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a Department of Pathology & Laboratory Medicine;
b Department of Internal Medicine, Division of Hematology/Oncology, University of Pennsylvania School of Medicine;
c Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
Key Words. Hematopoiesis • Chemokines • Chemokine receptors • CXCR4 • CCR5 • Stromal derived factor-1 (SDF-1) • HIV
Correspondence:
Mariusz Z. Ratajczak, M.D., Ph.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania, 405A Stellar Chance Labs, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. e-mail: mariusz{at}mail.med.upenn.edu
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Abstract |
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[MIP-1], MIP-1ß, and RANTES) did not activate calcium flux, MAPK and AKT phosphorylation or chemotaxis of CD34+KIT+ cells or cells isolated from the BFU-E colonies. Interestingly, none of the chemokines tested in this study had any effect on BFU-E colony formation. In conclusion, only CXCR4 is functional, and its specific ligand SDF-1 may therefore play an important role in the homing and/or retention of early erythroid precursors in the bone marrow environment.
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Introduction |
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The discovery that some chemokine receptors are coreceptors for HIV entry into cells offers new avenues for increasing our understanding of the mechanisms underlying HIV-1-associated marrow dysfunction [1, 4]. All HIV-1 strains studied to date use CCR5 (R5 strains), CXCR4 (X4) or both receptors (R5X4) to enter cells [1, 4]. It has been found that the CCR5 receptor binds macrophage inflammatory protein-1 (MIP-1), MIP-1ß, and RANTES, whereas the CXCR4 receptor binds stromal cell-derived factor-1 (SDF-1).
We previously reported that human erythroid progenitors and erythroblasts cannot be infected by HIV-1 envelope pseudotyped XR and R5 viruses and suggested that other indirect mechanisms are responsible for the pathogenesis of HIV-related anemia [7]. Since many different chemokines are elaborated in the body during HIV-1 infection [1, 2, 6] and some of these chemokines have been reported to inhibit erythropoiesis [8-10], we became interested in their potential role in the pathogenesis of HIV-related anemia. We approached this question from different points of view by: A) looking at the expression of chemokine receptors both on human CD34+ cells which include erythroid progenitors and on more mature erythroid cells; B) evaluating the functionality of the chemokine receptors in chemotaxis, mitogen-activated protein kinase (MAPK) p42/44 and AKT phosphorylation and calcium flux studies, and finally by C) studying the effect of selected chemokines on in vitro proliferation of human BFU-E progenitors.
We found that from all the chemokines tested, only SDF-1 induced calcium flux, MAPK p42/44, and AKT phosphorylation and chemotaxis in early human erythroid precursor cells. Therefore, as determined by the biological assays employed in this study, only CXCR4 appeared to be a functional chemokine receptor in cells of this lineage and SDF-1 may play an important role in the retention of early erythroid precursors in the bone marrow (BM) environment [11-17]. Finally, we did not observe any influence of chemokines on proliferation of human erythroid progenitor cells.
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Materials and Methods |
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Selection of CD34+KIT+ A–T– MNC by Fluorescence-Activated Cell Sorting (FACS)
The c-kit receptor positive (c-kit-R+) subset of CD34+ cells was isolated by FACS. Briefly, 2 x 107 human A–T– MNC were suspended in phosphate-buffered saline (PBS), supplemented with 5% bovine calf serum (BCS), and labeled for 30 min at 4°C with anti-Kit R mAb (clone #104D2) directly conjugated with Cy5 (Becton Dickinson; 20 µl/106 cells) and with an anti-CD34 mAb directly conjugated with phycoerythrin ([PE] anti-HPCA-2, Becton-Dickinson; 20 µl/106 cells). After the final incubation, cells were washed 3x in ice-cold PBS supplemented with 5% BCS, and then subjected to FACS using a FACS Star Plus II (Becton Dickinson).
Ex Vivo Expansion of Human Erythroid Cells
CD34+ cells were expanded in a serum-free liquid system as described [19]. Briefly, CD34+ A–T– MNC were resuspended in Iscove's Dulbecco's modified Eagle's medium (104/ml; GIBCO BRL; Grand Island, NJ) supplemented with 25% of artificial serum containing 1% delipidated, deionized, and charcoal-treated bovine serum albumin (BSA), 270 µg/ml iron-saturated transferrin, insulin (20 µg/ml), and 2 mmol/l L-glutamine (all from Sigma; St. Louis, MO). BFU-E growth was stimulated with recombinant human (rHu) erythropoietin (EPO) (2 U/ml), and rHu kit ligand (KL) (10 ng/ml). Cytokines were from R&D Systems, Inc., Minneapolis, MN. Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO2. Under these conditions, approximately 100% of expanded cells were glycophorin A (GPA-A) positive, CD33, and gpIIa/IIIb-negative after 14 days [19]. In our studies we employed erythroid cells isolated from these cultures which were expanded from 2 to 14 days.
Cell Cultures
Briefly, CD34+ A–T– MNC cells (1 x 104) were cloned in 1 ml of Iscove's modified Dulbecco's medium (IMDM) (GIBCO BRL) containing 0.8% methylcellulose (Methocel MC; Fluka, Switzerland) and supplemented with 1% delipidated, deionized, charcoal-absorbed BSA (Sigma), 270 µg/ml iron-saturated transferrin (Sigma), 5.6 µg/ml cholesterol (Sigma), and 2 mmol/l L-glutamine as described [18]. rHu growth factors appropriate for growth of the colonies were added to the mixture which was then transferred to 3.5-cm plastic petri dishes and incubated (37°C, 95% air, 5% CO2 humidified atmosphere) for the appropriate times. Growth factors employed and utilized concentrations were as follows: EPO (5 U/ml) + KL (100 ng/ml) for optimal stimulation and EPO (1 U/ml) + KL (40 ng/ml) for suboptimal stimulation of BFU-E.
The various chemokines (SDF-1, MIP-1, MIP-1ß, RANTES, MIP-3, interleukin (IL)-8, Gro-, Gro-ß, Gro-
, NAP-2, ENA-78) in a dose of 100 ng/ml (R&D Systems Inc. and PeproTech Inc.; Rocky Hill, NY) were added as costimulators to some culture dishes. In all the experiments BFU-E colonies were counted with the aid of an inverted microscope on day 14.
FACS Analysis of Chemokine Receptor Expression
The expression of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CXCR2, CXCR3, and CXCR4 on expanded ex vivo erythroid cells from CD34+ BMMC under serum-free conditions and mature peripheral blood erythrocytes were evaluated by FACS as described [7]. mAbs against CCR1 (Clone #53504), CCR2 (Clone #48607), CCR3 (rat mAbs Clones #61856, 61832, 61837, 61834), CCR5 (Clone #45529), CCR6 (Clone #53113), CXCR2 (Clone #48301), CXCR3 (Clone #49801), and CXCR4 (Clone #44701) were obtained from R&D Systems. The CCR4 mAb was a kind gift from Dr. Patrick Gray (ICOS Corp.; Seattle, WA) and the rabbit polyclonal against CCR8 was a kind gift from Dr. Richard Horuk (Berlex; San Rafael, CA) to Dr. Robert Doms (University of Pennsylvania; Philadelphia, PA). Before we performed our studies, the specificity of these antibodies against their cognate receptors was confirmed on transfected 293T cells. Flow cytometric staining and analysis of the receptors were performed as described [7]. Briefly, the cells were stained in PBS (Ca and Mg-free) supplemented with 5% BCS (Hyclone; Logan, Utah). Primary mAbs were detected with secondary phycoerythrin or fluorescein isothiocyanate (FITC)-conjugated goat antimouse mAbs (Sigma) (1:100) or antirat antibodies. After the final wash, cells were fixed in 1% paraformaldehyde prior to FACS analysis using FACscan (Becton Dickinson). Similarly, we have stained expanded ex vivo human erythroid cells under serum-free conditions [19] and mature erythrocytes isolated from peripheral blood. In addition, we have also been looking for an expression of chemokine receptors on CD34+ BMMC. In brief, BMMC were double stained with chemokine receptors, and specific mAbs were detected with PE and anti-CD34 mAb directly conjugated with FITC anti-HPCA-1, (20 µl/106 cells; Becton Dickinson) as described [7]. Data analysis was performed using the Cell Quest (Becton Dickinson).
Chemotaxis Studies
All experiments were performed in triplicate on purified CD34+ or serum-free expanded erythroid cells. Briefly, after isolation the cells were resuspended in serum-free medium (106/ml) and equilibrated for 10 min at 37°C. In the meantime 600 µl/point of prewarmed serum-free medium containing appropriate ligand was added to the lower chamber of the Costar Transwell 24-well plate, 6.5 mm diameter, 5 µM pore filter (Costar Corning Co.; Cambridge, MA). Subsequently, 100 µl aliquots of the cell suspension were distributed to the upper chambers, and cultures were incubated at 37°C, 95% humidity, 5% CO2 for 4 h. After 4 h of incubation, the plates were evaluated under an inverted microscope and subsequently, cells from the lower chambers were collected and cell number was scored by FACscan (Becton Dickinson) as described [13]. Briefly, the cells were gated according to their forward scatter (FSC) and sideward scatter (SSC) parameters and counted during a 20-sec acquisition. Data are demonstrated as a percentage of the input number of the cells. If required, samples of cells from the lower chambers have been plated in vitro and stimulated to growth erythroid colonies as described above.
Calcium Flux Studies by FACS
Sorted by FACS CD34+ KIT+, isolated by magnetic beads CD34+ and expanded ex vivo under serum-free conditions, erythroid cells were employed for Ca2+ flux studies. Cells were loaded with Indo-1AM (Molecular Probes; Eugene, OR) as described [22]. Subsequently, 2 x 105 sorted cells or 5 x 105 expanded cells were spun down at room temperature and resuspended in 1 ml of prewarmed loading medium (1 x Hank's balanced salt solution, 1 mM MgCl2, 1 mM CaCl2, 1% fetal bovine serum [FBS]), and 40 µl of 100 mM Probenecid + 10 µl of Indo-1AM mixture (50 µg of Indo-1 AM, 25 µl of Pluronic acid 127, 113 µl of FBS) were added. Cells were incubated for 30-45 min at 30°C and were then gently mixed every 10 min. After incubation, the cells were spun down at room temperature, supernatant was discarded and cells were resuspended in 300-500 µl of fresh loading medium. The cells were then equilibrated for 10 min at 37°C and analyzed by FACS StarPlus as described [22].
Calcium Flux Studies by Spectrophotofluorimeter
Calcium flux studies on expanded ex vivo CD34+ cells were also performed by a spectrophotofluorimeter, as previously described [21]. Briefly, the CD34+ cells were loaded with Fura-2/AM (Molecular Probes) for 30 min at 37°C. Fluorescence was recorded with an Aminco-Bowman Series-2 Luminescence Spectrophotofluorimeter (SLM Instruments, Inc.; Urbana IL). Cells (1 ml aliquots) were stirred continuously in a warmed holder during the period of changes in fluorescence recording. Fluorescence was monitored at 340 nm and 380 nm for excitation and 510 nm for emission. The data were recorded as the relative ratio of fluorescence excited at 340 nm and 380 nm.
Detection of MAPK and AKT Phosphorylation
1 x 106 of purified CD34+KIT+ cells were put in RPMI medium containing 0.5% BCS over night in order to make the cells quiescent. After 12 h, cells were transferred to the fresh RPMI without serum for an additional 2 h. After that time, cells were divided and stimulated with appropriate ligands for 1 min at 37°C, and subsequently they were spun down in order to stop the reaction. Supernatants were removed immediately and the cells were lysed in the lysing buffer (150 mM NaCl/50 mM Tris HCl/1% Triton-X-100 plus protease and phosphatase inhibitors) for 5 min on ice. Subsequently, the equal amounts of 2x loading buffer were added and the lysates were boiled for 3 min. The extracted proteins were separated by 15% SDS-PAGE gel and transferred to the nitrocellulose membrane—Hybond ECL (Amersham Life Sciences; Little Chalfont, England). Phosphorylation of 44/42 MAPK and AKT was detected by protein immunoblotting using monoclonal 44/42 phospho-specific MAPK or rabbit polyclonal AKT phospho-specific AKT antibodies (New England Biolabs; Beverly, MA) with horseradish peroxidase-conjugated goat anti-mouse IgG as secondary antibody (Santa Cruz Biotech; Santa Cruz, CA) as described [23]. The membranes were developed with ECL reagent (Amersham Life Sciences) and subsequently dried and exposed to film. Equal loading in the lanes was evaluated by stripping the blot and reprobing with an anti-MAPK antibody clone #9102 and an anti-AKT antibody clone #9272 (New England Biolabs).
Isolation of mRNA from BFU-E
Cells from expanded ex vivo BFU-E colonies growing in serum-free methylcellulose cultures were analyzed for CXCR4, CCR5, and ß-actin expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Briefly, colonies were visualized with the aid of an inverted microscope and plucked from serum-free cultures on day 6 through day 9 using a Pasteur pipette. Randomly selected colonies (~20) from each culture dish were utilized for each assay. Colonies were resuspended in 10 ml of IMDM and incubated for 1 h at 37°C to dissolve the methylcellulose. The cells were washed 2x in PBS after which their mRNA was extracted as detailed below.
RT-PCR
Cells from 20 colonies were lysed in 200 µl of RNAzol (Biotecx Labs; Houston, TX) + 22 µl of chloroform (Sigma). The aqueous phase was collected and mixed with 1 volume of isopropanol (Sigma). RNA was precipitated overnight at –20°C. The RNA pellet was washed in 75% ethanol and resuspended in 3x-autoclaved H2O. RT-PCR was carried out as described [7, 18, 20]. Briefly, mRNA (0.5 µg) was reverse-transcribed with 500 U of Moloney murine leukemia virus reverse transcriptase and 50 pmol of an oligodeoxynucleotide (ODN) primer complementary to the 3' end of the following sequence of CXCR4 (5'-CAA GGA AGC TGT TGG CTG AAA-3') or CCR5 (5'-GAA AAT GAG AGC TGC AGG TGT-3'), according to the reported cDNA sequences as described [7]. The resulting cDNA fragments were amplified using 5 U of Thermus aquaticus (Taq) polymerase and primers specific for the 5' end of CXCR-4 (5'-CGA GGC AAG TGA CGC CGA GGG GCT G-3') or for the 5' end CCR5 (5'-GCT GTC CAC ATC TCG TTC TCG-3'). ß-actin mRNAs were amplified simultaneously using specific primers as described respectively [18]. Amplified products (10 µl) were electrophoresed on a 2% agarose gel, and transferred to a nylon filter and documented photographically. Specificity of the amplified products was further confirmed by Southern blotting as described [18].
Statistical Analysis
Arithmetic means and standard deviations were calculated on a MacIntosh computer using Instat 1.14 (GraphPad; San Diego, CA) software. Experiments were performed on cells obtained from 15 healthy donors. Cell cultures were carried out in quadruplicate. Data were analyzed using the Student's t-test for unpaired samples. Statistical significance was defined as p < 0.05.
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In this paper, we extended these observations by looking for the presence of eight other chemokine receptor proteins on human CD34+ cells. We found that in addition to CXCR4, a small percentage of CD34+ cells (~5%-10%) express the CCR6 receptor. At the same time, we were unable to demonstrate the presence of CCR1, CCR2, CCR3, CCR4, CCR5, CCR8, CXCR2, or CXCR3 receptors on the same cells using flow cytometry. Figure 1
demonstrates the coexpression of selected chemokine receptors (CCR1, CCR3, CCR5, CCR6, and CXCR4) and CD34 antigen on human BMMC in the lymphocyte gate. We then phenotyped more differentiated erythroid cells for expression of these chemokine receptors. First, we looked for the presence of major HIV coreceptors (CXCR4, CCR5) on human erythroid cells grown in serum-free liquid cultures, (Fig. 2A-D). Figure 2B shows that the CXCR4 protein was still detectable on ~15% of human erythroid cells cultured ex vivo for four days and became undetectable on more differentiated erythroid cells which were cultured for six days (Fig. 2D). By employing a more sensitive RT-PCR assay, we were still able to detect the presence of CXCR4 mRNA in cells isolated from day 11 erythroid colonies (Fig. 3). Similarly, while the CCR5 protein was not detected by FACS on human CD34+ cells (Fig. 1D), or cultured erythroid cells (Fig. 2A, C), CCR5 mRNA was easily detected by RT-PCR (Fig. 3). Therefore, we postulated that the apparent lack of expression of major chemokine receptors on the differentiated erythroid cells was the result of limited FACS detection sensitivity.
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). We also found that CCR6 was still present on ~20% erythroid cells, expanded for 11 days (not shown).
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Functional Studies on Chemokine Receptor-Chemokine Axes
Flow cytometric detection of chemokine receptors on cell surfaces is sensitive to the level of ~200-500 molecules. If a protein is expressed at a lower level, a definitive statement about expression cannot be made. Further, the absence of a particular protein as assessed by FACS does not allow us to conclude that this receptor is not present on the cell surface. In fact, mRNAs for many chemokine receptors have been found in early human erythroid cells [9, 10]. We assumed that various chemokine receptors that were not detectable by FACS might still be functional even though they are expressed at low levels on the cell surface. To examine this possibility we performed functional studies, looking specifically for the ability of selected chemokines to activate chemotaxis in CD34+ progenitors and differentiating erythroid cells, induce calcium flux and phosphorylate MAPK p42/44 (ERK-1, ERK-2) and AKT.
Functional Analysis of Chemokine Receptors on CD34+ Cells by Chemotaxis Assay
As demonstrated above, ~50% of human CD34+ cells coexpress X4 HIV-binding CXCR4 coreceptor, while ~5%-10% coexpress the CCR6 protein. In addition, human CD34+ cells express at least CCR5 mRNA for the R5 HIV cell surface coreceptor [4]. The biological significance of CXCR4, CCR6 expression and potential presence of CCR5 receptor was investigated by employing various functional studies. First, we asked whether any of the chemokines show a chemotactic activity against human CD34+ cells. We found that only SDF-1 (CXCR4 ligand), but not MIP-1, MIP-1ß, RANTES (CCR5 ligand) or MIP-3 (CCR6 ligand) were able to attract human CD34+ cells (Fig. 5). It has been reported that ~25%-45% of human BM CD34+ cells show chemotaxis to SDF-1 [11, 12, 16]. We found in our studies that ~ 40% of the human CD34+ cells migrated to SDF-1 in the chemotaxis assay (Fig. 5). In addition, we noticed that among CD34+ cells, which migrated in chemotaxis assay to SDF-1, BFU-E were present, which formed erythroid colonies after replating to the semisolid methylcellulose cultures. We calculated that 47% ± 12% of input BFU-E migrated during 4 h in the chemotaxis assay to SDF-1 (300 ng/ml).
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, MIP-1ß and RANTES activate chemotaxis in malignant hematopoietic progenitor CTS cells first after addition of KL [26], we have supplemented medium containing chemokines with this growth factor. The addition of KL, however, did not influence chemotaxis of human CD34+ cells to MIP-1, MIP-1ß and RANTES (not shown).
Functional Analysis of Chemokine Receptors on Erythroid Cells Expanded Ex Vivo by Chemotaxis Assays
Next, we studied chemotaxis of the differentiated erythroid progenitor/precursor cells. We found that human early erythroid cells showed chemotaxis to SDF-1 after three and six days of culture. It is also worth mentioning that early erythroid cells from day 6 expansion cultures, collected from the lower chambers after chemotaxis assay, formed small colony-forming units-erythroid (CFU-E) colonies after replating in the methylcellulose cultures (not shown). We noticed that the chemotactic activity of early erythroid cells cultured ex vivo decreased after they started to differentiate and express GPA-A on their surface (Fig. 5
). Accordingly, by day 9, all the cultured cells were GPA-A+ and no longer migrated in response to SDF-1 (Fig. 5).
Surprisingly, even though early erythroid cells express CCR6 and CXCR2 proteins as well as CCR5 mRNA, the ligands which bind to those receptors (MIP-3, IL-8, MIP-1, MIP-1ß, RANTES) did not activate chemotaxis in those cells (Fig. 5).
Functional Analysis of Chemokine Receptors on CD34+ Cells by Calcium Flux Studies and Phosphorylation of MAPK (ERK1 and ERK2) and AKT
In order to learn more about the biological role of chemokine receptor expression on human CD34+ cells, we performed calcium flux studies. Since CD34 antigen marks early lymphoid cells as well as myeloid cells, we FACS sorted a population of CD34+KIT+ cells, which is enriched in BFU-E [26], to identify responding cells by lineage. Accordingly, CD34+KIT+ cells were loaded with Indo-1AM, and calcium flux was evaluated by FACS. We found that SDF-1 (Fig. 6A) but not MIP-1, MIP-1ß (Fig. 6B), or RANTES and MIP-3 (not shown) strongly induced calcium flux in this population of early stem/progenitor cells. As demonstrated in Figure 6A, SDF-1 induced calcium flux in almost 100% of CD34+KIT+ cells. Similarly, we found that SDF-1, but not MIP-1, MIP-1ß, MIP-3, or MCP-3, were able to phosphorylate ERK1 and ERK2 and AKT in human CD34+KIT+ cells (Fig. 7). This strongly supports our observation that, in contrast to CCR5, CCR6 and CCR2, CXCR4 receptor protein is not only detectable but functional on human CD34+ KIT+ cells.
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), and in more differentiated erythroid cells that had been expanded ex vivo for three to six days, was also evaluated (Fig. 9). Accordingly, CD34+ cells were isolated by magnetic beads and cultured serum-free for two days with KL + EPO (Fig. 8), and calcium flux was first evaluated by spectrophotofluorimeter. We found that SDF-1 (Fig. 8B) but not MIP-1 (Fig. 8A), MIP-1ß, RANTES, and MIP-3 (not shown) were able to induce calcium flux in this population of cells.
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). We chose this technique because FACS, in contrast to spectrophotofluorimetry, not only allows for detection of a calcium flux itself but also allows for an estimate of the percentage of the cells which respond in this assay. We noticed that a subpopulation of erythroid cells, which was expanded ex vivo for three (Fig. 9A) and four days (Fig. 9B) responded to stimulation with SDF-1. To our surprise ex vivo-expanded erythroid cells did not show calcium flux after stimulation with MIP-1, MIP-1ß, RANTES, IL-8, and MIP-3 (not shown) even though the corresponding chemokine receptors were expressed. Simultaneously, we did not observe any influence of those chemokines on MAPK and AKT phosphorylation in the same population of cells (not shown).
Influence of HIV-Related and Other Chemokines on BFU-E Growth Under Serum-Free Conditions
Data from the literature on the influence of chemokines on human hematopoiesis are inconsistent [6, 8-10, 27-32] and in our hands neither MIP-1 nor IL-8 inhibited human erythropoiesis in vitro [33]. We therefore reexamined these observations focusing on those chemokines, for which the receptors are expressed at a high level on the BFU-E (CXCR4) or on more differentiated erythroid cells (CCR6, CXCR2). Human CD34+ cells were isolated by magnetic beads and cultured serum-free in the presence or absence of different chemokines (Table 1). Transforming growth factor-ß1, a potent known inhibitor of the erythropoietic colony formation, was added to some cultures [34].
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, SDF-1 (CXCR4 ligand), MIP-3 (CCR6 ligand) and IL-8, Gro-, -ß, -, NAP-2, ENA-78 (CXCR2 ligands) all failed to affect human BFU-E growth in vitro. Similarly, MIP-1, MIP-1ß, and RANTES (CCR5 ligands) were without apparent effect. After addition of chemokines, the number of colonies, their hemoglobinization, and expression of GPA-A remain unchanged. At the same time, however, the growth of the erythroid colonies was strongly inhibited after an addition of TGF-ß1 to the cultures. Similar data also were obtained when BFU-E growth was stimulated with a suboptimal dose of EPO + KL (not shown). |
Discussion |
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We found that CXCR4 is highly expressed on human CD34+ cells enriched for BFU-E. In this study, we also phenotyped human CD34+ cells for the expression of eight other chemokine receptors using FACS. We found that a small population of CD34+ cells (5%-10%) expresses the CCR6 protein, which binds MIP-3. Other receptors were undetectable. Hence, CXCR4 remains the most highly expressed member of this receptor family on human CD34+ cells. This observation supports other reports demonstrating that CXCR4 plays an important and privileged function in homing and trafficking of human CD34+ progenitor/stem cells [11, 13, 15, 17, 35]. Our failure to detect CCR1, CCR3, and CCR5 proteins on CD34+ cells is intriguing because all these receptors bind chemokines which interfere with R5 HIV-1 entry into the cells (MIP-1, MIP-1ß, RANTES) [10]. In addition, CCR1 and CCR5 bind MIP-1, which has been found to inhibit proliferation of CD34+ cells [8].
To validate these observations on a population of pure erythroid cells, we phenotyped human erythroid cells expanded ex vivo as well as mature erythrocytes. We found that the expression of CXCR4 varied with the state of erythroid maturation and could not be detected on BFU-E-derived cells expanded for six days. Nevertheless, these more differentiated erythroid cells still contained CXCR4 mRNA. In contrast, human erythroid cells expressed CCR6 protein, and to a lesser degree CXCR2, after six days of culture. Expression of CCR6 and CXCR2 decreased as the erythroid cells matured. Finally, we looked for the presence of chemokine receptors on mature human peripheral blood erythrocytes but were unable to detect any of the chemokine receptors for which we tested.
In functional assays, we found that SDF-1 (CXCR4 ligand), but not MIP-1, MIP-1ß, RANTES (CCR5 ligand), IL-8 (CXCR2 ligand) or MIP-3 (CCR6 ligand) induced calcium flux and phosphorylated MAPK ERK-1 and ERK-2 and AKT in a FACS-purified CD34+KIT+ population. Moreover, only SDF-1-induced calcium flux in human erythroid cells expanded for two to four days. The response to SDF-1 disappeared when the cells began to differentiate and express GPA-A. In contrast, even though more differentiated erythroid cells expressed CCR6 and CXCR2 receptors, their corresponding ligands (MIP-3 and IL-8) did not activate calcium flux in those cells. We also confirmed that of all the chemokines tested, only SDF-1 is a strong chemotactic factor for human CD34+ cells [11, 13] as well as for human CD34+ BFU-E and for the more differentiated CD34– CFU-E progenitor cells. Of note, none of the chemokines evaluated in this study affected cloning efficiency or maturation of human BFU-E. These data are in apparent conflict with other reports which found that MIP-1 inhibits human erythroid colony formation [8, 10].
The contradictory responses to MIP-1 reported here might be explained by differences in MIP-1 protein purification, differences in the cloning system itself and in the formation of inactive dimers [29]. This latter possibility is unlikely because the same preparations of MIP-1-activated calcium flux in our study and chemotaxis in certain human hematopoietic cell lines (not shown). This suggests that the source of MIP-1 we utilized in our experiments was biologically active. In addition, the reported inhibitory effects of MIP-1 on hematopoiesis are often unclear [6]. Mice with MIP-1 knock-out do not show any hematopoietic defects [28]. In some reports MIP-1 was found to be only inhibitory if added at a very low dose [29], whereas in others was reported to inhibit cell growth if added at a very high dose and repetitively added doses [31].
Finally, after we recently demonstrated that human BM CD34+ cells secrete MIP-1 endogenously [25], we concluded that the role of this chemokine in regulating human hematopoiesis is more complex than we initially thought [6, 8, 29]. In light of our recent data we conclude that human BM CD34+ cells are exposed in vivo to low levels of secreted MIP-1 and other chemokines [25].
Based on the assays carried out in this study (chemotaxis, calcium flux, phosphorylation of MAPK and AKT), we concluded that only the CXCR4 receptor is functional on the earliest human erythroid cells. It appears to play an important role in regulating chemotaxis and perhaps in homing and retention of erythroid progenitors in the BM microenvironment. Therefore, the perturbation of the SDF-1/CXCR4 axis which occurs during HIV infection or when induced by therapeutic agents designed to block HIV cell entry (e.g., ALX40) [1, 36] may affect the homing and development of erythroid cells. Since we did not observe any effect of chemokines on erythroid colony formation by human CD34+ cells, we conclude that the influence of chemokines on human hematopoiesis is more complicated than initially thought. Further studies are needed to elucidate their role in human hematopoiesis, including examining their autocrine effect on individual hematopoietic lineages [25] as well as their indirect influence as mediated by interactions with hematopoietic accessory cells.
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Acknowledgments |
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References |
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by interacting mainly with a C-C chemokine receptor, CCR1. Blood 1997;90:605-611. and the expression of MIP-1 receptors on these immature cells. STEM CELLS 1998;16:349-356. and stem cell factor/kit ligand share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis. J Immunol 1998;161:3652-3658., is the endogenous chemokine that cooperates with TGF-ß to inhibit the cycling of primitive normal but not leukemic (CML) progenitors in long-term human marrow cultures. Blood 1998;92:2338-2344. for an inflammatory response to viral infection. Science 1995;269:1583-1585. inactivates its myelosuppressive effects in vitro: the active form is monomer. Proc Natl Acad Sci USA 1993;90:2232-2236. maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay. Blood 1998;91:4516-4522.
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 S. Chaisavaneeyakorn, J. M. Moore, L. Mirel, C. Othoro, J. Otieno, S. C. Chaiyaroj, Y. P. Shi, B. L. Nahlen, A. A. Lal, and V. Udhayakumar Levels of Macrophage Inflammatory Protein 1{alpha} (MIP-1{alpha}) and MIP-1{beta} in Intervillous Blood Plasma Samples from Women with Placental Malaria and Human Immunodeficiency Virus Infection Clin. Vaccine Immunol., July 1, 2003; 10(4): 631 - 636. [Abstract] [Full Text] [PDF]
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J. Libura, J. Drukala, M. Majka, O. Tomescu, J. M. Navenot, M. Kucia, L. Marquez, S. C. Peiper, F. G. Barr, A. Janowska-Wieczorek, et al. CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion Blood, September 18, 2002; 100(7): 2597 - 2606. [Abstract] [Full Text] [PDF]
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J.-J. Lataillade, D. Clay, P. Bourin, F. Herodin, C. Dupuy, C. Jasmin, and M.-C. Le Bousse-Kerdiles Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism Blood, February 15, 2002; 99(4): 1117 - 1129. [Abstract] [Full Text] [PDF]
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 E. A. de Wynter, C. M. Heyworth, N. Mukaida, E. Jaworska, A. Weffort-Santos, K. Matushima, and N. G. Testa CCR1 chemokine receptor expression isolates erythroid from granulocyte-macrophage progenitors J. Leukoc. Biol., September 1, 2001; 70(3): 455 - 460. [Abstract] [Full Text] [PDF]
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 J. Kijowski, M. Baj-Krzyworzeka, M. Majka, R. Reca, L. A. Marquez, M. Christofidou-Solomidou, A. Janowska-Wieczorek, and M. Z. Ratajczak The SDF-1-CXCR4 Axis Stimulates VEGF Secretion and Activates Integrins but does not Affect Proliferation and Survival in Lymphohematopoietic Cells Stem Cells, September 1, 2001; 19(5): 453 - 466. [Abstract] [Full Text] [PDF]
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M. Majka, A. Janowska-Wieczorek, J. Ratajczak, K. Ehrenman, Z. Pietrzkowski, M. A. Kowalska, A. M. Gewirtz, S. G. Emerson, and M. Z. Ratajczak Numerous growth factors, cytokines, and chemokines are secreted by human CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner Blood, May 15, 2001; 97(10): 3075 - 3085. [Abstract] [Full Text] [PDF]
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M. Majka, A. Janowska-Wieczorek, J. Ratajczak, M. A. Kowalska, G. Vilaire, Z. K. Pan, M. Honczarenko, L. A. Marquez, M. Poncz, and M. Z. Ratajczak Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis Blood, December 15, 2000; 96(13): 4142 - 4151. [Abstract] [Full Text] [PDF]
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