulocyte
colony-stimulating factor induces the
release in the bone marrow of proteases
that cleave c-KIT receptor (CD117) from
the surface of hematopoietic progenitor
cells. Exp Hematol. 2003;31(2):109-17.
23. Shen Y, Winkler IG, Barbier V, Sims NA,
Hendy J, Lévesque J-P. Tissue inhibitor of
metalloproteinase-3 (TIMP-3) regulates
hematopoiesis and bone formation in vivo.
PLoS ONE. 2010;5(9):e13086.
24. Harrell MI, Iritani BM, Ruddell A. Lymph
node mapping in the mouse. J Immunol
Methods. 2008;332(1-2):170-4.
25. Barbier V, Winkler IG, Wadley R, Levesque
JP. Flow cytometry measurement of bone
marrow perfusion in the mouse and sorting
of progenitors and stems cells according to
position relative to blood flow in vivo.
Methods Mol Biol. 2012;84445-63.
26. Shaposhnikov VL. Distribution of the bone
marrow cells in the skeleton of mice. Biull
Eksp Biol Med. 1979;87(5):483-5.
27. Ma Q, Jones D, Borghesani PR, Segal RA,
Nagasawa T, Kishimoto T, et al. Impaired
B-lymphopoiesis, myelopoiesis, and
derailed cerebellar neuron migration in
CXCR4- and SDF-1-deficient mice. Proc
Natl Acad Sci USA. 1998;95(16):9448-53.
28. Nagasawa T, Kikutani H, Kishimoto T.
Molecular cloning and structure of a pre-B-
cell growth-stimulating factor. Proc Natl
Acad Sci USA. 1994;91(6):2305-9.
29. von Freeden-Jeffry U, Vieira P, Lucian LA,
McNeil T, Burdach SE, Murray R.
Lymphopenia in interleukin (IL)-7 gene-delet-
ed mice identifies IL-7 as a nonredundant
cytokine. J Exp Med. 1995; 181(4):1519-26.
30. Ueda Y, Yang K, Foster SJ, Kondo M, Kelsoe
G. Inflammation Controls B
Lymphopoiesis by Regulating Chemokine
CXCL12 Expression. J Exp Med. 2004;
199(1):47-58.
31. Hardy RR, Carmack CE, Shinton SA, Kemp
JD, Hayakawa K. Resolution and character-
ization of pro-B and pre-pro-B cell stages in
normal mouse bone marrow. J Exp Med.
1991;173(5):1213-25.
32. Brunner C, Marinkovic D, Klein J,
Samardzic T, Nitschke L, Wirth T. B cell–
specific transgenic expression of Bcl2 res-
cues early B lymphopoiesis but not B cell
responses in BOB.1/OBF.1-deficient mice. J
Exp Med. 2003;197(9):1205-11.
33. Monni O, Franssila K, Joensuu H, Knuutila
S. BCL2 overexpression in diffuse large B-
cell lymphoma. Leuk Lymphoma. 1999;34
(1-2):45-52.
34. Tomita N. BCL2 and MYC dual-hit lym-
phoma/leukemia. J Clin Exp Hematop.
2011;51(1):7-12.
35. Egle A, Harris AW, Bath ML, O'Reilly L,
Cory S. VavP-Bcl2 transgenic mice develop
follicular lymphoma preceded by germinal
center hyperplasia. Blood. 2004;
103(6):2276-83.
36. Levesque JP, Hendy J, Takamatsu Y,
Williams B, Winkler IG, Simmons PJ.
Mobilization by either cyclophosphamide
or granulocyte colony-stimulating factor
transforms the bone marrow into a highly
proteolytic environment. Exp Hematol.
2002;30(5):440-9.
37. Donzella GA, Schols D, Lin SW, Este JA,
Nagashima KA, Maddon PJ, et al.
AMD3100, a small molecule inhibitor of
HIV-1 entry via the CXCR4 co-receptor.
Nat Med. 1998;4(1):72-7.
38. Cain D, Kondo M, Chen H, Kelsoe G.
Effects of acute and chronic inflammation
on B-cell development and differentiation. J
Invest Dermatol. 2009;129(2):266-77.
39. Ueda Y, Kondo M, Kelsoe G. Inflammation
and the reciprocal production of granulo-
cytes and lymphocytes in bone marrow. J
Exp Med. 2005;201(11):1771-80.
40. Sedger LM, Hou S, Osvath SR, Glaccum
MB, Peschon JJ, van Rooijen N, et al. Bone
marrow B cell apoptosis during in vivo
influenza virus infection requires TNF-
alpha and lymphotoxin-alpha. J Immunol.
2002;169(11):6193-201.
41. Tokoyoda K, Egawa T, Sugiyama T, Choi
B-I, Nagasawa T. Cellular niches controlling
B lymphocyte behavior within bone mar-
row during development. Immunity.
2004;20(6):707-18.
42. Sugiyama T, Kohara H, Noda M, Nagasawa
T. Maintenance of the hematopoietic stem
cell pool by CXCL12-CXCR4 chemokine
signaling in bone marrow stromal cell nich-
es. Immunity. 2006;25(6):977-88.
43. Bleul CC, Schultze JL, Springer TA. B lym-
phocyte chemotaxis regulated in associa-
tion with microanatomic localization, dif-
ferentiation state, and B cell receptor
engagement. J Exp Med. 1998;187(5):753-
62.
44. Honczarenko M, Douglas RS, Mathias C,
Lee B, Ratajczak MZ, Silberstein LE. SDF-1
responsiveness does not correlate with
CXCR4 expression levels of developing
human bone marrow B cells. Blood. 1999;
94(9):2990-8.
45. Vogler M, Dinsdale D, Sun XM, Young KW,
Butterworth M, Nicotera P, et al. A novel
paradigm for rapid ABT-737-induced apop-
tosis involving outer mitochondrial mem-
brane rupture in primary leukemia and
lymphoma cells. Cell Death Differ. 2008;
15(5):820-30.
46. Mason KD, Khaw SL, Rayeroux KC, Chew
E, Lee EF, Fairlie WD, et al. The BH3
mimetic compound, ABT-737, synergizes
with a range of cytotoxic chemotherapy
agents in chronic lymphocytic leukemia.
Leukemia. 2009;23(11):2034-41.
47. Del Gaizo Moore V, Brown JR, Certo M,
Love TM, Novina CD, Letai A. Chronic
lymphocytic leukemia requires BCL2 to
sequester prodeath BIM, explaining sensi-
tivity to BCL2 antagonist ABT-737. J Clin
Invest. 2007;117(1):112-21.
48. Nguyen M, Marcellus RC, Roulston A,
Watson M, Serfass L, Murthy Madiraju
SR, et al. Small molecule obatoclax
(GX15-070) antagonizes MCL-1 and over-
comes MCL-1-mediated resistance to
apoptosis. Proc Natl Acad Sci USA.
2007;104 (49):19512-7.
Effect of mobilizing agents on B-lymphopoiesis
haematologica | 2013; 98(3) 333
... To determine whether the increased splenic HSC pool observed at 14 days post-CSF1-Fc treatment was an indirect consequence of compensatory extramedullary hematopoiesis, myeloid progenitors [7,31] (Additional file 1: Fig. S7), lymphoid progenitors [32,33,53] (Additional file 1: Fig. S7), erythroblasts and reticulocytes [35,36] were assessed in BM, spleen and liver at day 14 postinitial CSF1-Fc or saline treatment. No effect on common myeloid progenitor (CMP) number was observed at this time point in either BM or spleen, whereas they were increased in the liver (Fig. 4b). ...
... CSF1-Fc treatment causes rapid expansion of boneresorbing osteoclasts [15], and osteal macrophages are CSF1-responsive [26,71], and paradoxically, systemic CSF1 treatment has an anabolic impact on bone [72]. Osteoblast-lineage cells in turn support B cell maturation [53]. Activation of this complex cellular feedback loop was not specifically examined in this study. ...
Article
Full-text available
Background Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation. Methods We administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging flow cytometry with functional validation by colony formation and competitive transplantation assay. Results CSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48 ⁻ CD150 ⁺ HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48 ⁻ CD150 ⁺ HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment. Conclusion These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.
... As these osteomacs are essential to osteoblast maturation and bone formation [67], G-CSF, LPS and sepsis also dramatically reduce osteoblast numbers and bone formation at endosteal surfaces indirectly via osteomacs [36,66,81]. As osteoblasts are the main source of key lymphopoietic cytokines IL-7 and CXCL12 to common lymphoid progenitors in the BM and are essential to B lymphopoiesis [15,82,83], loss of osteomacs indirectly leads to arrest in medullary B lymphopoiesis in response to G-CSF, LPS or sepsis [81,84] (Figure 1). ...
Article
Full-text available
The bone marrow (BM) contains a mosaic of niches specialised in supporting different maturity stages of haematopoietic stem and progenitor cells such as haematopoietic stem cells, myeloid, lymphoid and erythroid progenitors. Recent advances in BM imaging and conditional gene knock-out mice have revealed that niches are a complex network of cells of mesenchymal, endothelial, neuronal and haematopoietic origins, together with local physicochemical parameters. Within these complex structures, phagocytes such as neutrophils, macrophages and dendritic cells, all of which are of haematopoietic origin, have been shown to be important in regulating several niches in the BM, including haematopoietic stem cell niches, erythropoietic niches and niches involved in endosteal bone formation. There is also increasing evidence that these macrophages have an important role in adapting haematopoiesis, erythropoiesis and bone formation in response to inflammatory stressors and play a key part in maintaining the integrity and function of these. Likewise, there is also accumulating evidence that subsets of monocytes, macrophages and other phagocytes contribute to the progression and response to treatment of several lymphoid malignancies such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma as well as lymphoblastic leukemia, and may also play a role in myelodysplastic syndrome, myeloproliferative neoplasms associated with Noonan syndrome and aplastic anaemia. In this review, the potential functions of macrophages and other phagocytes in normal and pathological niches will be discussed, as well as the challenges to studying BM and other tissue-resident macrophages at the molecular level.
... Collectively, our data showed that the combination of rhIL-7-hyFc with G-CSF and AMD3100 synergistically enhanced HSC mobilization. Consistent with previous report [12], G-CSF treatment reduced pro-B cell number in the BM ( Supplementary Fig. S6b). However, the combination of rhIL-7-hyFc with G-CSF preserved pro-B cells in the BM, implying a rescue of B-lymphopoiesis ( Supplementary Fig. S6b). ...
Article
Bone remodeling occurs through the interactions of three major cell lineages, osteoblasts, which mediates bone formation, osteocytes, which derive from osteoblasts, sense mechanical force and direct bone turnover, and osteoclasts, which mediate bone resorption. However, multiple additional cell types within the bone marrow, including macrophages, T lymphocytes and B lymphocytes influence the process. The bone marrow microenvironment, which is produced, in part, by bone cells, forms a supporting network for B-lymphopoiesis. In turn, developing B-lymphocytes influence bone cells. Bone health during homeostasis depends on the normal interactions of bone cells with other lineages in the bone marrow. In disease state these interactions become pathologic and can cause the abnormal function of bone cells and the inadequate repair of bone after a fracture. This review summarizes what is known about the development of B lymphocytes and the interactions of B lymphocytes with bone cells in both health and disease.
Article
Murine norovirus (MNV), which can be used as a model system to study human noroviruses, can infect macrophages/monocytes, neutrophils, dendritic, intestinal epithelial, T and B cells, and is highly prevalent in laboratory mice. We previouslyshowed that MNV infection significantly reduces bone marrow B cell populations in a Stat1-dependent manner. We show here that while MNV-infected Stat1−/− mice have significant losses of bone marrow B cells, splenic B cells capable of mounting an antibody response to novel antigens retain the ability to expand. We also investigated whether increased granulopoiesis after MNV infection was causing B cell loss. We found that administration of anti-G-CSF antibody inhibits the pronounced bone marrow granulopoiesis induced by MNV infection of Stat1−/− mice, but this inhibition did not rescue bone marrow B cell losses. Therefore, MNV-infected Stat1−/− mice can still mount a robust humoral immune response despite decreased bone marrow B cells. This suggests that further investigation will be needed to identify other indirect factors or mechanisms that are responsible for the bone marrow B cell losses seen after MNV infection. In addition, this work contributes to our understanding of the potential physiologic effects of Stat1-related disruptions in research mouse colonies that may be endemically infected with MNV.
Article
Blockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab and obinutuzumab are of human IgG1 isotype. Here, we investigated the impact of the variable regions of these three CD20 antibodies, when they were expressed as human IgA2 isotype variants. We observed more effective direct tumor cell killing by OBI-IgA2 compared to RTX- and OFA-IgA2, which was caspase-independent and required a functional cytoskeleton. Furthermore, IgA2 variants of all three antibodies triggered complement dependent cytotoxicity, with OBI-IgA2 being less effective than RTX- and OFA-IgA2. All three IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by PMN. Both effector mechanisms were significantly enhanced in the presence of a CD47 blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, OBI-IgA2 was consistently more potent than RTX- and OFA-IgA2 in triggering ADCC. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX- or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells, and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which is potentially relevant in the context of CD47-SIRPα blockade.
Article
Full-text available
Background Osteosarcoma is the most common malignant solid tumor that affects bones, however, survival rates of patients with relapsed osteosarcoma have not improved in the last 30 years. Oncolytic virotherapy, which uses viruses designed to selectively replicate in cancer cells, has emerged as a promising treatment for solid tumors. Our group uses mesenchymal stem cells (MSCs) to transport oncolytic adenoviruses (OAds) to the tumor site, a therapeutic strategy called Celyvir. This treatment has been already applied in human patients, canine patients and different mouse models. In parallel, previous results have probed that administration of granulocyte-colony stimulating factor (G-CSF) increased immune infiltration in tumors. We then hypothesized that the mobilization of immune cells by G-CSF may increase the antitumor efficacy of Celyvir treatment by increasing the immune infiltration into the tumors. Methods In this study, we use a murine version of Celyvir consisting in murine MSCs carrying the murine OAd dlE102—here called OAd-MSCs—in an immunocompetent model of osteosarcoma. We tested the antitumoral efficacy of the combination of OAd-MSCs plus G-CSF. Results Our results show that treatment with OAd-MSCs or the union of OAd-MSCs with G-CSF (Combination) significantly reduced tumor growth of osteosarcoma in vivo. Moreover, treated tumors presented higher tumor infiltration of immune cells—especially tumor-infiltrating lymphocytes—and reduced T cell exhaustion, which seems to be enhanced in tumors treated with the Combination. The comparison of our results to those obtained from a cohort of pediatric osteosarcoma patients showed that the virotherapy induces immunological changes similar to those observed in patients with good prognosis. Conclusions The results open the possibility of using cellular virotherapy for the treatment of bone cancers. Indeed, its combination with G-CSF may be considered for the improvement of the therapy.
Article
Fibroblast growth factor-23 (FGF23) hormone is produced by bone-embedded osteocytes and regulates phosphate homeostasis in kidneys. We found that granulocyte colony-stimulating factor (G-CSF) administration in mice induced a rapid and tremendous increase in FGF23 mRNA in bone marrow (BM) cells. This increase mainly originated from CD45-Ter119+CD71+ erythroblasts. FGF23 protein in BM extracellular fluid was markedly increased during G-CSF-induced hematopoietic progenitor cell (HPC) mobilization but remained stable in the blood with no change in the phosphate level. Consistent with the BM hypoxia induced by G-CSF, low oxygen concentration induced FGF23 release from human erythroblast HUDEP-2 cells in vitro. The mobilization efficiency by G-CSF was drastically decreased in both FGF23-/- and chimeric mice with FGF23 deficiency only in hematopoietic cells but increased in osteocyte-specific FGF23-/- mice. This suggests that erythroblast-derived, but not bone-derived, FGF23 is required to release HPCs from BM to circulation. Mechanistically, FGF23 did not influence CXCL12 binding to CXCR4 on progenitors but interfered with their transwell migration toward CXCL12, which was canceled by FGF receptor inhibitors. These results suggest that BM erythroblasts facilitate G-CSF-induced HPC mobilization via FGF23 production as an intrinsic suppressor of chemoattraction.
Article
Full-text available
The interactions of leukemia cells with the bone marrow (BM) microenvironment is critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-(endothelial)-selectin is a key niche component that directly mediates acute myeloid leukemia (AML) chemo-resistance, revealing E-selectin as a promising therapeutic target. To understand how E-selectin promotes AML survival, we investigated the potential receptors on AML cells involved in E-selectin-mediated chemo-resistance. Using CRISPR-Cas9 gene editing to selectively suppress canonical E-selectin receptors CD44 or P-selectin glycoprotein ligand-1 (PSGL-1/CD162) from human AML cell line KG1a, we show that CD162, but not CD44, is necessary for E-selectin-mediated chemo-resistance in vitro. Using preclinical models of murine AML, we then demonstrate that absence of CD162 on AML cell surface leads to a significant delay in the onset of leukemia and a significant increase in sensitivity to chemotherapy in vivo associated with a more rapid in vivo proliferation compared to wild-type AML and a lower BM retention. Together, these data reveal for the first time that CD162 is a key AML cell surface receptor involved in AML progression, BM retention and chemo-resistance. These findings highlight specific blockade of AML cell surface CD162 as a potential novel niche-based strategy to improve the efficacy of AML therapy.
Article
Full-text available
Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein α subunit Gsα is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of Gsα early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking Gsα in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in Gsα-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that Gsα-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner. • B lymphocyte • G protein • osteoblast
Article
Full-text available
The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide (CYP) as adjuvant to granulocyte colony-stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. It has recently emerged that G-CSF mediates HSC mobilization and inhibits bone formation via specific bone marrow (BM) macrophages. We compared the effect of these three mobilizing agents on BM macrophages, bone formation, osteoblasts, HSC niches and HSC reconstitution potential. Both G-CSF and CYP suppressed niche-supportive macrophages and osteoblasts, and inhibited expression of endosteal cytokines resulting in major impairment of HSC reconstitution potential remaining in the mobilized BM. In sharp contrast, although AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, endosteal cytokine expression or reconstitution potential of HSC remaining in the mobilized BM. In conclusion, although G-CSF, CYP and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches and bone formation are distinct with both G-CSF and CYP targeting HSC niche function and bone formation, whereas AMD3100 directly targets HSC without altering niche function or bone formation.
Article
Full-text available
Identification of the precise location, where hematopoietic stem cells (HSCs) reside in the bone marrow, has made a great leap forward with the advance of live time-lapse video 2-photon fluorescent microscopy. These studies have shown that HSCs preferentially resides in the endosteal region of the BM, at an average of two cell diameters from osteoblasts covering endosteal bone surfaces. However, this equipment is very sophisticated and only a very few laboratories can perform these studies. To investigate functional attributes of these niches, we have developed a flow cytometry technique in which mice are perfused with the cell-permeable fluorescent dye Hoechst33342 in vivo before bone marrow cells are collected and antibody stained. This method enables to position phenotypic HSC, multipotent and myeloid progenitors, as well as BM nonhematopoietic stromal cells relative to blood flow in vivo. This technique enables prospective isolation of HSCs based on the in vivo perfusion of the niches in which they reside.
Article
Full-text available
Translocation of the BCL2 gene on the chromosome band 18q21.3 results in consistent expression of the Bcl2 protein, an apoptosis inhibitor. BCL2 usually translocates to the immunoglobulin (IG) heavy chain (IGH) gene as t(14;18)(q32;q21.3) and rarely to IG light chain (IGK, IGL) loci as t(2;18)(p11;q21.3) or t(18;22)(q21.3;q11). The t(14;18) translocation is observed in 70-95% of follicular lymphoma cases and 20-30% of diffuse large B-cell lymphoma (DLBCL) cases. The MYC gene on chromosome band 8q24 acts as an accelerator of cell proliferation. MYC translocates to 14q32/IGH as t(8;14)(q24;q32) or less commonly to 2p11/IGK as t(2;8)(p11;q24) or 22q11/IGL as t(8;22)(q24;q11). The 8q24/MYC translocation is detected in nearly all Burkitt lymphoma (BL) and up to 10% of DLBCL cases. Both translocations rarely occur in an identical cell and this lymphoid malignancy is termed BCL2 and MYC dual-hit lymphoma/leukemia (DHL). The pathological diagnosis in most cases of DHL with BCL2-IG and MYC-IG translocation is B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL, although DLBCL is most common in DHL with BCL2-IG and MYC-nonIG translocation. The frequency of DHL with BCL2 and MYC translocation is estimated at around 2% of all B-cell malignancies. The condition is characterized by elevated serum lactate dehydrogenase levels, the presence of B symptoms, bone marrow involvement, advanced disease stage, extranodal involvement, and central nervous system (CNS) involvement at presentation or disease progression. Despite treatment strategies including CNS-targeted therapy, the prognosis for DHL is extremely poor. In this review, the current knowledge of the clinicopathological status of DHL is summarized and discussed.
Article
Full-text available
Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1(hi) monocytes (MOs), Gr-1(lo) MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin(+) niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169(+) MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin(+) niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.
Article
We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFL16.1, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement, but no V-D-J. Finally, functional analysis demonstrates that the proliferative response to IL-7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A.
Article
Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.
Article
Specialized roles for the pro-inflammatory cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) were characterized in TNF/LTα−/− and TNF−/− mice established by direct gene targeting of C57BL/6 ES cells. The requirement for LT early in lymphoid tissue organogenesis is shown to be distinct from the more subtle and varied role of TNF in promoting correct microarchitectural organization of leukocytes in LN and spleen. Development of normal Peyer's patch (PP) structure, in contrast, is substantially dependent on TNF. Only mice lacking LT exhibit retarded B cell maturation in vivo and serum immunoglobulin deficiencies. A temporal hierarchy in lymphoid tissue development can now be defined, with LT being an essential participant in general lymphoid tissue organogenesis, developmentally preceeding TNF that has a more varied and subtle role in promotion of correct spatial organization of leukocytes in LN and spleen. PP development in TNF−/− mice is unusual, indicating that TNF is a more critical participant for this structure than it is for other lymphoid tissues.
Article
Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.