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Volume 272, Number 44, Issue of October 31, 1997
pp. 27529-27531
(Received for publication, July 31, 1997, and in revised form, September 10, 1997)
From the Department of Physiology & Biophysics and Sealy Center for
Molecular Science, University of Texas Medical Branch, Galveston, Texas
77555 and ABSTRACT Chemokines are cytokines that activate and
induce the migration of leukocytes. Stroma-derived factor-1 (SDF-1) is
a novel chemokine that blocks the entry of T-tropic HIV-1 mediated by fusin/CXCR4/LESTR (leukocyte-derived seven-transmembrane domain receptor). In this work we demonstrate that SDF-1 triggers increases in
intracellular calcium and inhibits the proliferation of myeloid progenitor cell line 32D. By contrast, SDF-1 neither triggers a calcium
response nor affects the proliferation of the myeloid progenitor cell
line 32D-GR that is deficient in CXCR4. Responsiveness to SDF-1 was
rescued by transfection of 32D-GR cells with a cDNA encoding the
human CXCR4. The data indicate that SDF-1 induces myelosuppression by
activation of CXCR4. The constitutive production of SDF-1 by bone
marrow stromal cells argues for a major role of SDF-1 on the regulation
of myelopoiesis.
INTRODUCTION Chemokines are peptides of 70-100 amino acids secreted by many
cell types in response to injury and infection. Two major subfamilies of chemokines are distinguished according to the position of the first
two cysteines, the CXC or CC. Chemokines activate and induce migration
of leukocytes in a cell-specific fashion (1).
SDF-11 is a CXC chemokine
secreted constitutively from several cell types. Two isoforms of SDF-1
have been identified, EXPERIMENTAL PROCEDURES The murine
IL-3-dependent 32D and 32D-GR cell lines were kindly
provided by Dr. J. Greenberger, University of Pittsburgh Medical School, Pittsburgh, PA. These cell lines were maintained in RPMI 1640 plus 15% heat-inactivated fetal bovine serum and 15% conditioned medium from the murine myelomonocytic cell line WEHI-3B as a source of
crude IL-3 (12). Cells were cultured at 37 °C in a 5%
CO2 atmosphere and maintained at a cell density of 0.5 × 106 cells/ml. 32D-GR cells (107 cells/ml)
were transfected by electroporation with the human CXCR4 cDNA
subcloned into the unique EcoRI site of the expression vector pMEXneo (13). Transfected cells were selected by growing the
cells in the presence of 500 µg/ml G418. Colonies were scored on days
7 and 14 of culture.
To prepare the murine
SDF-1 (mSDF-1) proteins, the cDNA fragment encoding mature
mSDF-1 Cultures of 32D or 32D-GR cells were
carried out as described by Metcalf (16). In brief, 300 cells were
seeded in 35-mm Petri dishes containing 1 ml of Iscove's modified
Dulbecco's medium supplemented with 10% heat-inactivated fetal bovine
serum, 0.3% agar, and IL-3 or 10% of conditioned medium from the cell
line WEHI-3B. SDF-1 resuspended in phosphate-buffered saline or an equal volume of phosphate-buffered saline was added to the empty culture dish prior to the addition of the cell suspension in agar medium. After 7 days of incubation clusters of 50 or more cells were
scored as colonies.
Exponentially
growing 32D or 32D-GR cells were harvested by centrifugation and
resuspended in a solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM NaHPO4,
5 mM glucose, 20 mM Hepes pH 7.4, 1 mg/ml
bovine serum albumin, and 1 mM Probenecid. Cells
(107/ml) were loaded with 5 µM of the
calcium-sensitive dye Indo-1AM for 1 h at room temperature as
described previously (17). Intracellular calcium levels were monitored
at 37 °C with a spectrofluorimeter (Perkin-Elmer 650-10S) using an
excitation wavelength of 330 nm and an emission wavelength of 405 nm.
Total RNA (10 µg) was fractionated
on denaturating formaldehyde gels and transferred onto nylon membranes.
RNA was hybridized with 32P-labeled cDNA encoding human
CXCR4. Blots were washed twice in 0.25 × SSC, 0.1% SDS at
55 °C for 30 min and once in 0.1 × SSC, 0.1% SDS at 65 °C
for 15 min. Blots were exposed to x-ray film at RESULTS AND DISCUSSION Since SDF-1 is constitutively expressed by bone marrow-derived
stromal cells (2) we investigated whether SDF-1 plays a role in
myelopoiesis. SDF-1-induced intracellular signals in myeloid precursor
cells were monitored by increases in the level of intracellular Ca2+. We found that SDF-1
[View Larger Version of this Image (17K GIF file)]
[View Larger Version of this Image (18K GIF file)]
To determine whether the calcium responses and suppression of
proliferation of 32D cells are mediated by activation of the HIV-1
coreceptor CXCR4 we first explored the expression of CXCR4 in 32D cells
by Northern blot analysis. Blots of RNA extracted from 32D cells,
Jurkat T cells, and white blood cells were probed with human CXCR4
cDNA. As shown in Fig. 3, all these
cells express the mRNA of CXCR4. Second, we found that 32D-GR cells
(19), a cell line derived from 32D cells, neither express the mRNA
of CXCR4 (Fig. 3) nor exhibit the typical
SDF-1
[View Larger Version of this Image (49K GIF file)]
[View Larger Version of this Image (12K GIF file)]
FOOTNOTES ACKNOWLEDGEMENT We thank Nancy Wilkinson for helping with the
agar colony assays.
REFERENCES
COMMUNICATION:
Activation of HIV-1 Coreceptor (CXCR4) Mediates
Myelosuppression*
and
Genetics Institute,
Cambridge, Massachusetts 02140
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
and 
, which are generated by differential
splicing (2). SDF-1 was initially characterized as a pre-B-cell
stimulatory factor (3). Recently, SDF-1 has been identified as a highly
efficient chemotactic factor for T-cells, monocytes (4), and
CD34+ human progenitor cells (5). The constitutive
expression of SDF-1 by many tissues has suggested that SDF-1 plays a
key role in homing T-cells and monocytes under basal conditions (4). Targeted disruption of the SDF-1 gene in mice was shown to be lethal
with severe abnormalities in B-cell lymphopoiesis, bone marrow
myelopoiesis, and development of the cardiac ventricular septum (6).
The signaling of SDF-1 is mediated by CXCR4/fusin/LESTR (leukocyte-derived seven-transmembrane domain receptor), a G
protein-coupled receptor expressed preferentially in leukocytes (4,
7-9). Recent studies have demonstrated that CXCR4 is the coreceptor for the entry of T-tropic HIV-1 (10) and HIV-2 (11) into
CD4+ and CD4
cells,
respectively. Of importance is the observation that SDF-1 blocked the
entry of T-tropic HIV-1 strains (7, 8). Since SDF-1 is constitutively
expressed by bone marrow cells we investigated whether SDF-1 also
regulates myelopoiesis. Our data indicate that SDF-1 induces
myelosuppression of bone marrow-derived cell line 32D by activation of
CXCR4.
or mSDF-1 was subcloned into the Escherichia
coli expression vector pAL781 (14) behind the translation initiation codon ATG. The protein expression was carried out in the
E. coli strain GI934 as described (15). Inclusion bodies were harvested, washed sequentially with buffers containing 1 M NaCl and 0.5% Triton X-100, solubilized in 6 M guanidine HCl, and then dialyzed against a pH 6.5 buffer
containing 15 mM sodium acetate, 15 mM sodium
phosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM p-aminobenzamidine. The refolded mSDF
proteins were then further purified from the clarified dialysates by
ion-exchange chromatography.
80 °C for 24 h.
or - increased the
intracellular Ca2+ in 32D cells (Fig.
1), a non-tumorigenic cell line that
exhibits features of normal myeloid progenitor cells (16). In contrast to leukemic cell lines, the proliferation of 32D cells requires the
presence of IL-3 (18). The Ca2+ response mediated by
SDF-1 was concentration-dependent, and a maximal
response was achieved with 100 nM SDF-1 (Fig. 1). Cells treated with SDF-1 or - were weakly responsive to subsequent additions of SDF-1 or - showing homologous desensitization. Other
chemokines including IL-8, MIP-2, neutrophil activating peptide-2,
platelet factor-4, melanocyte growth stimulatory activity, monocyte
chemotactic protein-1, or ATP did not desensitize the Ca2+
response mediated by SDF-1. The SDF-1-induced calcium responses in
32D cells were similar to those previously observed with T-cells and
monocytes (7). In addition, SDF-1 failed to induce calcium responses
in 32D cells pretreated with pertussis toxin (data not shown),
demonstrating that the SDF-1 receptor is coupled to Gi proteins. To evaluate the functional role of SDF-1 on myeloid progenitor cells we tested the effect of SDF-1 on the proliferation of 32D cells using agar colony assays (16). Like normal hematopoietic progenitor cells, 32D cells form colonies in semisolid culture that are
strictly dependent on the presence of IL-3. As shown in Fig.
2, SDF-1 inhibited colony formation of
32D cells, suggesting that SDF-1 is a myelosuppressor.
Fig. 1.
SDF-1 induces mobilization of intracellular
Ca2+. Increases of intracellular Ca2+ were
recorded upon addition of 25 nM SDF-1 or -
(A and B). Each experiment has been reproduced at
least three times. Concentration dependence of SDF-1-induced
mobilization of intracellular Ca2+ is shown (C
and D). Concentrations of SDF-1 added to 32D cells loaded
with Indo-1 were: 2, 5, 10, 20, 40, 60, 100, and 150 nM. Results are representative of three separate
experiments.
Fig. 2.
Inhibition of proliferation of progenitor
cells by SDF-1. Effect of SDF-1 on colony formation of 32D cells
(
), 32D-GR (
), and 32D-GR transfected with CXCR4 cDNA ().
Each point represents the average of two independent
experiments. The standard deviations of the responses of 32D-GR cells
transfected with CXCR4 cDNA are indicated by bars. The
variability of responses of 32D and 32D-GR cells is negligible.
-dependent Ca2+ response (Fig.
4). However, similarly to 32D cells,
32D-GR cells exhibit the typical ATP-dependent
Ca2+ responses (Fig. 4, A and C),
form colonies in semisolid culture, and require IL-3 for proliferation.
Both 32D and 32D-GR form a similar number of colonies; however,
SDF-1 failed to inhibit the proliferation of 32D-GR cells (Fig. 4).
Finally, responsiveness to SDF-1 was rescued by transfection of 32D-GR
with human CXCR4 cDNA. Figs. 2 and 4 show that SDF-1 triggers a
calcium response and inhibits proliferation of 32D-GR cells expressing
the human CXCR4. These data indicate that SDF-1 induces
myelosuppression by activation of CXCR4. Previously, several chemokines
including IL-8, platelet factor-4, MIP-1, and MIP-1 have shown
myelosuppressive activity in bone marrow myeloid progenitor cells;
however, the heterogeneity and low frequency of precursor cells from
bone marrow has precluded the identification of receptor systems that
mediate the effect of these chemokines (20-22). It is unlikely that
these chemokines mediate their myelosuppressive activity via CXCR4
since they do not activate CXCR4 or desensitize the calcium responses mediated by SDF-1. Probably, these chemokines bind specific receptors. Indeed, we have recently shown the endogenous expression of the murine
homolog of the human IL-8 receptor B in 32D
cells.2 The constitutive
expression of SDF-1 by bone marrow stromal cells strongly argues for a
major role of the HIV-1 coreceptor CXCR4 on myelopoiesis.
Fig. 3.
Expression of CXCR4 mRNA. Northern
blots were probed with a human cDNA encoding CXCR5 labeled by a
random priming procedure. Lanes: 32D cells; white blood
cells (WBC); Jurkat cells; 32D-GR cells.
Fig. 4.
Mobilization of intracellular calcium in
CXCR4-deficient cells and cells rescued by transfection with human
CXCR4 cDNA. SDF-1 (25 nM) or ATP (500 nM) were added to: A, 32D cells; B, 32D-GR cells; and C, 32D-GR cells transfected with human
CXCR4 cDNA. The experiments have been reproduced at least three
times.
§
To whom correspondence should be addressed: Dept. of Physiology & Biophysics, Rt 0641, University of Texas Medical Branch, Galveston, TX
77555-0641. Tel.: 409-772-5480; Fax: 409-772-3381; E-mail:
jnavarro{at}mspo2.utmb.med.edu.
1
The abbreviations used are: SDF-1,
stroma-derived factor-1; HIV, human immunodeficiency virus; IL,
interleukin; mSDF-1, murine SDF-1; MIP, macrophage inflammatory
protein.
2
X. Sanchez, K. Suetomi, B. Hodges, J. Horton,
and J. Navarro, unpublished results.
Volume 272, Number 44,
Issue of October 31, 1997
pp. 27529-27531
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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