Address correspondence to Dr. Bruno Azzarone, INSERM U542, Bâtiment Lavoisier, Hôpital Paul Brousse, 94807 Villejuif, France. Phone: 00-33-145595344; Fax: 00-33-145595343; E-mail: bazzarone{at}hotmail.com
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Abstract |
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Key Words: IL-15 GM-CSF IL-15R signal transduction CD34+ cells
In this study, we demonstrate that normal CD34+ cells, but not nonhematopoietic cells, express a novel functional hybrid receptor composed of the
Isolation and Culture of Normal Polyclonal NK Cells.
Cell Lines.
Proliferation Assays.
TF1ß cells (3 x 105 cells/ml) were also cultured for 4 d with MS9 stromal cells as the feeder layer. Sister cultures were treated with antiIL-15 (R&D Systems), antiIL-15R
Analysis of IL-15 Signal Transduction in the Human Hematopoietic TF1ß Cell Line and in CBPr CD34+/CD56- Precursors.
Immunoblotting: Western Blotting.
Primary antibody binding to the membrane was detected by incubation with peroxidase-conjugated secondary antibodies, followed by the enhanced chemiluminescence (ECL) system (Amersham Biosciences). The membrane was then subjected to densitometry, including correction for background, with analysis using NIH Image software. To correct for possible variations in the amount of protein loaded, values are expressed as pSTAT/STAT ratios. Results are expressed as increases (e.g., three times) with respect to the results obtained for untreated cells.
Confocal Microscopy.
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
The development of hematopoietic cells is controlled by hematopoietic growth factors, cytokines and chemokines produced in the bone marrow (BM)* by accessory cells and T lymphocytes (1, 2), and by several other mechanisms (3). Hematopoietic factors constitute a complex network, in which each cytokine may act at various stages: for example, myeloid growth factors such as G-CSF and GM-CSF may also affect the lymphoid compartment (4, 5). GM-CSF inhibits the expansion of the NK cell progenitor population from unprimed BM CD34+ cells, and the generation and functioning of NK cells both in vivo and in vitro (5, 6). IL-15, the most powerful factor inducing the differentiation of CD34+ hematopoietic progenitor cells into CD56+/CD3- NK cells in vivo (7), may also cause a large increase in the number of long-term culture-initiating cells (LTC-IC) when added to the IL-3/SCF/Flt3-L combination (8). Transgenic mice carrying a mutation in the cytoplasmic domain of the common gamma chain (c), or lacking the signaling kinase JAK3, both of which are necessary for IL-15mediated signaling, have very low lymphocyte counts and display dysregulated myelopoiesis (9, 10). Moreover, the erythroid burst-forming cells of these mice have a larger than normal fraction of hematopoietic precursors with the CD34+/
c- phenotype (11). These data suggest that there is cross talk between myeloid growth factors and IL-15, which may regulate the balance between the myeloid and lymphoid lineages during hematopoietic differentiation. This regulation may operate at the level of signal transduction because IL-15 and GM-CSF share signaling pathways (JAK2/STAT5 and NF-
B) heavily involved in the control of hematopoiesis (1216). Alternatively, the cross talk between these two cytokines may involve direct interactions between their receptor subunits, as suggested by the fact that GM-CSF almost totally inhibits IL-2 binding to IL-2/IL-15Rß/
c in the M07sb CD34+ hematopoietic cell line (17). We investigated whether this cross talk resulted from a physical interaction between the two receptors, possibly involving the
c chain and the GM-CSFRß chain. Indeed, these two subunits identify two families of cytokines, one with the
c chain (IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21; references 7 and 18) and the other, with the GM-CSFRß chain (IL-3, IL-5, and GM-CSF; reference 19).
c and GM-CSFRß chains, in which the GM-CSFRß chain may inhibit
c/JAK3 signaling. This complex is maintained in CD34+ myeloid and cord blood (CB)Pr CD34+/CD56- cells, but not in mature NK cells, suggesting that it is involved in controlling hematopoietic differentiation.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Purification of CD34+ Cells.
Mononuclear cells from CB or adult G-CSF mobilized peripheral blood (MPB) were isolated by centrifugation on a Ficoll gradient (Lymphoprep Nicomed Pharma SA). Adherent cells were isolated by adhesion to plastic Petri dishes for 2 h at 37°C. CD34+ cells were selected by an immunomagnetic method (Miltenyi Biotec), on nonadherent mononuclear cells labeled with a mAb specific for the QBEND10 epitope of the CD34 antigen, achieving a purity >97%, as previously described (20). CB CD34+ cells were used unprimed or after treatment with SCF/Flt3-L to expand the population of CD34+/CD56- primed progenitors (CBPr) in which the frequency of NK cell precursors may be 65 to 235 times higher than that in freshly prepared CD34+ HPCs (7).
Non-adherent PBMCs from healthy volunteers were isolated by centrifugation on a Ficoll gradient. CD3-/CD4-/CD8- cells were purified by negative depletion and cultured with 100 U/ml rIL-2 (Proleukin; Chiron Corp.) in the presence of 105 irradiated allogenic PBMCs/well and 104 221 lymphoblastoid cells/well. By day 15, almost all the proliferating cells expressed CD16 and CD56 antigens.
Human TF1ß (IL-15R/ß/
c) and M07sb (IL-15Rß/
c) are two IL-15dependent leukemic cell lines, whereas TF1 (IL-15R
/
c) is a GM-CSFdependent cell line. TF1 and TF1ß cells have the potential to display proerythroid differentiation whereas M07Sb cells display promegakaryocytic differentiation. NK-L is a functional human cytolytic NK cell line that is dependent on rIL-2. Raji is a B cell line (IL-15R
/
c) that does not depend on growth factors for its proliferation. The growth characteristics and phenotypes of these cells have been reported elsewhere (2125). Cells were maintained in RPMI medium (Eurobio) supplemented with 10% FCS (ATGC Biotechnologie), 2 mM glutamine, 1% antibiotics (GIBCO BRL), 10 ng/ml rIL-15, or rGM-CSF (R&D Systems) at 37°C, under an atmosphere of 5% CO2/95% air. Cells were subcultured twice per week by seeding fresh culture medium with 105 cells/ml. The human stromal fibroblast MS9 strain, isolated in culture from the spleen of a patient with a myeloproliferative syndrome, was used as a feeder layer for hematopoietic cells. MS9 fibroblasts express a bioactive membrane-bound IL-15 that induces the NK cell differentiation of CD34+ cells; they also secrete GM-CSF (26, 27). MS9 cells between passages 5 and 10 were cultured for 72 h at a density of 2 x 105 cells/ml in RPMI medium supplemented with 10% FCS in 6-well plates before being brought into contact with hematopoietic cells (5 x 105 cells/well). TF1ß and NK-L cells were kindly provided by Drs. Paul Sondel (Dept. of Human Oncology, University of Wisconsin, Madison, WI) and Alessandro Moretta (Department of Experimental Medicine, University of Genoa, Genoa, Italy), respectively.
TF1ß cells and Raji cells (3 x 105 cells/ml) were cultured for 4 d in complete growth medium supplemented with rIL-15 or rGM-CSF (10 ng/ml). Sister cultures were treated with neutralizing antiIL-15R M162 (developed by Immunex Corp., provided by Genmab A/S, Copenhagen, Denmark), rat anti-
c (TUGh4), and anti-GM-CSFRß (BD Biosciences/Becton Dickinson) mAbs.
M162, antiIL-15Rß (CF-1; a gift from Dr. Y. Jacques, U463 INSERM, Nantes, France), anti-
c (TUGh4), anti-GM-CSFRß, or anti-GM-CSFR
(S-20; Santa Cruz Biotechnology, Inc.) mAbs. In some inhibition experiments, we used low concentrations of antiIL-15Rß and anti-
c mAbs, because a combination of 1 µg/ml of each mAb is more efficient than a single dose of 10 µg/ml of each mAb at inhibiting IL-15 effects (23). Cells were counted in triplicate. The statistical significance of differences was determined using Student's t test, with P
0.05 considered significant.
For signal transduction analysis, cells were incubated overnight with a low concentration of rIL-15 (0.5 ng/ml) and were then deprived of growth factors for 3 h at 37°C. Cells were then stimulated by incubation with 10 ng/ml rIL-15 for 5 to 15 min. In some experiments, cells were initially treated for 1 h with either 10 µg/ml neutralizing antibody against IL-15, IL-15Rß, c, or GM-CSFRß chains or with the JAK3-specific inhibitor, WHI-P131 (Calbiochem), which has no effect on JAK1 and JAK2.
Experiments were performed as described previously (12, 14, 28, 29). Briefly, cultures were serum-starved to reduce basal phosphorylation levels. Cells were then washed twice and suspended in lysis buffer supplemented with 0.5% NP-40. For immunoprecipitation, lysates were incubated with anti-c (TUGh4), anti-JAK3 (Upstate Biotechnology) or polyclonal anti-TRAF2 (Santa Cruz Biotechnology, Inc.) antibody and immune complexes were captured by incubation with protein G-Sepharose beads (Amersham Biosciences) overnight at 4°C. The captured immune complexes were washed twice with lysis buffer. Complexes or lysates (for Western blotting) were then dissolved in Laemmli buffer, boiled, and separated by SDS-PAGE (7.5% or 12% polyacrylamide gels). The protein bands were transferred to PVDF membranes (NEN Life Science Products). Membranes were blocked by incubation with 5% BSA and probed with the following antibodies: anti-JAK1, anti-JAK2, and 4G10 anti-phosphotyrosine (Upstate Biotechnology/USA Euromodex), anti-pJAK1 (Tyr 1022/1023), anti-pJAK2 (Tyr 1007/Tyr 1008), anti-STAT3, anti-pSTAT3 (Tyr705), and anti-STAT6 (Santa Cruz Biotechnology, Inc.), anti-STAT5 (Transduction Laboratories/Becton Dickinson), anti-pSTAT5 (Tyr694), anti-pSTAT6 (Tyr641; Cell Signaling/New England Biolabs, Inc.) and anti-pI
B
(Calbiochem).
For the double staining of c and GM-CSFRß chains, human MPB and CB CD34+ cells, the leukemic cell lines (TF1, TF1ß, and M07sb), and MS9 cells were washed and permeabilized by incubation with ORTHOpermeafix (Ortho Diagnostic Systems Inc.) for 45 min at room temperature. Cells were then stained with anti-
c (TUGh4) mAb and with the biotinylated antiGM-CSFRß secondary mAb, and were then incubated at room temperature with Alexa Fluor594-GAR and streptavidin-Alexa Fluor594 (Molecular Probes). The extent of association between the two chains was assessed using the colocalization option of Methamorphe Software (Universal Imagine). We also used confocal microscopy to evaluate production of the activated transcription factor, pSTAT5, in TF1ß and M07sb cells. Cells were starved of growth factors overnight and were then stimulated with rGM-CSF, as described above. Some samples were pretreated with antiIL-15ß/
c or antiGM-CSFRß mAbs. Cells were then permeabilized and indirect immunofluorescence assessed by means of antibodies recognizing the phosphorylated form of the transcription factor STAT5 (pSTAT5). Samples were washed and incubated with Alexa Fluor488-GARa antibody. Nuclei were stained with 2 µg/ml propidium iodide (PI, red staining). All antibodies were dissolved in PBS supplemented with 10 mg/ml BSA to block nonspecific binding. The stained cells were washed with PBS, centrifuged in a Cytospin 3 (Shandon) onto glass slides, and mounted under a coverslip in Prolong Antifade (Molecular Probes) mounting medium. The slides were analyzed by laser scanning confocal microscopy, using a Leica TCS Confocal System.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Human Hematopoietic CD34+ Cells Express a Hybrid c/GM-CSFRß Receptor.
We investigated the possible interactions between IL-15R and GM-CSFR complexes in human hematopoietic and nonhematopoietic cells by Western blotting (Fig. 1
A), coimmunoprecipitation (Fig. 1 B), and confocal microscopy (Fig. 1 C). The Western blotting of total lysates (Fig. 1 A) showed that the cells analyzed (TF1ß, M07Sb, and MPB CD34+) expressed both the c chain (a single band of 64 kD) and the GM-CSFRß chain (a major band of 130 kD, corresponding to the mature protein, and a second band of
95 kD, corresponding to the nonglycosylated precursor protein; reference 30).
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The c/GM-CSFRß association was also detected both in the proerythroid TF1 and TF1ß cell lines, which expressed IL-15R
/
c and IL-15R
/ß/
c, respectively, and in the promegakaryocytic (M07sb) cells expressing IL-15Rß/
c, whereas it was not detected in the cytolytic NK-L cell line (Fig. 1 B, panel b). In addition, the
c/GM-CSFRß complex was not found in human skin myofibroblasts despite the presence of the
c and GM-CSFRß chains in these cells (Fig. 1 B, panel c). Reprobing the
c membrane with anti-GM-CSFR
or anti-gp130 mAbs did not give a specific signal (unpublished data).
We demonstrated, by confocal microscopy, the colocalization of the c (green staining) and GM-CSFRß (red staining) chains in MPB and CB CD34+ progenitors, as well as in the TF1, TF1ß and M07sb cell lines (Fig. 1 C). The images presented are overlay-compacted images from confocal analysis of 1 µm serial optical sections from the cell surface to the inner compartment.
In MPB CD34+ and unprimed CB CD34+ cells (Fig. 1 C, panels a1 and a2), all cells displayed strong colocalization of the two molecules (yellow staining), restricted to the membrane/submembrane compartment. In TF1 and TF1ß cells (Fig. 1 C, panels b1 and b2), we observed similar yellow staining of the membrane/submembrane compartment and spotted yellow staining in the cytoplasm, suggesting that the hybrid receptor was taken up by some of the cells. This punctate distribution of the c/GM-CSFRß complex in the cytoplasms was the predominant staining pattern in M07sb cells (Fig. 1 C, panel b3). No colocalization was observed in human MS9 myofibroblasts (Fig. 1 C, panel c). The specificity of
c and GM-CSFRß chain colocalization was further confirmed by the lack of colocalization of the
c chain and the IL-6R subunit gp130 (Fig. 1 C, panel d) or the ß1 integrin (Fig. 1 C, panel e) in normal CB CD34+ cells. Computerized quantification of the extent to which the
c chain and the GM-CSFRß chain were colocalized in normal and leukemic CD34+ cells showed that 74.9% (MPB CD34+), 66.3% (TF1ß), 50.2% (TF1), and 58.3% (M07sb) of the GM-CSFRß chain colocalized with the
c chain, whereas 94.1% (MPB CD34+), 99.6% (TF1ß), 82.1% (TF1), and 93.6% (M07sb) of the
c chain colocalized with the GM-CSFRß chain. Our results indicate that the
c chain is almost completely associated with the GM-CSFRß chain, with
3040% of the GM-CSFRß chain left free.
The expression of this hybrid receptor is constitutive and does not seem to be influenced by cytokine stimulation, as it is observed in CB hematopoietic progenitors, whether unprimed or treated with SCF/Flt3-L, in MPB CD34+ cells, and in CD34+ myeloid cell lines, which are dependent on GM-CSF (TF1) or IL-15 (TF1ß, M07sb) for growth.
IL-15 and GM-CSFdependent Proliferation in TF1ß Cells Is Modified by AntiGM-CSFRß and Anti-c mAbs, Respectively.
The presence of the hybrid receptor on CD34+ cells dependent on IL-15 or GM-CSF led us to investigate the role of this receptor in cell proliferation. As illustrated in Fig. 2
A, TF1ß cells proliferated in the presence of rIL-15 (+200%) or rGM-CSF (+300%). IL-15dependent proliferation was inhibited by antiIL-15R, but not by anti-
c mAbs. Anti-GM-CSFRß mAb slightly, but significantly increased (+2025%, P < 0.001) the rate of cell proliferation.
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We investigated whether the growth of TF1ß cells could be stimulated by contact with human stromal MS9 cells, which have been shown to secrete several hematopoietic growth factors (26), and whether the hybrid receptor was involved in these interactions. TF1ß cells proliferated when cultured in contact with MS9 cells, doubling in number after 4 d (Fig. 2 C). The proliferation induced by MS9 cells was totally inhibited by neutralizing mAbs against the GM-CSFR and GM-CSFRß chains. Neutralizing mAbs directed against IL-15 or the IL-15R
chain had no effect whereas antiIL-15Rß/
c mAbs inhibited the proliferation of TF1ß cells by 60%. Thus, TF1ß cell proliferation is completely dependent on GM-CSF produced by MS9 cells (26) and can be efficiently inhibited by antiIL-15Rß/
c mAbs, consistent with the existence of cross talk between the two cytokine receptors.
IL-15induced JAK/STAT Signaling Is Inhibited by AntiGM-CSFRß mAb.
The inability of the anti-c mAb to block IL-15dependent proliferation in TF1ß cells suggests that the
c chain cannot mediate the effects of IL-15 in these cells. We investigated the signal transduction triggered by IL-15 and the involvement of the various subunits of the IL-15R, in TF1ß cells.
Densitometric analysis revealed that rIL-15 (10 ng/ml) caused the levels of phosphorylation of JAK1 and STAT3 to increase by a factor of seven within 15 min (Fig. 3
A). Neutralizing antiIL-15Rß mAb totally inhibited the phosphorylation of JAK1 and inhibited that of STAT3 by 50%. In contrast, anti-c mAb had no effect on either pathway. These data confirm the results of the proliferation assays, demonstrating the incapacity of the
c chain to mediate IL-15dependent functions in TF1ß cells.
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In contrast, JAK2 phosphorylation was totally inhibited by antiIL-15Rß/c mAb but was only inhibited by 30% with the anti-GM-CSFRß mAb. The use of an isotype-matched anti-gp130 mAb had no significant effect on the level of phosphorylation of the various molecules. In contrast to what was observed in TF1ß cells, antiGM-CSFRß mAb did not affect IL-15 signaling in CD34+ promegakaryocytic M07sb cells expressing a low-affinity IL-15Rß/
c receptor (unpublished data).
GM-CSFinduced pSTAT5 Nuclear Translocation Is Inhibited by AntiIL-15Rß/c mAbs in TF1ß Cells.
We analyzed the effects of antiIL-15Rß/c mAbs on the signal transduction activated by rGM-CSF (Fig. 4
A). Densitometric analysis revealed that rGM-CSF (at a concentration of 10 ng/ml) doubled the phosphorylation of JAK2 and quadrupled that of STAT5 in TF1ß cells in a 15-min period. In experiments with antiIL-15Rß/
c mAbs, the specific JAK3 inhibitor WHI-P31 or the irrelevant anti-gp130 mAb, we observed no significant inhibition of either pathway, which demonstrated that neither of the two chains of the IL-15R affected these steps of GM-CSF signaling. The effects of rGM-CSF and IL-15Rß/
c mAbs on the localization of pSTAT5 were analyzed by confocal microscopy (Fig. 4 B). Cells were stained for pSTAT5 (green staining) and with propidium iodide (specific for nuclei; red staining); superimposition of the two stains gave a yellow coloration. In basal culture conditions, all cells displayed green staining of the cytoplasm, indicating the presence of pSTAT5 protein in this compartment. The intensity of the green staining increased after GM-CSF stimulation suggesting that the level of STAT5 phosphorylation increased. No inhibition was observed in the presence of neutralizing anti-IL-15Rß/
c mAbs, or of the specific JAK3 inhibitor WHI-P31 (unpublished data), confirming the results obtained by Western blotting.
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TF1 Cells Express Functional IL-15R/
c Complex, the Formation of which Is Controlled by the Hybrid
c/GM-CSFRß Receptor.
The absence of proliferation in IL-15R/
c-positive TF1 cells in response to IL-15 has been attributed to the lack of the IL-15Rß subunit (23), which is indispensable for IL-15 binding and
c chain signaling (7). However, it has recently been reported that the IL-15R
chain may transduce a signal in the absence of links with any other receptor subunit (12), leading to NF-
B activation via the cytoplasmic interaction of this molecule with the signaling molecule TRAF2. We therefore investigated the possible presence of a functional IL-15R
/
c receptor in TF1 cells. Analysis of total lysates showed that the TF1 and TF1ß cell lines expressed the
c chain (64 kD) and at least two IL-15R
isoforms, 58 and 38 kD in size (Fig. 5
A). The two cell lines were then incubated with an anti-
c mAb for immunoprecipitation. The
c membrane was reprobed with the anti-
c mAb. A strong specific signal was identified at 64 kD. In contrast, two major bands, 58 and 38 kD in size, were identified if the membrane was reprobed with antiIL-15R
mAb. Thus, in both TF1 and TF1ß cells, the
c chain is physically associated with the two IL-15R
isoforms.
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Total lysates of the TF1 and TF1ß cell lines were subjected to immunoprecipitation with an anti-JAK3 mAb, and the JAK3 membranes were probed with the anti-phosphotyrosine mAb 4G10. A single phosphorylated band was detected for the samples treated with an anti-GM-CSFRß mAb. Reprobing of the membrane with an anti-JAK3 mAb resulted in the detection of a single protein with migration characteristics identical to those of the phosphorylated band detected on the 4G10 Western blot, indicating that the phosphorylated protein is, indeed, JAK3. These data indicate that the c subunit of the IL-15R
/
c receptor is functional in TF1 cells, even in the absence of the IL-15Rß chain, but that signaling is controlled by GM-CSFRß. The hybrid receptor is, therefore, functional in these cells.
We analyzed whether the IL-15R/
c complex was associated with the signaling molecule TRAF2 (Fig. 5 C). Analysis of total lysates with anti-TRAF2, anti-
c, or antiIL-15R
mAbs showed that TF1 cells expressed TRAF2 (55 kD), the 58 kD IL-15R
isoform and the
c chain (64 kD). After immunoprecipitation with an anti-TRAF2 antibody, reprobing of the TRAF2 membranes with mAbs recognizing the IL-15R
and
c chains resulted in the detection of specific 58 and 64 kD bands, respectively, indicating that TRAF2 is physically associated with the IL-15R
58 kD standard isoform and the
c chain.
We also showed that rIL-15, but not rIL-7, triggered phosphorylation of the p65 NF-B subunit inhibitor I
B
(42 kD) within 15 min in TF1 and TF1ß cells (Fig. 5 D). Reprobing the membranes with an antiß-actin mAb identified a band of similar intensity at 42 kD in each sample, indicating that the gel had been evenly loaded in terms of amount of protein. We also showed by confocal microscopy that rIL-15, but not rIL-7, induced the translocation to the nucleus of the p65 NF-
B subunit (unpublished data). These data indicate that, in TF1 cells, the IL-15R
chain is functional and able to activate the NF-
B factor. Moreover, not only does its association with the
c chain not interfere with NF-
B activation, it also probably renders the
c chain functional even in the absence of the IL-15Rß chain, as shown by the induction of JAK3 phosphorylation by rIL-15.
IL-15induced STAT5 Signaling Is Inhibited by AntiGM-CSFRß mAb in Normal CBPr CD34+/CD56- Cells.
Finally, we investigated the functional significance of the hybrid receptor in normal CB hematopoietic progenitors and the properties of this receptor in these cells. We used populations of CB progenitors that had been expanded for 5 d in the presence of SCF and Flt3-L (CBPr CD34+/CD56-). This facilitates proliferation of the hematopoietic progenitors, which maintain a CD34+/CD56- phenotype with no expression of lineage-specific markers (7). We found that rIL-15 quadrupled levels of STAT3 phosphorylation, which was not significantly affected by antiGM-CSFRß or isotype-matched control anti-gp130 mAbs (Fig. 6)
. As expected, rGM-CSF did not induce STAT3 phosphorylation. In contrast, anti-c, but not anti-gp130 mAbs inhibited constitutive STAT3 phosphorylation, suggesting interference with an autocrine IL-15dependent loop. Indeed, unprimed hematopoietic precursors secrete several hematopoietic cytokines that regulate normal hematopoiesis (3) by autocrine/paracrine loops, and IL-15 belongs to this group (20).
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Discussion |
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On the basis of these results, we propose a model for the activation of STAT5 by rGM-CSF via the hybrid receptor c/GM-CSFRß. We suggest that STAT5 tyrosine phosphorylation and nuclear translocation are regulated independently and that the translocation pathway is controlled by the
c/JAK3 complex, which, in cells with type I IL-4R, is necessary for the translocation of STAT5 to the nucleus (35).
The hybrid receptor is functional not only in leukemic cell precursors, but also in normal progenitors, although its functions appear more limited in normal cells. Indeed, in normal CBPr CD34+/CD56- progenitors, the hybrid receptor controls the IL-15dependent phosphorylation of STAT5 but not of STAT3 and STAT6. Moreover, levels of GM-CSFdependent STAT5 phosphorylation are decreased by anti-c mAb in CBPr CD34+/CD56- progenitors, but not in TF1ß cells. These differences may be due to differences in origin (normal versus pathological) and/or degree of primitiveness of the two types of CD34+ cell. In any case, our results emphasize the involvement of the hybrid receptor in the control of normal and pathological hematopoiesis, with this receptor probably displaying various functions during hematopoietic development.
The absence of the hybrid receptor on nonhematopoietic cells strengthens the evidence for its "specific" involvement in hematopoiesis. The distribution of this receptor in cells of various lineages and functions suggests that it is involved in the development of hematopoiesis, probably affecting engagement in myeloid, rather than lymphoid, lineages. Indeed, the inhibition of c/JAK3 signaling by the GM-CSFRß chain should be interpreted in light of the inhibition of lymphocyte maturation and myelopoiesis dysregulation observed in mice and humans bearing
c or JAK3 deletions or mutations (911).
In erythrocytic TF1 cells, we describe, for the first time, a novel IL-15R/
c/TRAF2 complex that triggers specific IL-15 signaling, even though these cells do not express the IL-15Rß chain23, a subunit indispensable for the
c chain functions (7). Both subunits of the IL-15R
/
c heterodimer are functional, although they behave differently from what would be expected based on the results obtained for other cell types. It has been reported that IL-15R
, associating with the signaling molecule TRAF2, activates the transcription factor NF-
B, in the absence of a link with the
c chain (12, 24). In contrast, in TF1 cells, IL-15R
and
c are associated but we nevertheless observed the phosphorylation, not only of I
B
, but also of JAK3 factors. However, the phosphorylation of JAK3 factor was only observed in samples treated with rIL-15 and antiGM-CSFRß mAb, indicating that the
c/JAK3 pathway is functional in the absence of the IL-15Rß chain but is controlled by the hybrid receptor.
The association of IL-15R/
c/TRAF2 may play a regulatory role at certain stages of hematopoiesis, associating NF-
B activation, which is involved in the control of erythropoiesis (15), with the conditional activation of JAK3-dependent pathways.
Finally, we showed that rIL-15 induced the phosphorylation of STAT6 both in leukemic TF1ß proerythroid precursors, and in normal CBPr CD34+/CD56- progenitors. Our data suggest that this unusual IL-15dependent pathway, the activation of which in mast cells was attributed to the high-affinity p65 IL-15RX receptor (28, 29), is also triggered in other types of CD34+ precursor. However, in TF1ß cells, this pathway is controlled by the classical IL-15R/ß/
c receptor and/or by the
c/GM-CSFRß hybrid receptor, whereas in normal CBPr, CD34+/CD56- IL-15dependent STAT6 phosphorylation is not controlled by the hybrid receptor. Overall, these results suggest the possible and complex involvement of STAT6 in the control of hematopoiesis (36) and provide further evidence that IL-15/GM-CSF are involved in cross talk.
Recent data have shown that unprimed hematopoietic progenitors secrete several mediators activating autocrine/paracrine regulatory loops (3). For instance, CD34+ cells may express GM-CSF transcripts (3) and a biologically active IL-15 that controls expression of the c chain, but is not competent for inducing the spontaneous differentiation of these cells into NK cells (20). Thus, IL-15 and GM-CSF may contribute, by means of the hybrid receptor, to the autocrine/paracrine cross talk that controls the development of hematopoietic cells (3), perhaps by inhibiting the spontaneous engagement of these cells in the lymphoid lineage. In contrast, in leukemic hematopoiesis, the action of the hybrid receptor is probably irreversible and may be involved in the molecular mechanisms responsible for the inhibition of differentiation in certain leukemic clones.
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Acknowledgments |
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Submitted: January 29, 2002
Revised: January 27, 2003
Accepted: January 28, 2003
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Footnotes |
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References |
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