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Departments of
* Microbiology/Immunology,
Medicine (Hematology/Oncology), and
Pediatrics (Neonatology),
Walther Oncology Center, and
¶ Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202;
|| Walther Cancer Institute, Indianapolis, IN 46208; and
# Laboratory of Immunology and Hematopoiesis, Department of Veterinary Pathology, Purdue University, West Lafayette, IN 47907
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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i proteins as determined by ELISA, an
antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin.
Transgenic effects of low SDF-1/CXCR4 may be due to synergy of
SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes
with low concentrations of other cytokines to enhance survival of
normal mouse myeloid progenitors. Consistent with in vitro results,
progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow
and splenic myelopoiesis: greatly increased progenitor cell cycling and
significant increases in progenitor cell numbers. These results
substantiate survival effects of SDF-1/CXCL12, now extended to
progenitors engineered to endogenously produce low levels of this
cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition
to cell trafficking. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These gene-knockout studies, as well as other in vitro analyses implicated SDF-1/CXCL12 in chemotaxis/migration/homing of mature leukocytes and of hemopoietic stem and myeloid progenitor cells (reviewed in Refs. 5, 6, 7, 8). However, many cytokines have more than one effect (7). Recently, SDF-1/CXCL12 has been shown to have survival-enhancing effects for myeloid progenitors (9, 10, 11) and other more mature cell types (12, 13, 14, 15), although these effects have not been noted by all groups for different cell types and even for the same cell types in different tissues (9, 16, 17, 18, 19). In an effort to learn more about the multifaceted effects of SDF-1/CXCL12, we generated transgenic mice expressing SDF-1/CXCL12 under a Rous sarcoma virus (RSV) promoter. During characterization of the myeloid progenitor cells from these mice, we noted enhanced survival of the transgenic cells subjected to delayed addition of growth factors in vitro and enhanced myelopoiesis in vivo at the level of the myeloid progenitors. The studies presented in this article document these effects and demonstrate the potent activity of low level transgene expression of SDF-1/CXCL12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse cells were obtained from the femurs and spleens of RSV-SDF-1 transgenic mice and their littermate controls, which we generated, and also from C3H/HeJ mice purchased from The Jackson Laboratory (Bar Harbor, ME). c-kit+lin- mouse marrow cells were obtained as previously described (20).
Generation of SDF-1/CXCL12 transgenic mice
Full length mouse SDF-1/CXCL12 cDNA was cloned out by RT-PCR
amplification from a mouse stromal cell line M2-10B4 using the
following primers: TGG TGG TAC CAT GGA CGC CAA GGT CGT CGC CGT G
(forward) and CTG CGG CCG CCT ATT ACT TGT TTA AAG CTT TCT CC (reverse).
The amplified mouse SDF-1/CXCL12 gene was ligated
into the KPNI and NotI sites of Prep4 (Invitrogen, San
Diego, CA) so that SDF-1/CXCL12 was expressed under the control of
pRSV. Prep4-SDF-1/CXCL12 clones were verified for correct mouse
SDF-1/CXCL12 sequence (GenBank accession number L12029). This
Prep4-SDF-1/CXCL12 construct was transfected into 293/EBNA
(Invitrogen), and culture medium was tested for expression of
functional SDF-1/CXCL12 protein by Transwell chemotaxis assay of MO7e
cells as previously described (21). Significant
chemotactic activities (typically 
5% specific migration rate over
background, which is equal to 5–10 ng/ml/day of SDF-1/CXCL12
production) were detected in the 24-h-incubated culture medium of
293/EBNA cells transfected with Prep4-SDF-1/CXCL12, but not (0%
specific migration rate) with the parental vector Prep4, suggesting
functional expression of SDF-1/CXCL12 protein from the
pRSV-SDF-1/CXCL12 expression vector construct. Chemotaxis of MO7e cells
with standards for SDF-1 is quantitative with a linear dynamic range
between 5 and 50 ng/ml SDF-1. The SDF-1 produced by the 293 cells was
highly reproducible using the MO7e chemotaxis assay.
SalI-digested pRSV-SDF-1/CXCL12-poly(A) fragment was
microinjected into inbred C3HeB/FeJ (The Jackson Laboratory) zygotes.
Microinjected embryos were cultured in vitro to the two-cell stage, and
then reimplanted into pseudopregnant SW/Taconic (Taconic Farms,
Germantown, NY) female mice by the Indiana University Cancer Center
(Indianapolis, IN) core facility directed by Dr. L. Fields. For
all surgeries, mice were anesthetized with 2.5% Avertin (0.015 ml/g
body weight, i.p.; Fluka Chemical, Ronkonkoma, NY). All
manipulations were performed according to National Institutes of Health
and Institutional Animal Care and Use Guidelines. Pups derived from the
microinjected embryos were screened for the presence of the transgene
by PCR using the following primers: GTG CCT AGC TCG ATA CAA TAA ACG CC
(promoter specific forward) and CTG CGG CCG CCT ATT ACT TGT TTA AAG CTT
TCT CC (SDF-1 specific reverse). Tail DNAs were used for genomic PCR
analysis. Transgenic animals were then bred with wild-type littermates
to maintain SDF-1 transgenic mouse lineages. RSV-SDF-1/CXCL12
transgenic mouse lineages were maintained in a C3He/FeJ background.
RT-PCR analysis
Transgenic mice and wild-type littermate controls were sacrificed and bone marrow, thymus, liver, and spleen were obtained. Tissues were homogenized by passing through syringe needles or iron meshes to get single-cell suspension. Total mRNA was isolated using TRIzol solution (Life Technologies, Rockville, MD), and cDNA was synthesized by an mRNA reverse transcription kit (Life Technologies). Expression of SDF-1/CXCL12 transgene was examined as previously described (22) by specifically amplifying the transgenic, but not the natural SDF-1, messages using two primers: ACC AAG ATC TAT GGA CGC CAA GGT CGT CGC CGT G (forward) and GGT TTG TCC AAA CTC ATC AAT G (reverse). Also, RT-PCR was performed in the presence of [32P]dCTP so that only amplified products, but not genomic DNA, could be detected.
Cytokines, Abs, and other agents
Purified recombinant preparations of SDF-1 and SDF-1
were
purchased from R&D Systems (Minneapolis, MN), and purified chemically
synthesized SDF-1 was obtained from I. Clark-Lewis (University of
British Columbia, Vancouver, British Columbia, Canada). The effects and
dose-response curves for all three preparations were exactly the same
in our assays. Most of the studies used the chemically synthesized
SDF-1. Purified recombinant preparations of murine (mu)GM-CSF
and steel factor (SLF) and human (hu)Flt3-ligand (FL) were kind
gifts of Immunex (Seattle, WA), and purified recombinant human
erythropoietin (Epo) was purchased from Amgen (Thousand Oaks,
CA). Purified recombinant thrombopoietin (TPO) was purchased from R&D
Systems. AMD3100 was a kind gift of AnorMed (Langley, British Columbia,
Canada). Ab to mouse CXCR4 (23) was a kind gift from
Millenium (Cambridge, MA). Anti-c-kit, anti-activated
caspase-3, and Abs to CD3E, CD4, CD8a, Gr-1,
B220, Mac-1, and isotype controls were purchased from BD PharMingen
(San Diego, CA).
Myeloid progenitor cell assays
Sorted c-kit+lin- bone marrow (1000 cells/ml) or unseparated mouse bone marrow (5 x 104 cells/ml) or spleen (5 x 105 cells/ml) cells from mice were plated in 0.9% methylcellulose culture medium with 30% FBS (HyClone, Logan, UT) with the combination of 5% v/v pokeweed mitogen mouse spleen cell conditioned medium (PWMSCM), muSLF (50 ng/ml), huEpo (1 U/ml) and hemin (0.1 mM; Sigma-Aldrich, St. Louis, MO). In some experiments, CFU-granulocyte-macrophage (CFU-GM) colonies were scored after plating cells in 0.3% agar culture medium with 10% FBS and growth factors. More details can be found elsewhere (24). For the delayed addition of growth factors, cells were plated with or without SDF-1/CXCL12 at time 0, and the growth factors were added, respectively, to murine cells at either time 0, 24, or 48 h after the start of the cultures (time = 0 h). Colonies were scored at 7 days after the addition of growth factors. Plates were incubated at 5% CO2 in lowered (5%) oxygen in a humidified atmosphere.
Apoptosis
These studies were done as described in Fig. 5
using activated
caspase-3 (25) as a measure of apoptosis.
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Statistical significance for the colony assays was determined using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Delayed addition of growth factors results in decreased survival
of myeloid progenitor cells in vitro, effects due completely or in
large part to growth-factor deprivation-induced apoptosis
(26, 27, 28). Exogenously added SDF-1/CXCL12 acts as a
survival/antiapoptotic factor for myeloid progenitor cells in the
context of delayed addition of growth factors (9, 10). To
further evaluate the survival-enhancing effects of SDF-1/CXCL12 on
myeloid progenitor cells, and to elucidate the effects of endogenous
production of SDF-1/CXCL12 on these cells, we used cells from mice that
we produced that expressed a mouse SDF-1/CXCL12 transgene under an RSV
promoter. Identification of transgenic RSV-SDF-1/CXCL12 pups by genomic
PCR and tissue expression of the transgene as evaluated by RT-PCR are
shown in Fig. 1, A and
B, respectively. mRNA expression of the SDF-1 transgene was
seen in the bone marrow, thymus, liver, and spleen of the transgenic,
but not wild-type control, mice (Fig. 1B). As seen in Figs. 2 and 3,
myeloid progenitors from unseparated marrow of SDF-1 transgenic mice
survived better in the presence of serum in vitro with delayed addition
of growth factors compared with cells from wild-type littermate
controls. Similar results were seen with progenitors from the spleens
of the SDF-1 transgenic and littermate control mice (data not shown).
Enhanced survival of the progenitors was also seen for a highly
purified population of
c-kit+lin- cells
(>99% c-kit+; <99%
lin-; a greatly enriched source of myeloid
progenitor cells and also stem cells) from SDF-1 transgenic mice
separated by FACS compared with these cells from wild-type littermate
control mice; soluble SDF-1 prolonged survival of
c-kit+lin- myeloid
progenitors from littermate control mice to the same level as that of
c-kit+lin- SDF-1
transgenic cells in the absence of added SDF-1 (Fig. 4). As previously noted, the effects of
SDF-1/CXCL12 on normal myeloid progenitors is dose dependent.
Exogenously added SDF-1 at concentration 
100 ng/ml had maximum
survival-enhancing effects on normal progenitors, while concentrations
of 
10 ng/ml had no significant effects compared with control medium.
Addition of 100–500 ng/ml SDF-1 did not further enhance the survival
of myeloid progenitors from SDF-1 transgenic mice (see next section),
suggesting that the survival-enhancing effects of endogenous SDF-1 in
the SDF-1 transgenic mouse myeloid progenitor cells were maximally
effective.
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To determine whether the SDF-1/CXCL12 transgene survival-enhancing
effects on the myeloid progenitors reflected antiapoptotic effects, we
evaluated apoptosis (as assessed by activated caspase-3) in populations
of c-kit+ bone marrow cells, using
multivariate flow cytometric analysis. As shown in Fig. 5
(for a representative experiment),
c-kit+ cells from SDF-1/CXCL12 transgenic
mice demonstrated significantly less apoptosis than these cells from
wild-type mice in the absence of added growth factors. The percentage
of apoptosis averaged 40 for wild-type cells and 22 for SDF-1/CXCL12
transgenic cells (p < 0.03; n
= 6 experiments) in the absence of growth factors. Addition
of growth factors to transgenic cells resulted in 19% apoptosis
(p > 0.05 compared with transgenic cells
without growth factors or to 15% apoptosis of wild-type cells with
growth factors). Thus, addition of growth factors, which decreased
apoptosis of wild-type cells, did not significantly alter the decreased
apoptosis of SDF-1 transgenic cells. As reported previously for normal
human progenitors (10), SDF-1/CXCL12 significantly
decreased apoptosis of control cells from wild-type mice by at least
40% (p < 0.01).
To determine whether the enhanced survival of SDF-1/CXCL12
transgene-expressing myeloid progenitor cells was being mediated
through CXCR4, the receptor for SDF-1, bone marrow cells from the
transgenic mice were pretreated for 30 min before plating and then the
cells were plated with final concentrations of either 1 µM, 0.1 µM,
or 0.01 µM AMD-3100, a specific antagonist of SDF-1 binding to CXCR4
(29) that we previously demonstrated blocks
SDF-1/CXCL12-induced activation of mitogen-activated protein
kinase activity in the human factor-dependent cell line MO7e
(10), or 10, 1, or 0.1 µg/ml of anti-mouse CXCR4 Abs
(23). As shown in Fig. 6, AMD-3100 at 1 µM and 0.1 µM, but not at 0.01 µM, and
anti-CXCR4 at 10 µg and 1 µg/ml, but not at 0.1 µg/ml,
blocked the survival-enhancing effects of the SDF-1/CXCL12 transgenic
myeloid progenitor cells. Addition of 100 ng/ml soluble SDF-1/CXCL12, a
maximally effective concentration for enhancing survival of myeloid
progenitors, did not further enhance the survival of SDF-1/CXCL12
transgene-expressing progenitors. Higher concentrations of AMD-3100 and
anti-CXCR4 were needed to counter the survival enhancement of
SDF-1/CXCL12 transgene-expressing progenitors cultured with, compared
to without, exogenously added SDF-1/CXCL12. It took 1 µM AMD-3100 and
10 µg/ml anti-CXCR4 to counterbalance the survival-enhancing
effects of exogenously added SDF-1/CXCL12 to the transgenic cells,
compared with 0.1 µM AMD-3100 and 1 µg/ml anti-CXCR4 to
counterbalance the survival-enhancing effects of transgenic cells in
the absence of added SDF-1/CXCL12. Concentrations of AMD-3100 at 1 µM
and anti-CXCR4 at 10 µg/ml were needed to counteract the
survival-enhancing effects of 100 ng/ml exogenously added SDF-1/CXCL12
to wild-type littermate control cells (data not shown). Thus, while the
enhanced survival of SDF-1/CXCL12 transgene-expressing myeloid
progenitor cells, compared with the survival of wild-type progenitors,
is not further enhanced by exogenously added SDF-1/CXCL12, titration
experiments with AMD-3100, anti-CXCR4, and anti-SDF-1/CXCL12
demonstrate that the transgenic cells can respond to exogenously added
SDF-1/CXCL12, and both endogenous and exogenous effects of SDF-1/CXCL12
on the transgenic cells are mediated through CXCR4.
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, anti-SDF-1 at 10 and 1 µg/ml, but not at 0.1 µg/ml,
blocked the survival-enhancing effects of the SDF-1/CXCL12 transgenic
myeloid progenitor cells. Only the 10 µg/ml concentrations of
anti-SDF-1/CXCL12 completely blocked the survival-enhancing effects
of transgenic cells in the presence of 100 ng/ml exogenously added
SDF-1/CXCL12. In comparison, 10 µg/ml, but not 1 or 0.1 µg/ml,
concentrations of anti-SDF-1 blocked the exogenously added
SDF-1/CXCL12 enhancing effects on wild-type littermate control myeloid
progenitors (data not shown). These results demonstrate that the
enhanced survival of SDF-1/CXCL12 transgene-expressing myeloid
progenitor cells is mediated by SDF-1 released by these cells. Also,
because much lower concentrations of anti-SDF-1 are needed to
counter the survival-enhancing effects of the SDF-1/CXCL12
transgene-expressing progenitors in the absence of exogenously added
SDF-1/CXCL12 or transgenic cells cultured with exogenously added
SDF-1/CXCL12, compared with that of wild-type progenitors cultured with
100 ng/ml exogenously added SDF-1/CXCL12, this suggests that the
transgenic cells are releasing and responding to very low
concentrations of endogenously produced transgenic SDF-1/CXCL12. This
is consistent with our attempts to quantitate SDF-1/CXCL12 in the
lysates or medium conditioned for 24 h by 1 x
106 bone marrow or spleen cells per milliliter
from RSV-SDF-1/CXCL12 transgenic mice. The amounts of SDF-1/CXCL12
detected from these cell lysates or conditioned medium were at or
slightly above the level of detection of the ELISA (0.1 ng/ml). No
SDF-1/CXCL12 was found in the lysates or conditioned medium from bone
marrow or spleen cells of the wild-type littermate control
mice.
Chemokine receptors are linked to heterotrimeric G proteins, and a
number of chemokine functions are Gi linked and PTX sensitive
(5, 6, 7, 8). As shown in Fig. 6, the enhanced survival of
myeloid progenitors from SDF-1/CXCL12 transgenic mice in the absence of
exogenously added SDF-1/CXCL12 is blocked by pretreatment of these
cells with PTX, and exogenous addition of SDF-1 to SDF-1/CXCL12
transgenic progenitors does not overcome the blocking effect of PTX on
SDF-1/CXCL12-enhanced survival. This, along with the above data,
demonstrates that the enhanced survival of SDF-1/CXCL12
transgene-expressing myeloid progenitors acts through endogenously
produced and released SDF-1/CXCL12 acting on surface CXCR4 and is
mediated through Gi proteins.
Low concentrations of SDF-1/CXCL12 act synergistically with low concentrations of other cytokines in vitro to enhance survival of mouse bone marrow progenitor cells
The information presented above suggested that the amount
of transgenic SDF-1/CXCL12 that was produced, released, and active in
the enhancement of the survival of the SDF-1/CXCL12
transgene-expressing myeloid progenitor cells was much lower than the
amounts of exogenously added SDF-1/CXCR4 necessary to enhance the
survival of wild-type littermate control mice. The titration studies
above with AMD-3100, anti-CXCR4, and anti-SDF-1 also
demonstrated that the RSV-SDF-1 transgenic progenitors were capable of
responding to exogenously added SDF-1/CXCR4. Thus, we did not have
evidence that there is a unique interaction of transgenic vs
exogenously added SDF-1/CXCL12 with the CXCR4 receptors expressed on
the cells from the transgenic mice that would explain the potent effect
of the low amounts of transgenic SDF-1/CXCL12 on the transgenic cells.
We have recently reported that low concentrations of SDF-1/CXCL12,
which by themselves are inactive as survival-enhancing factors,
synergize with low concentrations of other cytokines such as GM-CSF,
TPO, SLF, or FL, which by themselves at low concentrations
provide no survival-enhancing activity, to enhance the survival of
huCD34+++ myeloid progenitors subjected to
delayed addition of growth factors (10). This synergistic
effect was also noted for human MO7e cells (10). With this
in mind, we assessed whether SDF-1/CXCL12 could also synergize with
other cytokines to enhance the survival of mouse myeloid progenitors
from the marrow of C3H/HeJ mice. As seen in Fig. 7, low inactive concentrations of SDF-1
synergized with low inactive concentrations of FL, SLF, GM-CSF, or TPO,
to enhance the survival of murine marrow CFU-GM. Thus, the possibility
exists that enhancement of cell survival noted with the RSV transgenic
myeloid progenitors is due to a synergistic activity with low
concentrations of other cytokines present either in the serum or
released by the cells. Although the exact cytokine(s) involved here are
not known, it is clear that transgenic SDF-1 is involved because the
enhanced survival of the transgenic myeloid progenitors was greatly
decreased with AMD-3100, anti-CXCR4, or anti SDF-1/CXCL12 treatment
(Fig. 6).
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To evaluate in vivo effects of transgenic SDF-1, mice were
evaluated for cycling status (percentage of cells in S phase) and
absolute numbers of myeloid progenitor cells in marrow and spleen. As
shown in Fig. 8, the cycling status
of multi-growth factor-responsive CFU-GM, burst-forming unit-erythroid
(BFU-E), and CFU-granulocyte-erythrocyte-megakaryocyte-monocyte
(CFU-GEMM), and GM-CSF-responsive CFU-GM in marrow and spleen of SDF-1
transgenic mice was significantly greater than in those of littermate
controls, with the effects most apparent in the spleen where the
usually slow or noncycling myeloid progenitors were in rapid cell cycle
in the SDF-1 transgenic mice. The cycling status of
M-CSF-responsive CFU-macrophage (CFU-M) was significantly
enhanced in the marrow, but not in the spleen of the SDF-1 transgenic
mice compared with littermate controls. This enhanced progenitor cell
cycling was associated with significantly enhanced absolute numbers of
CFU-GM, BFU-E, and CFU-GEMM in the spleen, and to a lesser extent in
the marrow, where only GM-CSF-responsive CFU-GM and M-CSF-responsive
CFU-M were significantly increased in SDF-1 transgenic, compared with
littermate control, mice (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The survival-enhancing effects of SDF-1/CXCL12 transgene-expressing
myeloid progenitor cells were noted in bone marrow (
Figs. 2–4) and
spleen (data not shown) in the absence of exogenously added
SDF-1/CXCL12, when these cells were compared with those from littermate
control mice. The survival of RSV-SDF-1/CXCL12 transgenic progenitors
in the absence of exogenously added SDF-1/CXCL12 was equal to that of
the enhanced survival of wild-type littermate control mice cultured in
the presence of exogenously added SDF-1/CXCL12. Most interestingly,
addition of exogenous SDF-1/CXCL12 to RSV-SDF-1/CXCL12 transgenic
progenitors did not further enhance the survival of these cells, except
in the context of titrations of anti-CXCR4, anti-SDF-1/CXCL12,
and an antagonist to CXCR4 (Fig. 6), suggesting that survival
enhancement due to endogenous SDF-1/CXCL12 was already at a maximal
level in terms of SDF-1/CXCL12 effects. However, the amount of
endogenous SDF-1/CXCL12 was far below the concentrations of exogenously
added SDF-1/CXCL12 needed for survival enhancement of littermate
control cells or cells from a number of other mouse strains.
Exogenously added SDF-1/CXCL12 was maximally effective at a
concentration of 100 ng/ml, and concentrations of 10 ng/ml were
minimally active or inactive in this effect. Although the
RSV-SDF-1/CXCL12 transgenic cells clearly expressed the SDF-1/CXCL12
transgene as determined by RT-PCR analysis, and the RSV-SDF-1/CXCL12
construct was verified to express functional SDF-1/CXCL12 before it was
injected into the mice embryos, the amount of protein produced in the
transgenic mice appears to be very low. Serum SDF-1/CXCL12
concentrations for both the RSV-SDF-1/CXCL12 transgenic and wild-type
littermate controls were <2 ng/ml, as assessed by ELISA analysis.
SDF-1/CXCL12 amounts in lysates or medium conditioned by 1 x
106 bone marrow or spleen cells per milliliter
from RSV-SDF-1/CXCL12 transgenic mice were at or slightly above the
level of detection of the ELISA (0.1 ng/ml). That the
survival-enhancing effect was due to released SDF-1/CXCL12 and acting
through CXCR4 was confirmed by blocking the survival-enhancing effects
of the SDF-1/CXCL12 transgenic cells with Abs to SDF-1/CXCL12 and with
Abs and an antagonist to muCXCR4. Moreover, the use of PTX clearly
demonstrated that the survival-enhancing effect was mediated through
Gi proteins. SDF-1/CXCL12 as well as other chemokines are known to
mediate their chemotactic effects for myeloid progenitors and other
more mature cells through Gi proteins (5, 6, 7, 8, 21). To establish additional controls for the enhanced survival
of RSV-SDF-1/CXCL12 transgenic myeloid progenitors, we tested the
survival of bone marrow and spleen cells from other transgenic mice we
produced in which SDF-1/CXCL12 transgene expression was under the
control of an LCK promoter (30), which limited expression
of the SDF-1/CXCL12 transgene to lymphoid cells (data not shown).
LCK-SDF-1/CXCL12 transgene expression was confirmed by RT-PCR analysis
in thymus, lymph nodes, and spleen, but not bone marrow or liver. No
SDF-1/CXCL12 transgene expression was detected in the tissues of the
LCK-SDF-1/CXCL12 littermate control mice. Contrary to results with
RSV-SDF-1/CXCL12 transgenic progenitor cells, the myeloid progenitor
cells from the marrows and spleens of the LCK-SDF-1/CXCL12 transgenic
mice were not enhanced in survival compared with LCK-SDF-1/CXCL12
littermate control mice (data not shown). Addition of exogenous
SDF-1/CXCL12 (100 ng/ml) significantly enhanced survival of both
LCK-SDF-1/CXCL12 transgenic and littermate control myeloid progenitors
to the same extent as that of RSV-SDF-1/CXCL12 littermate control
progenitors and progenitors from other strains of mice (data not
shown). Because there is no endogenous SDF-1/CXCL12 expression in
myeloid progenitors from LCK-SDF-1/CXCL12 transgenic mice and the
progenitors from these mice were not enhanced in survival, this
information complements our other studies with Abs to SDF-1/CXCL12 and
CXCR4 and with an antagonist to CXCL12 that demonstrates that
endogenous SDF-1/CXCL12 expression from the RSV-SDF-1/CXCL12 transgene
is the reason for the enhanced survival of these cells.
It is not clear why so little SDF-1/CXCL12 was found in the serum and cells from the RSV-SDF-1/CXCL12 transgenic mice. Deficient SDF-1/CXCL12 or CXCR4 expression caused the perinatal lethality seen in SDF-1/CXCL12-/- and CXCR4-/- mice (2, 3, 4). However, the possibility exists, which we currently favor although we have not yet proven, that too much SDF-1/CXCL12 may also be detrimental to development. In this context, we were able to identify only two RSV-SDF-1/CXCL12 transgenic mice of over 140 births in contrast to high birth rates (20–30%) of other transgenic animals expressing different genes. Results with offspring of the two founder RSV-SDF-1/CXCL12 transgenic mice were similar in terms of survival enhancement of myeloid progenitor cells in response to transient growth factor withdrawal from these cells in vitro. Reasons for the low birth rate of RSV-SDF-1/CXCL12 transgenic mice are of interest and will require further study.
The survival-enhancing effects of SDF-1/CXCL12 transgene-expressing progenitors was shown to be antiapoptotic by evaluation of activated caspase-3 in populations of mouse c-kit+lin- bone marrow cells, which are highly enriched for myeloid progenitor cells (20). However, our studies do not rule out the possibility that some of the SDF-1/CXCL12 survival-enhancing effects involve actions other than antiapoptosis. For example, the survival-enhancing effects of SDF-1/CXCL12 on myeloid progenitor cells, as assessed by detection of colony formation, only allows us to detect whether or not the progenitor cell divided enough times to form a colony. Those cells that did not form a colony may have died by apoptotic or another form of cell death, or perhaps they remained alive but were unable to proliferate even in the presence of the potent combination of growth factors that we used to stimulate colony formation. At the population cell level, we have not been able to demonstrate SDF-1/CXCL12 stimulation of colony formation, but it has been reported that SDF-1/CXCL12 promotes G0/G1 transition in nonmobilized human peripheral blood CD34+ cells (9). Whether or not SDF-1/CXCL12, alone or in combination with other growth factors, promotes more than a G0/G1 transition will likely have to be evaluated at the single-cell level.
Our studies show that endogenous transgene expression of SDF-1/CXCL12
results in enhanced myelopoiesis in vivo at the level of myeloid
progenitor cells (Figs. 8 and 9), factors most certainly not due to
potential abnormalities in cell migration within these mice, because
the enhanced cycling was seen in progenitors in marrow and spleen, and
absolute numbers of progenitors in the marrow and spleen of the
transgenic mice were either increased in number or not significantly
different from the numbers seen in the marrow and spleen of the
littermate control mice.
Because the amounts of SDF-1/CXCL12 found in and released from the transgenic cells were lower than those that can enhance survival of myeloid progenitors, and SDF-1/CXCL12 has been shown previously with human cells (10), and in this study with mouse cells, to synergize with other growth factors such as GM-CSF, SLF, FL, and TPO, we consider it likely that the survival-enhancing effects we are noting in the SDF-1/CXCL12 transgene-expressing cells are due to synergism of the transgenic SDF-1/CXCL12 and one or more other survival-enhancing cytokines. Others have reported expression and release of numerous growth factors and cytokines from CD34+ enriched populations of progenitor cells and immature blast cells (31). It is possible that such synergistic possibilities are also operative in the SDF-1/CXCL12 transgenic mice and responsible for the enhanced myelopoiesis noted in our transgenic mice. Intracellular signaling events mediating this possible synergism may be similar to those noted in vitro with human progenitor cells, in which synergism between SDF-1/CXCL12 and other cytokines in the enhancement of progenitor cell survival was associated with synergism in intracellular events known to be involved in cell survival and antiapoptosis (10). Alternatively, SDF-1 working in an autocrine manner may be more potent in survival-enhancing effects than SDF-1 working in a paracrine way or exogenously added SDF-1.
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2 Address correspondence and reprint requests to Dr. Hal E. Broxmeyer, Walther Oncology Center, Indiana University School of Medicine, 1044 West Walnut Street, R4-302, Indianapolis, IN 46202. E-mail address: hbroxmey{at}iupui.edu
3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; CXCL, CXC chemokine ligand; RSV, Rous sarcoma virus; CFU-GM, CFU-granulocyte-macrophage; CFU-GEMM, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte; CFU-M, CFU-macrophage; BFU-E, burst-forming unit-erythroid; SLF, steel factor; FL, Flt3 ligand; TPO, thrombopoietin; Epo, erythropoietin; PWMSCM, pokeweed mitogen mouse spleen cell conditioned medium; PTX, pertussis toxin; hu, human; mu, murine.
Received for publication September 4, 2002. Accepted for publication October 23, 2002.
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