Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gupta, S. K.
Right arrow Articles by Aiyar, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gupta, S. K.
Right arrow Articles by Aiyar, N.
(Journal of Leukocyte Biology. 2001;70:431-438.)
© 2001 by Society for Leukocyte Biology

CXCR4 undergoes complex lineage and inducing agent-dependent dissociation of expression and functional responsiveness to SDF-1{alpha} during myeloid differentiation

Shalley K. Gupta, Kodandaram Pillarisetti and Nambi Aiyar

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CXC chemokine SDF-1 and its receptor CXCR4 mediate myelopoiesis, presumably by regulating the homing of hematopoietic progenitor cells. We used the inducible HL-60 cell line as a model system for comparative analysis of CXCR4 expression during differential maturation into the granulocytic or monocytic phenotypes. Five different measures of CXCR4 expression and functional coupling: mRNA and surface expression, SDF-1-mediated [35S]GTP{gamma}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]GTP{gamma}S 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]GTP{gamma}S 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular mechanisms that regulate hematopoietic progenitor cell homing and terminal differentiation into granulocytic or monocytic lineages are not fully characterized [1 , 2 ]. These precursor cells communicate with stromal cells by chemoattraction, adhesion, and cytokine secretion [3 ], eventually leading to the production of mature granulocytes, T-cells, B-cells, monocytes, and antigen-presenting macrophages. Stromal cells secrete various soluble mediators [4 ], such as stem-cell factor, interleukin-3 (IL-3), IL-7, granuloctye colony-stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF, which are essential for growth and differentiation of these precursors. The importance of chemokines and their receptors in mediating these events has also been established recently [5 ], although poorly understood.

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){gamma}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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
DMSO and sodium butyrate were from Sigma Chemical Co. (St. Louis, MO), and vitamin D3 was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Recombinant SDF-1{alpha}- 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]GTP{gamma}S (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]GTP{gamma}S 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]GTP{gamma}S. 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{alpha} 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CXCR4 transcription is regulated in a sinusoidal manner during granulocytic and monocytic differentiation of HL-60 cells
To help understand the kinetics of differentiation-related regulation of CXCR4, as an initial measure, we performed Northern blot analysis of total RNA prepared from HL-60 cells induced with DMSO, sodium butyrate, and vitamin D3 for the indicated time periods. HL-60 cells differentiate in the presence of DMSO to manifest the nuclear and cytoplasmic characteristics of neutrophilic granulocytes; a monocytic phenotype is conferred upon growth in the presence of sodium butyrate and vitamin D3 [1 , 2 ]. As shown in Figures 1 and 2, expression of CXCR4 mRNA was modulated in a sinusoidal manner during the course of HL-60 cells differentiation into both pathways. Specifically, DMSO-induced granulocytic differentiation of HL-60 cells was marked by an initial fourfold decrease in steady-state levels of CXCR4 transcripts within 3 h. However, CXCR4 mRNA levels were restored steadily upon prolonged incubation with DMSO and subsequently increased to more than twofold above those seen in control undifferentiated HL-60 cells (Fig. 1) .



View larger version (50K):
[in this window]
[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.

 
The differentiation of HL-60 cells along the monocytic lineage also had a profound impact on CXCR4 transcription in a time-dependent manner. In this case, with sodium butyrate, there was an initial, rapid twofold up-regulation of CXCR4 mRNA levels that peaked in 30–60 min (Fig. 2) , followed by a decline to pretreatment levels within 3–5 h. A marked fourfold to sixfold induction in steady-state levels of CXCR4 mRNA was observed in HL-60 cells treated with sodium butyrate for the longer duration of 2–3 days. In contrast, treatment with vitamin D3 failed to modulate CXCR4 mRNA expression in HL-60 cells in a significant manner (unpublished results).



View larger version (48K):
[in this window]
[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).

 
Regulation of CXCR4 surface expression during the differentiation of HL-60 cells
To correlate the modulation of CXCR4 transcription with surface expression during HL-60 cells differentiation over the 3-day period, we next evaluated CXCR4 expression with flow cytometric analysis using the specific, anti-CXCR4 mAb 12G5 [31 ]. As shown in Figures 3 and 4, control, undifferentiated HL-60 cells demonstrated robust CXCR4 expression on their cell surface. A comparison of the mean fluorescence values between control and DMSO-treated, granulocytic HL-60 cells (Fig. 3 and Table 1 ) revealed that following an initial decline within 5 h, steady-state levels of CXCR4 surface expression recovered, were induced in a time-dependent manner similar to the regulation of its mRNA levels, and peaked with a 59% increase after 3 days. Significantly, in parallel experiments, however, we were unable to detect CXCR4 surface expression in peripheral blood neutrophils (unpublished results), an observation that is indicative of the highly variable CXCR4 expression among circulating polymorphonuclear neutrophils (PMNs) [16 , 18 19 20 , 24 25 26 27 ].



View larger version (21K):
[in this window]
[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.

 

View this table:
[in this window]
[in a new window]
  		Table 1. HL-60 Differentiation

 
In the case of monocytic differentiation, the effect of sodium butyrate followed an identical pattern to that observed with DMSO-treated cells, with a slight, initial decline at 3 h, followed by a time-dependent increase of up to 49.2% after 3 days (Fig. 4 and Table 1 ). In contrast, vitamin D3 treatment caused a significant down-regulation (up to 38.4%) in CXCR4 surface expression after 3 days (Fig. 4 and Table 1 ).



View larger version (42K):
[in this window]
[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.

 
SDF-1 activated [35S]GTP{gamma}S binding in control and differentiated HL-60 cells
The [35S]GTP{gamma}S 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{alpha}-mediated [35S]GTP{gamma}S binding. Initially, experiments were done to work out the optimal condition for SDF-1{alpha}-activated, specific [35S]GTP{gamma}S binding. SDF-1{alpha} at 100 nM increased [35S]GTP{gamma}S binding by a maximal 21.7% (35.95 fmol [35S]GTP{gamma}S 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{alpha} (100 nM)-mediated [35S]GTP{gamma}S binding by threefold to fourfold. Basal [35S]GTP{gamma}S 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 this window]
[in a new window]
  		Figure 5. Effect of differentiation inducers on agonist (SDF-1{alpha})-mediated stimulation of [35S]GTP{gamma}S 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{alpha}-induced [35S]GTP{gamma}S binding was assayed as described in Materials and Methods. The data are expressed as fold-change in specific SDF-1{alpha}-induced [35S]GTP{gamma}S 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.

 
SDF-1-mediated calcium mobilization in control and differentiated HL-60 cells
To further study the modulation of the CXCR4-activated signal-transduction pathway during the differentiation of HL-60 cells, mobilization of intracellular calcium in response to SDF-1{alpha} was measured using FURA-2 fluorescence. In control cells, 10 nM SDF-1{alpha} stimulated intracellular Ca+2 flux by 200 nM. However, in sharp contrast to the uniform increase of SDF-1{alpha}-mediated [35S]GTP{gamma}S 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{alpha} was significantly attenuated by more than twofold. On the other hand, SDF-1{alpha}-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{alpha} was used (unpublished results), a concentration known to give maximal, functional response in HL-60 cells [31 ]. These data indicate that SDF-1{alpha}-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 this window]
[in a new window]
  		Figure 6. Modulation of SDF-1{alpha}-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{alpha} 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.

 
SDF-1 mediated chemotactic response in control and differentiated HL-60 cells
Given the fundamental role of SDF-1 in chemotaxis, we next determined whether the modulation of early signal transduction events during HL-60 differentiation had a functional impact on subsequent cell migration. In the present study, SDF-1{alpha} 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{alpha}-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 this window]
[in a new window]
  		Figure 7. DMSO-induced granulocytic differentiation of HL-60 cells up-regulates SDF-1{alpha}-mediated chemotaxis. HL-60 cells were treated with DMSO (1.25% v/v) for the indicated time periods. Indicated amounts of SDF-1{alpha} 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{alpha}-mediated migration with differentiated HL-60 cells relative to control cells (n=2).

 
Maturation of HL-60 into monocyte-like cells by sodium butyrate also significantly enhanced the migration efficacy (>65% input cells migrated) and potency (EC50=5.5 nM) of SDF-1 in a sustained, time-dependent manner, with maximum effect seen from the 1-day timepoint onward (Fig. 8) . However, in sharp contrast, vitamin D3 exhibited a distinct, time-dependent, negative impact on the efficacy (only 25% input cells migrated) and potency (EC50=174 nM) after 3 days of treatment on SDF-1-induced HL-60 migration.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 8. Monocytic differentiation modulates SDF-1{alpha}-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{alpha} 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).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In contrast with other chemokines that are released from cells upon stimulation with pro-inflammatory cytokines, SDF-1 and CXCR4 are constitutively expressed in a variety of vascular tissues and leukocyte subsets [15 , 35 ]. Furthermore, SDF-1 mRNA is known to be refractory to stimulation with potent inflammatory mediators such as lipopolysaccharide (LPS) in bone-marrow, stromal cell lines [35 ]. This atypical property of SDF-1 led to the suggestion that rather than inflammation, it is involved in leukocyte recirculation and the homing of T-cells and monocytes [18 ] during hematopoiesis. To clarify this role of SDF-1, it is important to analyze the regulation of CXCR4 expression and functional response at various stages of cell maturation during differentiation. We have shown previously that CXCR4 transcription and functional activity are modulated during the differentiation of human monocytes into macrophages and foam cells in the presence of GM-CSF and oxidized low-density lipoprotein (Ox-LDL) [31 ]. In these studies, after an initial rapid decline, CXCR4 was shown to be transcriptionally and functionally re-expressed in mature macrophages. In the present study, we have made a detailed comparison of CXCR4 expression in an HL-60 cell-line model of granulocyte and monocyte differentiation and simultaneously correlated differential CXCR4 expression (Northern and FACS analysis) with responsiveness to SDF-1{alpha} using three functional assays: chemokine-dependent GTP{gamma}S 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]GTP{gamma}S 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]GTP{gamma}S 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]GTP{gamma}S 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]GTP{gamma}S 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{alpha}, 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{alpha}-mediated functional responsiveness measured by [35S]GTP{gamma}S 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.


   ACKNOWLEDGEMENTS
 
We thank Roberta Thomas for help with the [35S]GTP{gamma}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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tenen, D. G., Hormas, R., Licht, J. D., Zhang, D-E. (1997) Transcription factors, normal myeloid development, and leukemia Blood 90,489-519[Free Full Text]
  2. Berliner, N. (1998) Molecular biology of neutrophil differentiation Curr. Opin. Hematol 5,49-53[Medline]
  3. Melchers, F., Rolink, A. G., Schaniel, C. (1999) The role of chemokines in regulating cell migration during humoral immune responses Cell 99,351-354[Medline]
  4. Dorshkind, K. (1990) Regulation of hemopoiesis by bone marrow stromal cells and their products Annu. Rev. Immunol 8,111-137[Medline]
  5. Zlotnik, A., Morales, J., Hedrick, J. A. (1999) Recent advances in chemokines and chemokine receptors Crit. Rev. Immunol 19,1-47[Medline]
  6. Rollins, B. J. (1997) Chemokines Blood 90,909-928[Free Full Text]
  7. Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S., Kitamura, Y., Yoshida, N., Kikutani, H., Kishimoto, T. (1996) Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1 Nature 382,635-638[Medline]
  8. Tachibana, K., Hirota, S., Iizasa, H., Yoshida, H., Kawabata, K., Kataoka, Y., Kitamura, Y., Matsushima, K., Yoshida, N., Nishikawa, S., Kishimoto, T., Nagasawa, T. (1998) The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract Nature 393,591-594[Medline]
  9. Zou, Y-R., Kottman, A. H., Kuroda, M., Taniuchi, I., Littman, D. R. (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development Nature 393,595-599[Medline]
  10. Ma, Q., Jones, D., Borghesan, P. R., Segal, R. A., Nagasawa, T., Kishimoto, T., Bronson, R. T., Springer, T. A. (1998) Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice Proc. Natl. Acad. Sci. USA 95,9448-9453[Abstract/Free Full Text]
  11. Ma, Q., Jones, D., Springer, T. A. (1999) The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment Immunity 10,463-471[Medline]
  12. Cacalano, C., Lee, J., Kikly, K., Ryan, A. M., Pitts-Meek, S., Hultgren, B., Wood, W. I., Moore, M. W. (1994) Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog Science 265,682-684[Abstract/Free Full Text]
  13. Forster, R., Mattis, A. E., Kremmer, E., Wolf, E., Brem, G., Lipp, M. (1996) A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen Cell 87,1037-1047[Medline]
  14. Rothenberg, M. E., MacLean, J. A., Pearlman, E., Luster, A. D., Leder, P. (1997) Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia J. Exp. Med 185,785-790[Abstract/Free Full Text]
  15. Nagasawa, T., Kikutani, H., Kishimoto, T. (1994) Molecular cloning and structure of a pre-B-cell growth-stimulating factor Proc. Natl. Acad. Sci. USA 91,2305-2309[Abstract/Free Full Text]
  16. Loetscher, M., Geiser, T., O’Reilly, T., Zwahlen, R., Baggiolini, M., Moser, B. (1994) Cloning of a human seven-transmembrane receptor, LESTR, that is highly expressed in leukocytes J. Biol. Chem 269,232-237[Abstract/Free Full Text]
  17. Oberlin, E., Amara, A., Bachelerie, F., Bessia, C., Virelizier, J-L., Arenzan-Seisdedos, F., Schwartz, O., Heard, J-L., Clark-Lewis, I., Legler, D. F., Loetscher, M., Baggiolini, M., Moser, B. (1996) The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell line-adapted HIV-1 Nature 382,833-835[Medline]
  18. Bleul, C. C., Fuhlbrigge, R. C., Casanovas, J. M., Aiuti, A., Springer, T. A. (1996) A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1) J. Exp. Med 184,1101-1109[Abstract/Free Full Text]
  19. Ueda, H., Siani, M. A., Gong, W., Thompson, D. A., Brown, G. G., Wang, J. M. (1997) Chemically synthesized SDF-1{alpha} analogue, N33A, is a potent chemotactic agent for CXCR4/fusin/LESTR-expressing human leukocytes J. Biol. Chem 272,24966-24970[Abstract/Free Full Text]
  20. Forster, R., Kremmer, E., Schubel, A., Breitfeld, D., Kleinschmidt, A., Nerl, C., Bernhardt, G., Lipp, M. (1998) Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusin on various leukocyte subsets: rapid internalization and recycling upon activation J. Immunol 160,1522-1531[Abstract/Free Full Text]
  21. Gupta, S. K., Lysko, P. G., Pillarisetti, K., Ohlstein, E., Stadel, J. M. (1998) Chemokine receptors in endothelial cells. Functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines J. Biol. Chem 273,3282-4287
  22. Aiuti, A., Webb, I. J., Bleul, C., Springer, T. A., Gutierrez-Ramos, J. C. (1997) The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood J. Exp. Med 185,111-120[Abstract/Free Full Text]
  23. Honczarenko, M., Douglas, R. S., Mathias, C., Lee, B., Ratajczak, M. Z., Silberstein, L. E. (1999) SDF-1 responsiveness does not correlate with CXCR4 expression levels of developing human bone marrow B cells Blood 94,2990-2998[Abstract/Free Full Text]
  24. Kowalska, M. A., Ratajczak, J., Hoxie, J., Brass, L. F., Gewirtz, A., Poncz, M. (1999) Megakaryocyte precursors, megakaryocytes and platelets express the HIV co-receptor CXCR4 on their surface: determination of response to stromal-derived factor-1 by megakaryocytes and platelets Br. J. Haematol 104,220-229[Medline]
  25. Hori, T., Sakaida, H., Sato, A., Nakajima, T., Shida, H., Yoshie, O., Uchiyama, T. (1998) Detection and delineation of CXCR-4 (fusin) as an entry and fusion cofactor for T cell-tropic HIV-1 by three different monoclonal antibodies J. Immunol 160,180-188[Abstract/Free Full Text]
  26. Heesen, M., Berman, M. A., Benson, J. D., Gerard, C., Dorf, M. E. (1996) Cloning of the mouse fusin gene, homologue to a human HIV-1 co-factor J. Immunol 157,5455-5460[Abstract]
  27. Nomura, H., Nielsen, B. W., Matsushima, K. (1993) Molecular cloning of cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide receptors Int. Immunol 5,1239-1249[Abstract/Free Full Text]
  28. Collins, S. J. (1987) The HL60 promyelocytic leukemia cell line: proliferation, differentiation and cellular oncogene expression Blood 70,1223-1244
  29. Rosmarin, A. G., Weil, S. C., Rosner, G. L., Arnaout, M. A., Tenen, D. G. (1989) Differential expression of CD11b/CD18 (Mo1) and myeloperoxidase genes during myeloid differentiation Blood 73,131-136[Abstract/Free Full Text]
  30. Berquin, I. M., Yan, S., Katiyar, K., Huang, L., Sloane, B. F., Troen, B. R. (1999) Differentiating agents regulate cathepsin B gene expression in HL-60 cells J. Leukoc. Biol 66,609-616[Abstract]
  31. Gupta, S. K., Pillarisetti, K., Lysko, P. G. (1999) Modulation of CXCR4 expression and SDF-1{alpha} functional activity during differentiation of human monocytes and macrophages J. Leukoc. Biol 66,135-143[Abstract]
  32. Pregenzer, J. F., Alberts, G. L., Im, W. B. (1997) Agonist-induced [35S]-GTP{gamma}S binding in the membranes of Spodoptera frugiperda insect cells expressing the human Di3 dopamine receptor Neurosci. Lett 226,91-94[Medline]
  33. Malmberg, A., Mikaels, A., Mohell, N. (1998) Agonist and inverse agonist activity at the dopamine Di3 receptor measured by guanosine 5'-[{gamma}-thio]triphosphate-[35S]binding J. Pharmacol. Exp. Ther 285,119-126[Abstract/Free Full Text]
  34. Newman-Tancredi, A., Cussac, D., Audinot, V., Pasteau, V., Gavaudan, S., Millan, M. J. (1999) G protein activation by human dopamine D3 receptors in high-expressing Chinese hamster ovary cells: a guanosine-5'-O-(3-[35S]thio)-triphosphate binding and antibody study Mol. Pharmacol 55,564-574[Abstract/Free Full Text]
  35. Shirozu, M., Nakano, T., Inazawa, J., Tashiro, K., Tada, H., Shinohara, T., Honjo, T. (1995) Structure and chromosomal localization of the human stromal cell-derived factor1 (SDF1) gene Genomics 28,495-500[Medline]
  36. Jordan, N. J., Kolios, G., Abbot, S. E., Siani, M. A., Thompson, D. A., Petraki, K., Westwick, J. (1999) Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells J. Clin. Investig 104,1061-1069[Medline]
  37. Fregeau, C. J., Helgason, C. D., Bleackley, R. C. (1992) Two cytotoxic cell proteinase genes are differentially sensitive to sodium butyrate Nucleic Acids Res 20,3113-3119[Abstract/Free Full Text]
  38. Park, C., Chamberlin, M. E., Pan, C. J., Chou, J. Y. (1996) Differential expression and butyrate response of human alkaline phosphatase genes are mediated by upstream DNA elements Biochemistry 35,9807-9814[Medline]
  39. Moriuchi, M., Moriuchi, H., Turner, W., Fauci, A. (1997) Cloning and analysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry J. Immunol 159,4322-4329[Abstract]
  40. Pan, P., Reddy, K., Lee, S., Studzinski, G. P. (1991) Differentiation-related regulation of 1, 25-dihydroxyvitamin D3 receptor mRNA in human leukaemia cells HL-60 Cell Prolif 24,159-170[Medline]
  41. Haribabu, B., Zhelev, D., Pridgen, B. C., Richardson, R. M., Ali, H., Snyderman, R. (1999) Chemoattractant receptors activate distinct pathways for chemotaxis and secretion: role of G protein usage J. Biol. Chem 274,37087-37092[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Virol.Home page
G. Ghaffari, D. L. Tuttle, D. Briggs, B. R. Burkhardt, D. Bhatt, W. A. Andiman, J. W. Sleasman, and M. M. Goodenow
Complex Determinants in Human Immunodeficiency Virus Type 1 Envelope gp120 Mediate CXCR4-Dependent Infection of Macrophages
J. Virol., November 1, 2005; 79(21): 13250 - 13261.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. J. C. Rombouts, B. Pavic, B. Lowenberg, and R. E. Ploemacher
Relation between CXCR-4 expression, Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia
Blood, July 15, 2004; 104(2): 550 - 557.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. T. Suratt, J. M. Petty, S. K. Young, K. C. Malcolm, J. G. Lieber, J. A. Nick, J.-A. Gonzalo, P. M. Henson, and G. S. Worthen
Role of the CXCR4/SDF-1 chemokine axis in circulating neutrophil homeostasis
Blood, July 15, 2004; 104(2): 565 - 571.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gupta, S. K.
Right arrow Articles by Aiyar, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gupta, S. K.
Right arrow Articles by Aiyar, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS