during myeloid differentiation
Department of Cardiovascular Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania
Correspondence: Dr. Shalley K. Gupta, Department of CV Biology, Mail Code UW2511, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406. E-mail: Shalley_K_Gupta{at}sbphrd.com
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S binding, calcium flux, and
chemotaxis were examined simultaneously. Granulocytic
differentiation with dimethyl sulfoxide induced surface expression of
CXCR4 as well as SDF-1-mediated [35S]GTPS binding and
chemotaxis, whereas calcium flux was attenuated by twofold to threefold
in HL-60 cells. Conversely, monocytic differentiation with vitamin
D3 inhibited surface expression and SDF-1-mediated
chemotaxis, even as it induced [35S]GTPS binding and
calcium flux by more than twofold. Sodium butyrate up-regulated all
parameters of CXCR4 expression studied. Together, these results
demonstrate that CXCR4 expression undergoes complex regulation at
multiple checkpoints, with the likely involvement of different
G-proteins for signal transduction during cellular differentiation and
following activation with SDF-1.
Key Words: chemokines • cellular differentiation • granulocytes • monocytes
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Chemokines are a large superfamily of inducible, secreted proteins that mediate their functions through a family of G-protein-coupled receptors. The main function of chemokines, ostensibly, is to mediate the recruitment of circulating leukocytes to the sites of infection and inflammation [6 ]. In addition, recent studies with genetic knockouts in mice that targeted specific chemokines, such as stromal cell-derived factor-1 (SDF-1) and eotaxin, and chemokine receptors, such as BLR-1, CXCR2, and CXCR4 [7 8 9 10 11 12 13 14 ], have also provided conclusive evidence of their critical role as mediators of cellular trafficking during hematopoiesis and embryogenesis.
SDF-1 is a member of the C-X-C chemokine subfamily [15
]
and mediates its biological action through its specific
G-protein-coupled receptor, CXCR4 [16
, 17
].
It is known to cause migration of peripheral blood leukocytes,
monocytes, and endothelial cells [18
19
20
21
]. SDF-1 is also
a chemoattractant for uncommitted and committed hematopoietic
progenitor cells, pre-B cells, myeloid cells, and megakaryocytes
[22
23
24
]. CXCR4- and SDF-1-deficient mice die
perinatally with similar defects in cardiogenesis, neuron migration,
hematopoiesis, and vascularization [7
8
9
10
11
]. Although
T-lymphopoiesis is unaffected, B-lymphopoiesis and myelopoiesis are
impaired severely in these deficient mice. Myelopoiesis is decreased
quantitatively in fetal liver [10
] and virtually absent
in bone marrow [7
8
9
10
]. Although it is a C-X-C
chemokine, the role of SDF-1 in neutrophil migration and expression of
CXCR4 in these cells is uncertain, and contrasting studies have
appeared. Initial studies indicated lack of CXCR4 expression in
neutrophils [18
, 25
], and others found
significant levels of CXCR4 mRNA and functional expression on the
surface of neutrophils [16
, 19
,
20
, 24
, 26
, 27
].
Thus, although CXCR4 and SDF-1 have been demonstrated to regulate
myelopoiesis, the underlying mechanisms remain unclear. The ability of
promyelocytic human leukemia HL-60 cells [28
] to undergo
in vitro differentiation and acquire the phenotypic characteristics of
either mature neutrophils or monocytes offers a valuable model system
for comparative analysis of CXCR4 gene regulation and functions that
are specifically activated during different stages of differentiation
into the two hematopoietic pathways. HL-60 cells were committed to
become neutrophil-like upon induction with dimethylsulfoxide (DMSO) and
into monocytes following treatment with sodium butyrate and vitamin
D3. Under similar conditions of HL-60 differentiation, it
has been shown that cathepsin B and CD11b/CD18 (Mac1) mRNA levels are
increased and myeloperoxidase expression is markedly decreased
[29
, 30
]. In the present study, multiple
assays ranging from Northern blot and FACS analysis to assess CXCR4
mRNA and surface-antigen expression were deployed along with
experiments to measure SDF-1-mediated functional parameters such as
[35S]guanosine 5'-triphosphate (GTP)S binding, calcium
mobilization, and chemotaxis. The data provide evidence that CXCR4
expression and its correlation with functional responsiveness to SDF-1
are regulated in a complex manner that is selective for the type of
differentiation pathway involved and the inducing agent used.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and
CXCR4-specific 12G5 monoclonal antibody (mAb) were obtained from R & D
Systems (Minneapolis, MN). mAbs used for flow cytometry analysis were
purchased from Biosource International (Camarillo, CA).
[35S]GTPS (1250 Ci/mmol, Cat. No. NEG-03H) was
obtained from NEN/DuPont (Wilmington, DE). The promyelocytic, human HL-60 cell line was purchased from American Type Culture Collection (ATCC; Manassas, VA) and cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) and 2 mM glutamine. Cells were passaged after every 3 days. HL-60 cells were induced toward neutrophil granulocytic differentiation upon treatment with 1.25% DMSO (v/v) for the indicated time periods. HL-60 cells were differentiated toward the monocytic lineage by incubating with 1 mM sodium butyrate and 600 nM vitamin D3 for the indicated time periods.
Northern blot and flow cytometric analysis
Total RNA was extracted from control and differentiated HL-60
cells by using the acid phenol, single-extraction procedure with
TriReagent (Molecular Research Center, Inc., Cincinnati, OH). Total RNA
(20 µg/lane) was fractionated on 1.2% agarose formaldehyde gels.
After transfer to a nylon membrane, RNA was linked covalently with a UV
cross-linker (Stratagene Inc., La Jolla, CA). For Northern analysis, a
515-bp CXCR4 cDNA probe [21
] was used under
high-stringency hybridization conditions. The glyceraldehyde
3-phosphate dehydrogenase (GAPDH) gene probe (Clontech, Palo Alto, CA)
was used to normalize data for RNA sample differences.
Cell-surface expression of CXCR4 on undifferentiated HL-60 cells and following differentiation toward granulocytic and monocytic lineages were determined by flow cytometric analysis as previously described [31 ]. Cells were incubated on ice for 30 min with the primary CXCR4-specific 12G5 mAb; washed twice with ice-cold phosphate-buffered saline (PBS) and 0.1% bovine serum albumin (BSA); and labeled with isotype-matched second-stage fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) from Biosource International. Cells were subjected to fluorescein-activated cell sorter (FACS) analysis with a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). Intercellular adhesion molecule (ICAM)-specific antibodies were used as independent control for cell differentiation.
SDF-1-mediated [35S]GTPS binding assay
HL-60 cell membranes were prepared from cell pellets homogenized
in buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2,
and 1 mM ethylenediaminetetraacetate (EDTA) with a dounce
homogenizer. Homogenates were freeze-thawed, and then protein
concentrations were measured using the Bradford method. Assays were
carried out in buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM
MgCl2, and 1 mM EDTA. Agonist (SDF-1), 5 µM guanosine
5'-diphosphate (GDP), 30 mM NaCl, and 30 µM GTP (for measurement of
nonspecific binding) were added, followed by the addition of 25 µg
membrane protein and 970 pM [35S]GTPS. Final reaction
volume was 120 µL, and incubation took place for 60 min at 25°C.
Assays were terminated by the addition of 3 mL ice-cold 0.9% NaCl
followed by rapid filtration through Skatron GF filter membranes
(Skatron Instruments, Inc., Sterling, VA) with a Skatron 12-tube cell
washer. Filters were immersed in scintillant and counted on an LS
6000TA beta-counter (Beckman Instruments, Inc., Fullerton, CA).
Calcium mobilization assay
For measurements of intracellular calcium, HL-60 cells were
loaded for 30 min with 2 µM fura-2/acetoxymethylester (AM;
Molecular Probes, Eugene, OR). Cells were centrifuged and resuspended
into fresh-growth medium for 15 min and then centrifuged and
resuspended into Krebs-Ringer Henseleit (KRH) buffer, pH 7.4,
containing 0.1% gelatin. Cells were stored on ice at a concentration
of 20 x 106 cells/ml and diluted for use 1:10 with
fresh KRH buffer at 37°C. Fura-2-induced fluorescence of cells was
measured with a University of Pennsylvania Biomedical Instruments Group
dual-channel fluorometer. Data were captured with the aid of a PC,
running the Lab Windows application (National Instruments, Austin, TX),
and analyzed by Igor version 1.28 software (WaveMetrics, Lake Oswego,
OR). SDF-1 was added from 20-µM stocks in water.
Cell migration assay
HL-60 cell migration was performed as demonstrated earlier
[31
]. Briefly, 5 x 105 control or
differentiated HL-60 cells were added (suspended in RPMI-1640 with
0.25% BSA) in the top well of a 6.5-mm diameter, 5-µM pore
polycarbonate Transwell culture insert (Costar, Cambridge, MA). Cells
were incubated for migration at 37°C in 5% CO2 for
4 h. After incubation, migrated cells in the lower chamber were
counted with a ZM Coulter counter (Coulter Diagnostics, Hialeah, FL).
Percent migration was based on the total initial input cells per well.
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 View larger version (50K): [in a new window] |
Figure 1. Kinetics of CXCR4 transcriptional regulation during granulocytic
differentiation of HL-60 cells, which were induced to differentiate
toward neutrophilic lineage by treatment with DMSO (1.25% v/v) for the
indicated time periods. Total RNA was prepared for Northern blot
analysis. The mRNA units measure the ratio of signal intensity from
densitometric readings after normalization with the GAPDH probe
(n=3). Note the significant, time-dependent modulation of
CXCR4 expression (*, P<0.05 at 99% confidence interval,
according to the two-tailed t-test) in DMSO-treated cells
compared with the undifferentiated controls.
|
 View larger version (48K): [in a new window] |
Figure 2. Biphasic regulation of CXCR4 mRNA expression during monocytic
differentiation of HL-60 cells. Logarithmic-phase HL-60 cells were
treated with 1 mM sodium butyrate for the indicated time periods, and
total RNA was prepared for Northern blot analysis. Note the initial,
rapid up-regulation of CXCR4 mRNA levels within 0.5–1 h of treatment.
The data were analyzed for statistical significance (*,
P<0.05 at 99% confidence interval) with the GraphPad Prism
software two-tailed t-test. The mRNA units measure the ratio
of signal intensity from densitometric readings after normalization
with the GAPDH probe (n=3).
|
 View larger version (21K): [in a new window] |
Figure 3. Effect of DMSO-induced, granulocytic differentiation on surface
expression of CXCR4 in HL-60 cells. CXCR4 surface expression was
measured by FACS analysis with the CXCR4-specific 12G5 mAb. The shift
in the mean fluorescence values of HL-60 cells specifically stained
with 12G5 is indicative of levels of CXCR4-receptor surface expression.
The data for mean fluorescence values (n=2) are summarized
in Table 1
. A distinct shift toward increased CXCR4 expression is
evident after 24 h of treatment with DMSO. An anti-ICAM antibody
was used as a positive control.
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View this table: [in a new window] |
Table 1. HL-60 Differentiation
|
 View larger version (42K): [in a new window] |
Figure 4. Effect of monocytic differentiation on surface expression of
CXCR4 in HL-60 cells. HL-60 cells were incubated in the presence of 1
mM sodium butyrate and 600 nM vitamin D3 for the indicated
time periods. CXCR4 surface expression was measured by FACS analysis
with the CXCR4-specific 12G5 mAb. The shift in the mean fluorescence
values of HL-60 cells specifically stained with 12G5 is indicative of
levels of CXCR4-receptor surface expression. The data for mean
fluorescence values (n=2) are summarized in Table 1
. Note
the divergent effects of sodium butyrate (up-regulation) and vitamin
D3 (down-regulation) treatments with time on CXCR4 surface
expression. An anti-ICAM antibody was used as a positive control.
|
S binding in control and
differentiated HL-60 cellsS binding assay was used to evaluate
the effect of differentiation-mediated modulation of CXCR4 expression
on the CXCR4 receptor-G-protein-coupling. This corresponds to the first
step of the intracellular-activation cascade and directly reflects
ligand-binding events at the G-protein-coupled receptor itself
[32
33
34
]. Membranes were prepared from HL-60 cells after
treatment with different agents and were used to measure changes in
SDF-1-mediated [35S]GTPS binding. Initially,
experiments were done to work out the optimal condition for
SDF-1-activated, specific [35S]GTPS binding.
SDF-1 at 100 nM increased [35S]GTPS binding by a
maximal 21.7% (35.95 fmol [35S]GTPS bound per mg
membrane protein) over basal (Fig. 5
) in control, undifferentiated HL-60 cells. Compared with controls,
the HL-60 cells treated with DMSO, sodium butyrate, and vitamin
D3 increased SDF-1 (100 nM)-mediated
[35S]GTPS binding by threefold to fourfold. Basal
[35S]GTPS binding was not altered significantly under
these conditions. The data show that the functional coupling of CXCR4
receptors with ligand-specific G-proteins is stimulated to a similar
extent during differentiation of HL-60 cells into either the
granulocytic or monocytic pathways.
 View larger version (17K): [in a new window] |
Figure 5. Effect of differentiation inducers on agonist (SDF-1)-mediated
stimulation of [35S]GTPS binding to CXCR4 in HL-60
cells. HL-60 cell membranes were prepared from control cells, following
their treatment with DMSO (1.25% v/v), sodium butyrate (1 mM), and
vitamin D3 (600 nM) for 24 h. SDF-1-induced
[35S]GTPS binding was assayed as described in
Materials and Methods. The data are expressed as fold-change in
specific SDF-1-induced [35S]GTPS binding over
untreated, control HL-60 cells. The data (n=3) were analyzed
for statistical significance (*, P<0.05 at 99% confidence
interval) with the GraphPad Prism software two-tailed
t-test.
|
was
measured using FURA-2 fluorescence. In control cells, 10 nM SDF-1
stimulated intracellular Ca+2 flux by 200 nM.
However, in sharp contrast to the uniform increase of SDF-1-mediated
[35S]GTPS binding, we noted heterogeneity in the
calcium response of these differentiated HL-60 cells (Fig. 6
). In cells pretreated with DMSO for 24 h, calcium
mobilization in response to 10 nM SDF-1 was significantly attenuated
by more than twofold. On the other hand, SDF-1-mediated calcium
mobilization was induced by more than twofold in cells differentiated
into the monocytic pathway with both sodium butyrate and vitamin
D3. Identical results were obtained when 100 nM SDF-1
was used (unpublished results), a concentration known to give maximal,
functional response in HL-60 cells [31
]. These data
indicate that SDF-1-activated Ca+2
mobilization is potentiated during monocytic differentiation of HL-60
cells by butyrate or vitamin D3 treatments. Furthermore,
the results also suggest that calcium mobilization may not be the
principle signal transduction for CXCR4 receptors in granulocytic HL-60
cells differentiated with DMSO.
 View larger version (17K): [in a new window] |
Figure 6. Modulation of SDF-1-mediated calcium flux in HL-60 cells during
differentiation. HL-60 cells were differentiated with DMSO (1.25%
v/v), sodium butyrate (1 mM), and vitamin D3 (600 nM) for
24 h. SDF-1 elicits transient elevation of intracellular
calcium in HL-60 cells. Note the significant and unexpected attenuation
of such functional response in DMSO-treated, granulocytic HL-60 cells
compared with the control and monocytic-differentiated HL-60 cells. The
data (n=3) were analyzed for statistical significance (*,
P<0.05 at 99% confidence interval) with the GraphPad Prism
software two-tailed t-test.
|
caused an efficacious
migration of control HL-60 cells (>40% input cells migrated with 100
nM SDF-1) in a concentration-dependent manner with an EC50
value of 50 nM (Figs. 7
and 8). As
described, HL-60 cells were treated separately with DMSO, sodium
butyrate, or vitamin D3 for the indicated time periods and
then assessed for SDF-1-mediated migration in a dose-dependent
manner. As shown in Figure 7
, DMSO induced a pronounced, time-related increase in the maximal
efficacy (>60% input cells migrated after 1-day treatment with DMSO)
and potency (EC50=14 nM) of the SDF-1-mediated chemotactic
response among granulocytic HL-60 cells. Furthermore, an increase in
the chemotactic response was maintained during the extended
differentiation with DMSO for up to 3 days, albeit with a decrease in
the efficacy after the second day.
 View larger version (33K): [in a new window] |
Figure 7. DMSO-induced granulocytic differentiation of HL-60 cells up-regulates
SDF-1-mediated chemotaxis. HL-60 cells were treated with DMSO
(1.25% v/v) for the indicated time periods. Indicated amounts of
SDF-1 were added in the lower chamber of 5 µM pore Transwell
filters, and HL-60 cells (5x105 input cells) added in the
upper chamber were subjected to chemotaxis. Cells that migrated to the
lower chamber after 4 h incubation were counted with a Coulter
counter. Note the significant shift in the dose response of
SDF-1-mediated migration with differentiated HL-60 cells relative to
control cells (n=2).
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 View larger version (37K): [in a new window] |
Figure 8. Monocytic differentiation modulates SDF-1-mediated chemotaxis of
HL-60 cells. HL-60 cells were induced to differentiate with sodium
butyrate (1 mM) and vitamin D3 (600 nM) for the indicated
time periods. Indicated amounts of SDF-1 were added in the lower
chamber of 5 µM pore Transwell filters, and HL-60 cells
(5x105 input cells) added in the upper chamber were
subjected to chemotaxis. Cells that migrated to the lower chamber after
4 h incubation were counted with a Coulter counter. Note the
significant, time-dependent, inhibitory effect of vitamin
D3 treatment on HL-60 cells chemotaxis. Conversely, sodium
butyrate was a powerful inducer of the chemotactic response in HL-60
cells (n=2).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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using three functional assays: chemokine-dependent GTPS binding,
calcium flux, and chemotaxis.
Data concerning expression of CXCR4 in human neutrophils are highly
controversial, and in line with earlier studies [18
,
25
], we also observed the absence of cell-surface CXCR4
on neutrophils by staining with 12G5 mAb and FITC-conjugated reagents.
However, our results using DMSO-mediated granulocytic differentiation
of HL-60 cells show induction of CXCR4 expression in a biphasic manner,
with a significant, time-dependent increase of 2.6-fold in mRNA levels
and 1.6-fold in surface-receptor expression measured as mean
fluorescence index after the maximal 3-day treatment (Figs. 1
and 3
and
Table 1
). Furthermore, CXCR4 receptors expressed on
DMSO-differentiated, granulocytic HL-60 cells are functionally coupled
to G-proteins, as is shown by the threefold to fourfold increase in
SDF-1-mediated [35S]GTPS binding over control cells
(Fig. 5)
. The GDP-GTP exchange reaction at the level of G-proteins is
the primary reaction in the signaling pathway of G-protein-coupled
receptors and leads to the final cellular and tissue responses.
Therefore, this result lends strong support to evidence of high CXCR4
expression in neutrophil granulocytes along with their functional
responsiveness [16
, 19
, 20
,
24
, 26
, 27
]. Certainly, the
marked shift in the potency and efficacy of the chemotactic response to
SDF-1 seen with HL-60 cells after DMSO treatment (especially pronounced
after 1-day treatment; Fig. 7
) is also consistent with its role as an
efficient chemoattractant for granulocytes. Indeed, SDF-1 and its
chemically synthesized analogue N33A have been shown to induce
migration of neutrophils in a dose-dependent manner
[19
]. The differential attenuation of calcium-flux
response seen with DMSO here, although paradoxical, is also noteworthy
and underscores the dissociation of SDF-1-mediated, G-protein coupling
and signal-transduction pathways involved in calcium mobilization when
compared with chemotaxis.
In the present study, two known inducers of HL-60 monocytic
differentiation were also comprehensively studied: sodium butyrate and
vitamin D3. Of these, sodium butyrate had a uniquely
positive influence on CXCR4 expression and function. Sodium butyrate is
a short-chain, fatty acid and fermentation product of dietary fiber. It
caused up-regulation in all parameters of CXCR4 expression and
functional responsiveness studied (SDF-1-mediated
[35S]GTPS binding, calcium flux, and chemotaxis) in a
time-dependent manner. This is surprising, because in the case of
colonic epithelium HT-29 cells, which also differentiate upon treatment
with sodium butyrate, CXCR4 mRNA expression was shown to be completely
inhibited upon treatment [36
]. In the case of HL-60
cells, sodium butyrate may stimulate CXCR4 mRNA levels directly,
because it is known to enhance transcription via discrete, regulatory
elements [37
, 38
]. However, insofar as the
up-stream promoter region of the CXCR4 gene is concerned
[39
], the existence and role of discrete butyrate
response elements are not apparent. Moreover, the increased functional
coupling of CXCR4 to G-proteins as indicated by the induction of
[35S]GTPS binding (Fig. 5)
also most likely reveals a
direct action of sodium butyrate on the signal-transduction process.
The effect of vitamin D3 was equally complex because we
failed to detect any measurable changes in CXCR4 mRNA (unpublished
results). Vitamin D3 may act by binding to its receptor
known as VDR, a member of the steroid/nuclear-receptor family of
transcription factors expressed in HL-60 cells [40
].
However, although CXCR4 surface expression was decreased upon treatment
with vitamin D3, it enhanced the functional G-protein
coupling of receptors in HL-60 cells as demonstrated by the increase in
SDF-1-mediated [35S]GTPS binding (Fig. 5)
. Moreover,
similar to the differential activation of distinct, signal-transduction
pathways seen with DMSO treatment, vitamin D3 selectively
stimulated the calcium response to SDF-1, even as the chemotactic
ability of treated HL-60 cells was attenuated. In our studies, the
differences in the kinetics of CXCR4 expression and function between
sodium butyrate- and vitamin D3 (Figs. 2
4
and 8)
-differentiated, HL-60 monocytic cells may be attributed to the
different stages of myeloid differentiation accomplished with these
specific, inducing agents [28
].
In summary, we observed a lineage- and differentiation-inducing,
agent-dependent lack of correlation between CXCR4 expression and
SDF-1-mediated functional responsiveness measured by
[35S]GTPS binding, calcium flux, and chemotaxis.
Although, the G-protein-usage requirements for activating chemotaxis
versus calcium mobilization are presently unknown, our observation of
the selective uncoupling of these two ligand-stimulated, functional
responses reinforces the emerging notion that chemoattractant receptors
may activate distinct G-protein signaling pathways [41
]
in hematopoietic cells undergoing differentiation. Such an asymmetrical
response to modulation of CXCR4 function during differentiation may
also reflect on the homing pathways involved in hematopoiesis and
ultimately impact the differential recruitment of leukocyte subsets to
target sites in vascular tissues.
S binding assay. We also acknowledge Dr. John
White for critical discussions during preparation of this manuscript. Received December 10, 2000; revised April 25, 2001; accepted April 26, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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analogue, N33A, is a potent chemotactic agent for CXCR4/fusin/LESTR-expressing human leukocytes J. Biol. Chem 272,24966-24970 functional activity during differentiation of human monocytes and macrophages J. Leukoc. Biol 66,135-143[Abstract]S binding in the membranes of Spodoptera frugiperda insect cells expressing the human Di3 dopamine receptor Neurosci. Lett 226,91-94[Medline]-thio]triphosphate-[35S]binding J. Pharmacol. Exp. Ther 285,119-126This article has been cited by other articles:
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