CXCR3 Expression on CD34ⴙ Hemopoietic Progenitors Induced by Granulocyte-Macrophage Colony-Stimulating Factor: II. Signaling Pathways Involved1 Tan Jinquan,2*‡ Liu Anting,*‡ Henrik H. Jacobi,* Christian Glue,* Chen Jing,*‡ Lars P. Ryder,† Hans O. Madsen,† Arne Svejgaard,† Per S. Skov,* Hans-Jørgen Malling,* and Lars K. Poulsen2* CXCR3, known to have four ligands (IFN-␥ inducible protein 10 (␥ IP-10), monokine induced by IFN-␥ (Mig), I-TAC, and 6Ckine), is predominately expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34ⴙ hemopoietic progenitors, in which ␥ IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34ⴙ hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34ⴙ progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. ␥ IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34ⴙ progenitors by means of CXCR3. ␥ IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34ⴙ hemopoietic progenitors, whereas SDF-1␣ induces both chemotaxis and adhesion in these cells. Interestingly, ␥ IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34ⴙ hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for ␥ IP-10- and Mig-induced chemotaxis and adhesion in CD34ⴙ hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/␥ IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells. The Journal of Immunology, 2001, 167: 4405– 4413.
A
t early stage, chemokines and their receptors were characterized mainly as inflammatory mediators. It is now known that the chemokine system is involved in many physiological and pathological processes, such as inflammation, hemopoiesis, tumorigenesis, embryogenesis, and HIV infection (1– 4). CXCR3 is expressed on activated/memory T cells at a high level, as well as on B cells, natural killer cells, and plasmacytoid monocytes (5–7). We recently reported that CXCR3 is also expressed on GM-CSF-stimulated, but not freshly isolated, CD34⫹ hemopoietic progenitors from human cord blood (CB)3 (8). Its *Laboratory of Medical Allergology, Allergy Unit, and †Laboratory for Tissue Typing, Department of Clinical Immunology, National University Hospital, Copenhagen, Denmark; and ‡Department of Immunology, Anhui Medical University, People’s Republic of China Received for publication April 24, 2001. Accepted for publication August 20, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was partially supported by the Simon Fougner Hartmanns Foundation and the National Science Foundation of China (no. 39870674). T.J. is supported by the Danish Allergy Research Center. C.J. is supported by a generous grant from Directionen af Hovedstadens Sygehusfællensskab, Denmark. C.G. is supported by Danish Environmental Research Program 1998 –2001. 2 Address correspondence and reprint requests to Dr. Tan Jinquan or Dr. Lars K. Poulsen, Laboratory of Medical Allergology, Finsen Center 7542, National University Hospital, 9 Blegdamsvej, DK-2200 Copenhagen Ø., Denmark. E-mail addresses:
[email protected] or
[email protected] 3
Abbreviations used in this paper: CB, cord blood; Cbl, Casitas B-lineage lymphoma; ␥ IP-10, IFN-␥ inducible protein 10; Mig, monokine induced by IFN-␥; PNA, peptide nucleic acid; SDF-1␣, chemokine stromal cell-derived factor-1␣; ZAP-70, -associated protein-70. Copyright © 2001 by The American Association of Immunologists
ligands IFN-␥ inducible protein 10 (␥ IP-10) and monokine induced by IFN-␥ (Mig) induce chemotaxis and adhesion of the cells (8). Structurally, chemokine receptors belong to a class of seven transmembrane domain receptors and are associated with heterotrimeric Gi proteins. Although they have structural similarity and couple to the same type of G protein, chemokine receptors can activate specific signal transduction pathways leading to diverse physiological and pathophysiological responses in different cells or organs. However, the association of the receptors with distinct signal transduction pathways is poorly understood. The tyrosine kinase Syk plays critical roles in signaling through immune receptors (9). A number of studies have identified the cell types (T, B, and NK cells) that require Syk for development and function and the receptors (TCR and B cell receptor) that use Syk as well as their downstream signaling effectors (9). Casitas Blineage lymphoma (Cbl), also known as c-Cbl and one of major substrates for Syk, is a 120-kDa adaptor protein that forms complexes with a wide range of signaling partners in response to various growth factors, and functions as a negative regulator of the tyrosine kinases in cells (10). Cbl-b, a newly described member of the Cbl family, operates and regulates the threshold of T and B cell signaling leading to the development of autoimmunity (11, 12). To date it is still unclear how GM-CSF induces CXCR3 to express on CD34⫹ hemopoietic progenitors, and which signaling pathway CXCR3 and its ligands (␥ IP-10 and Mig) use in CD34⫹ hemopoietic progenitors. The present study aims to investigate whether tyrosine kinase Syk family and its downstream members of the Cbl family play a role in the differentiation of CD34⫹ hemopoietic progenitors. 0022-1767/01/$02.00
SIGNALING AND CXCR3 IN CD34⫹ STEM CELLS
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Materials and Methods
Real time quantitative RT-PCR assay
CD34 hemopoietic progenitor cell purification
All real time quantitative RT-PCRs were performed as described elsewhere (8, 17, 18). Briefly, total RNA from CD34⫹ hemopoietic progenitors (1 ⫻ 106, purity ⱖ96%) was prepared by using a Quick Prep Total RNA Extraction Kit (Pharmacia Biotech, Uppsala, Sweden), and any potential contaminating chromosomal DNA was digested with DNase I according to the manufacturer’s instructions. For reverse transcription, the RNA was reverse transcribed by using oligo(dT)12–18 and Superscript II reverse transcriptase (Life Technologies, Grand Island, NY), according to the manufacturer’s instructions. Reverse transcription was performed for 60 min at 37°C, and any potential contaminating protein was denatured by incubation for 10 min at 95°C. The real time quantitative PCR was performed in special optical tubes in a 96-well microtiter plate (Applied Biosystems) with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems), according to the manufacturer’s instructions. By using SYBR Green PCR Core Reagents Kit (P/N 4304886; Applied Biosystems), fluorescence signals were generated during each PCR cycle via the 5⬘ to 3⬘ endonuclease activity of AmpliTaq Gold (17) to provide real time quantitative PCR information. The CXCR3 genes were generated by connecting the following sequences of the specific primers (purchased from DNA Technology, Aarhus, Denmark): sense, 5⬘-GGAGCTGCTCAGAGTAAATCAC-3⬘; and antisense, 5⬘-GCACGAGTCACTCTCGTTTTC-3⬘. All unknown cDNAs were diluted to contain equal amounts of -actin cDNA. The standards, “no template” controls, and unknown samples were added in a total volume of 50 l per reaction. PCR retain conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles with 15 s at 95°C, 60 s at 60°C for each amplification. Potential PCR product contamination was digested by uracil-N-glycosylase because dTTP is substituted by dUTP (17). All PCR experiments were performed with a hot start. In the reaction system, uracil-N-glycosylase and AmpliTaq Gold (Applied Biosystems) were applied according to the manufacturer’s instructions (17, 18). To analyze data of PCR products, two terms were used to express the results: ⌬Rn, representing the normalized reporter signal minus the baseline signal established in the first few cycles of PCR; and CT (threshold cycle), representing the PCR cycle at which an increase in reporter fluorescence signal above a baseline can first be detected.
⫹
CD34⫹ hemopoietic progenitor cells were purified as described elsewhere (13). Briefly, umbilical CB samples were collected according to institutional guidelines. CD34⫹ hemopoietic progenitors were isolated from mononuclear cells from CB. A positive selection procedure of anti-CD34 mAb-coated Dynabeads M-450 (Dynal Biotech, Oslo, Norway) were performed according to manufacturer’s instruction. The purity of CD34⫹ hemopoietic progenitors ranged from 92% to 98% as determined by flow cytometry.
Immunoprecipitation and immunoblotting For immunoprecipitation, as previously described (14), the pretreated cells (5 ⫻ 106) were solubilized in 1 ml of cold TNE buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Nonidet P-40 containing 20 mM EDTA, 10 g/ml aprotinin, 0.4 mM sodium vanadate, and 10 mM sodium pyrophosphate. The cell lysates were centrifuged at 10,000 ⫻ g for 5 min, and the supernatants were precleared with protein G-Sepharose. The lysates were then incubated with 5 g of rabbit anti-Syk (c-20), goat antiCbl (C-15), or goat anti-Cbl-b (C-20) (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA), at 4°C for 1 h, and the immune complexes were precipitated with protein G-Sepharose. For blotting, the immune complexes were washes five times with TNE buffer. For immunoblotting, as previously described (14), proteins in immunoprecipitates were resolved by SDS-PAGE under reducing conditions and then transferred to polyvinylidene difluoride microporous membrane (Schleicher & Schuell Life Science, Dassel, Germany). The membrane was blocked in 5% BSA-TBS (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl), and then incubated with antiSyk, anti-Cbl, or anti-Cbl-b (5 g/ml). Immunoblots were incubated with ␥-125I-labeled protein A (NEN Life Science Products, Boston, MA). After the incubation the membrane was washed with TBS containing 0.1% Tween 20 and followed by autoradiography.
Immune complex kinase assay As previously described (14), the immune complexes precipitated with protein G-Sepharose were washed four times with TNE buffer and four times with kinase buffer (50 mM HEPES-NaOH, pH 7.4, and 10 mM MnCl2). The immunoprecipitates were suspended in 20 l of kinase buffer containing 10 Ci of [␥-32P]ATP and incubated at 30°C for 30 min. The reaction was stopped by the addition of 15 l of 3⫻ sample buffer (195 mM Tris-HCl, pH 6.8, 9% SDS, 15% 2-ME, and 30% glycerol). Then the mixture was boiled for 5 min and subjected to 8% SDS-PAGE under reducing conditions, followed by autoradiography.
Peptide nucleic acid (PNA) antisense procedure As previously described with a modification (15), purified CB CD34⫹ hemopoietic progenitor cells were permeabilized with a buffered solution containing a relatively low concentration of detergent (20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 68 mM KCl, 0.05% Tween 20, 1 mM ethylenebis (oxyethlene-nitrilo) tetraacetic acid, 5.0% glycerol, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). The cells were then cultured in RPMI 1640 with 10% FCS in the presence of antisense PNA (Applied Biosystems, Foster City, CA) at 2 M with or without stimuli for the time period indicated. PNA sequences used were as follows: Syk antisense, (933)5⬘-ATTTTTTGA CATGGGA-3⬘(918); Cbl antisense, (745)5⬘-TATTGTCTTTTCCC-3⬘(732); and Cbl-b antisense, (980)5⬘-TTGTTGATTTTAGA-3⬘(967). For further assaying, the cells were extensively washed before the procedures. The viability of the cells was ⬎95% checked by a trypan blue exclusion test. More than 97% of cells were still CD34 positive detected by flow cytometry.
Flow cytometry As previously described (8, 16), for detection of CXCR3, CD34⫹ hemopoietic progenitors either freshly isolated or stimulated with cytokines indicated were first incubated with a mouse anti-human CXCR3 FITC-labeled mAb (clone no. 49801.111; R&D Systems Europe, Abingdon, U.K.) at 5 g/ml or 5 g/ml matched isotype mouse IgG1 (DAKO, Glostrup, Denmark) in PBS containing 2% BSA and 0.1% sodium azide for 20 min, followed by washing twice in staining buffer. The cells were incubated with a mouse anti-human CD34 class I PE-labeled mAb (Immu409; Coulter-Immunotech, Margency, France) at 5 g/ml in PBS containing 2% BSA and 0.1% sodium azide for 20 min, followed by washing twice. All procedures were conducted at 4°C. The cells were then fixed with 1% paraformaldehyde. The analyses were performed with a flow cytometer (COULTER XL; Coulter, Miami, FL).
Chemotaxis assay As previously described by Kim and Broxmeyer (19), chemotaxis and chemokinesis were assayed by a modification of a checkerboard assay. Fifty microliters of chemotaxis buffer (RPMI 1640, 0.5% BSA, and antibiotics) suspension with 2 ⫻ 105 cells/ml was added to the upper well of the chamber, which was separated from the lower well by a 5-m pore size, polycarbonate, polyvinylpyrolidone-free membrane without collagen coating (Nuclepore, Pleasanton, CA). Chemotaxis buffer was added to the lower chamber. Various amounts of chemoattractants as indicated were added to the chemotaxis buffer in the upper and/or lower chamber to form various chemoattractant concentration gradients (positive gradient, 0/⫹; negative gradient, ⫹/0; and zero gradient, ⫹/⫹ or 0/0). All tests were performed in triplicate. Chambers were incubated at 37°C, 5% CO2 for 4 h. Cells migrating into the three lower chambers were collected and counted using either a flow cytometer (COULTER XL) for 20 s at a high flow rate or a light microscope, in which identical results could be obtained. The cell migration was determined by calculating the percentage of input cells migrated into the lower chamber.
Adhesion assays Adhesion assays were performed as described previously (8, 16). Briefly, microtiter plates (96-well) were coated with laminin (20 g/ml; Sigma, St. Louis, MO) in PBS for 1 h at 37°C. Plates were washed with PBS and incubated with medium containing 0.2% BSA for 1 h to block nonspecific adhesion. Thereafter, single-cell suspensions were prepared in RPMI 1640 medium with 0.2% BSA at 4 ⫻ 105 cells/ml, and ␥ IP-10, Mig, or chemokine stromal cell-derived factor-1␣ (SDF-1␣) at 100 ng/ml were added. The cell suspension was added 100 l/well in triplicate to 96-well plates, and incubated for 60 min at 37°C. To remove nonadherent cells, an eighttip manifold was used to aspirate all but ⬃50 l of liquid from wells by suspending the manifold at a uniform distance from the well bottom. The wells were then washed by directing a careful stream of 0.2% BSA in PBS along the sides of the wells using eight-tip manifold, followed by careful aspiration. Subsequently the adherent cells were fixed with 1% formaldehyde and stained with 1% crystal violet. Crystal violet was then extracted by the addition of a 1/1 mixture of 0.1 M sodium citrate and ethanol (pH 4.2); absorbency was then read at 540 nm. Cells bound to collagen I (10 g/ml) on separate wells were used to represent 100% attachment. Background cell adhesion to 2% BSA-coated wells was subtracted from all
The Journal of Immunology readings. For inhibition assays, cells were preincubated with different Abs (at 4°C) for 30 min before assays.
Results Involvement of Syk in GM-CSF-induced CXCR3 expression on CD34⫹ progenitors We previously reported that CXCR3 is expressed on GM-CSFstimulated (98%), but not freshly isolated (⬍3%) CD34⫹ hemopoietic progenitors. Likewise, a high level CXCR3 mRNA (4.3 ⫻ 104 copies) is expressed on GM-CSF-stimulated CD34⫹ hemopoietic progenitors, but low level (6.3 ⫻ 101 copies) in the freshly isolated cells. Moreover, CXCR3 ligands ␥ IP-10 and Mig induce GM-CSF-stimulated CD34⫹ progenitor chemotaxis (⬃60 and 57% of input cells, respectively) and adhesion (⬃68 and 75% of input cells, respectively) by means of CXCR3 (8). To address which signaling transduction pathways might be involved in the CXCR3 expression induced by GM-CSF stimulation, we have examined the activation of Syk protein kinases and its downstream substrates Cbl and Cbl-b in CD34⫹ hemopoietic progenitors from CB upon the stimulation with GM-CSF. Immunoprecipitation, immunoblotting, and immune complex kinase assays revealed that the freshly isolated CD34⫹ cells did not have Syk protein kinase phosphorylation activity. However, stimulation with GM-CSF caused phosphorylation of Syk protein kinase in CD34⫹ cells within 60 min (Fig. 1A), and the Syk activation persisted for
FIGURE 1. The activation of Syk (A), Cbl (B), and Cbl-b (C) kinase (upper panels) and their protein contents (lower panels) in the CB CD34⫹ hemopoietic progenitors. The cells were freshly isolated, stimulated with GM-CSF (10 ng/ml) as indicated, or precultured with a-CD116 (5 g/ml, blocking mAb toward GM-CSF receptor) for 2 h and subsequently with GM-CSF as indicated. KA, Immune complex kinase assay; IB, immunoblotting. The cells were stimulated with GM-SCF for different time intervals as indicated, then lysed and immunoprecipitated with rabbit anti-Syk pAb, goat anti-Cbl pAb, or goat anti-Cbl-b pAb as described in Materials and Methods. The immunoprecipitates were subjected to kinase reactions or immunoblotted with each Ab indicated as described in Materials and Methods.
4407 24 h (data not shown). Preculturing cells with anti-CD116 mAb for 2 h, GM-CSF is unable to induce Syk protein kinase phosphorylation in these cells (Fig. 1A). Interestingly, neither Cbl nor Cbl-b can be activated to phosphorylation by GM-CSF within 60 min in CD34⫹ cells (Fig. 1, B and C); both of them were reported to be down-stream kinase substrate proteins (10 –12). Prolonging the stimulation time with GM-CSF to 24 h, Cbl or Cbl-b phosphorylation still could not be seen (data not shown). To further investigate the specificity of GM-CSF/Syk protein kinase phosphorylation, we adopted PNA antisense assays to generate specific Syk-blocking CD34⫹ cells. After 6-day culture with PNA Syk antisense, Syk kinase activation was totally abolished in CD34⫹ hemopoietic progenitors from CB that were subsequently stimulated with GM-CSF (10 ng/ml) (Fig. 2). We next examined the CXCR3 expression in CD34⫹ progenitors under different culture conditions. In Fig. 3A, there rarely are CXCR3⫹ cell fractions in freshly isolated human CB CD34⫹ hemopoietic progenitors (10.4%) (Fig. 3Ab). GM-CSF has significantly up-regulated the expression of CXCR3 on CD34⫹ progenitors up to 97.8% (Fig. 3Ac) (8). After 7-day incubation with cytokine-free medium, there has been no significant change of CXCR3⫹ cell fraction compared with freshly isolated cells (15%) (Fig. 3Ad). The CXCR3⫹ cell fractions almost totally vanished in the cells cultured with Syk PNA antisense for 6 days and subsequently stimulated with GM-CSF for 36 h (2%) (Fig. 3Ae), whereas there are 90% and 97% CXCR3⫹ cell fractions in the cells cultured with Cbl PNA antisense ( Fig. 3Af ) or Cbl-b PNA antisense (Fig. 3Ag) for 6 days and subsequently stimulated with GMCSF for 36 h, respectively. The results in Fig. 3B show that CXCR3 mRNA has been detected at low levels in cultured CD34⫹ hemopoietic progenitors with cytokine-free medium for 7 days. There are ⬃1.6 ⫻ 102 copies of CXCR3 mRNA in the tested samples of cultured CD34⫹ progenitors. GM-CSF stimulation can significantly up-regulate the expression of CXCR3 mRNA in CD34⫹ progenitors (4.3 ⫻ 104 copies) (8). There are ⬃4.5 ⫻ 102 copies of CXCR3 mRNA in the tested samples of CD34⫹ progenitors cultured with Syk PNA antisense for 6 days and subsequently stimulated with GM-CSF for 36 h; 1.4 ⫻ 104 copies and 6.2 ⫻ 104 copies of CXCR3 mRNA in the cells cultured with Cbl PNA antisense or Cbl-b PNA antisense for 6 days and subsequently stimulated with GM-CSF for 36 h, respectively. A linear relationship between CT and log starting quantity of standard DNA template or target cDNA (CXCR3) has been detected (data not shown). In all experiments, the correlation coefficients are ⬃0.94. Thus, GM-CSF binds to its receptor CD116 and subsequently activates Syk phosphorylation, and this induces CXCR3 expression. In contrast, Cbl and Cbl-b activation does not
FIGURE 2. The blocking effects of PNA Syk antisenses on the activation of Syk kinase in CB CD34⫹ hemopoietic progenitors. The cells were freshly isolated from CB or permeabilized before further assays, and then cultured in the presence or absence of PNA antisense indicated as described in Materials and Methods for 6 days and subsequently stimulated with GM-CSF (10 ng/ml) for different time intervals as indicated. As described in Fig. 1, the cells were then lysed, immunoprecipitated, and subjected to kinase reactions (KA) or immunoblotted (IB).
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FIGURE 3. A, Double-color flow cytometric analysis of CXCR3 expressions on human CB CD34⫹ hemopoietic progenitors. The cells were either freshly isolated (b) or cultured under the conditions as follows; with GM-CSF (10 ng/ml) for 36 h (c), with cytokine-free medium for 7 days (d), with Syk PNA antisense for 6 days and subsequently stimulated with GMCSF for 36 h (e), with Cbl PNA antisense for 6 days and subsequently stimulated with GM-CSF for 36 h (f), with Cbl-b PNA antisense for 6 days and subsequently stimulated with GM-CSF for 36 h (g), respectively. a, Isotype Ab control. The cells were then stained with FITC-labeled antiCXCR3 mAb as described in Materials and Methods. The percentages of CXCR3⫹ cells are indicated in Results. The data are from a single experiment, which is a representative of six similar experiments performed. B, Plots of the real time detection and amplification of mRNA of CXCR3 in CD34⫹ progenitors from human CB. The cells were cultured under the conditions as follows: with cytokine-free medium for 7 days (red plots, CT ⫽ 29.5), with Syk PNA antisense for 6 days and subsequently stimulated with GM-CSF (10 ng/ml) for 36 h (orange plots, CT ⫽ 28.1), with Cbl PNA antisense for 6 days and subsequently stimulated with GM-CSF for 36 h (blue plots, CT ⫽ 23.9), and with Cbl-b PNA antisense for 6 days and subsequently stimulated with GM-CSF for 36 h (green plots, CT ⫽ 21.8), respectively. Black plots represent the amplification of standard DNA template (2.0 ⫻ 104 copies, CT ⫽ 23.6) with a housekeeping gene (-actin). The showing plots are representatives of two similar experiments conducted.
seem to be involved in terms of GM-CSF-induced CXCR3 expression in CD34⫹ progenitors. We have further examined the migration and adhesion of CD34⫹ hemopoietic progenitors induced by ␥ IP-10, Mig, or SDF-1␣ (ligand for CXCR4) after the cells had been cultured under different conditions. As expected, the cells cultured with Syk PNA antisense for 6 days and subsequently stimulated with GMCSF for 36 h (non-CXCR3 expression cells) showed neither chemotaxis (11.2 ⫾ 2.1% of input cells at 100 ng/ml; Fig. 4A) nor adhesion (12.2 ⫾ 1.7% of input cells at 100 ng/ml; Fig. 4B) toward
SIGNALING AND CXCR3 IN CD34⫹ STEM CELLS
FIGURE 4. The migration (A) and adhesion (B) of CD34⫹ hemopoietic progenitors toward ␥ IP-10 (black bars), Mig (gray bars), or SDF-1␣ (open bars). The cells were cultured with Syk PNA antisense for 6 days and subsequently stimulated with GM-CSF (10 ng/ml) for 36 h before chemotaxis and adhesion assays. All results were determined as described in Materials and Methods and expressed as migration or adhesion of percentage of input cells (mean ⫾ SD) (n ⫽ 3), and based on triplicate determinations of chemotaxis or adhesion on each concentration of chemokines as indicated. ⴱ, Significant difference from controls (medium).
␥ IP-10. Likewise, the cells showed neither chemotaxis (12.4 ⫾ 1.8% of input cells at 100 ng/ml; Fig. 4A) nor adhesion (14.3 ⫾ 1.5% of input cells at 100 ng/ml; Fig. 4B) toward Mig. The cells have been detected to express CXCR4 (data not shown), and interestingly, the cell can respond to SDF-1␣ in terms of chemotaxis (42.4 ⫾ 7.5% of input cells at 100 ng/ml; Fig. 4A) and adhesion (51.4 ⫾ 11.5% of input cells at 10 ng/ml; Fig. 4B). All enhanced migrations of these cells were due to chemotaxis rather than chemokinesis (data not shown). To be sure of the effects of Syk PNA antisense on chemotaxis and adhesion of CD34⫹ hemopoietic progenitors, we used Syk DNA oligo antisense as a control. The results showed that Syk DNA antisense had not affected the chemotaxis and adhesion of CD34⫹ hemopoietic progenitors induced by ␥ IP-10 and Mig (data not shown). Thus, Syk-blocked cells maintain chemotactic and adherent abilities if these cells are exposed to appropriate chemokines (MIP-1␣) to which they express receptors. The reason for nonresponding to ␥ IP-10 and Mig in these cells might be due to lack of CXCR3 expression (this point will be further demonstrated below). ␥ IP-10 and Mig induce Syk, Cbl, and Cbl-b activation in CD34⫹ cells by means of CXCR3 We have next examined signaling transduction pathways involved in that ␥ IP-10 and Mig induce GM-CSF-stimulated CD34⫹ progenitor chemotaxis and adhesion. In Fig. 5A, the results of immunoprecipitation, immunoblotting, and immune complex kinase assays revealed that ␥ IP-10 induce Syk protein kinase phosphorylation in CD34⫹ progenitors stimulated with GM-CSF, in which CXCR3 were expressed (8), whereas activation of Syk
The Journal of Immunology
4409 we used GM-CSF to induce CXCR3 expression on CD34⫹ progenitors, then to generate specific Syk-, Cbl-, and Cbl-b-blocked CD34⫹ progenitors with PNA antisense, respectively. After a 6-day culture with PNA antisense, these cells still expressed CXCR3 at same level (data not shown), in the other words, PNA culture did not interfere with CXCR3 expression, but specifically blocked the signaling pathway. We subsequently used CXCR3 ligands ␥ IP-10 and Mig to stimulate the cells. As shown in Fig. 6A,
FIGURE 5. The activation of Syk (A), Cbl (B), and Cbl-b (C) kinase (upper panels) and their protein contents (lower panels) in the CB CD34⫹ hemopoietic progenitors. The cells were either freshly isolated or stimulated with GM-CSF (10 ng/ml) for 36 h and subsequently with ␥ IP-10 (100 ng/ml) as indicated, or stimulated with GM-CSF for 36 h and followed preculture with anti-CXCR3 mAb (5 g/ml, blocking mAb toward CXCR3) for 2 h and subsequently with ␥ IP-10 as indicated. The cells were stimulated with ␥ IP-10 for different time intervals as indicated, lysed, and immunoprecipitated with rabbit anti-Syk pAb, goat anti-Cbl pAb, or goat anti-Cbl-b pAb as described in Materials and Methods. The immunoprecipitates were subjected to kinase reactions or immunoblotted with each Ab indicated as described in Materials and Methods.
protein kinase was not detected in freshly isolated CD34⫹ progenitors, in which CXCR3 were absent (8). Preculture with blocking anti-CXCR3 mAb could block ␥ IP-10-induced Syk protein kinase phosphorylation. Likewise, ␥ IP-10 induced activation of Cbl in CD34⫹ cells stimulated with GM-CSF, whereas Cbl phosphorylation was not detected in freshly isolated CD34⫹ cells, in which CXCR3 were absent. Preculture with blocking anti-CXCR3 mAb could block ␥ IP-10-induced Cbl phosphorylation (Fig. 5B). Similarly, ␥ IP-10 induces Cbl-b phosphorylation in CD34⫹ progenitors stimulated with GM-CSF, whereas preculture with blocking anti-CXCR3 mAb could block ␥ IP-10-induced Cbl-b phosphorylation (Fig. 5C). We have also observed similar abilities of Mig in terms of Syk protein kinase, Cbl, and Cbl-b phosphorylation in CD34⫹ hemopoietic progenitors stimulated with GM-CSF (data not shown). Thus, ligands ␥ IP-10 and Mig bind to CXCR3 and cause Syk protein kinase, Cbl, and Cbl-b phosphorylation in GMCSF-stimulated CD34⫹ progenitors. Cbl-b, but not Syk and Cbl, phosphorylation is essential for ␥ IP-10- and Mig-induced chemotaxis and adhesion in CD34⫹ cells Because we detected that ␥ IP-10 and Mig can activate Syk, Cbl, and Cbl-b in GM-CSF-stimulated CD34⫹ progenitor by means of CXCR3, we next examined the importance of activation of Syk, Cbl, and Cbl-b in ␥ IP-10- and Mig-induced chemotaxis and adhesion of GM-CSF-stimulated CD34⫹ progenitors. To do so, first
FIGURE 6. In A, the blocking effects of PNA Syk antisenses on the activation of Syk kinase in CB CD34⫹ hemopoietic progenitors. The cells were freshly isolated from CB or stimulated with GM-CSF (10 ng/ml) for 36 h, then permeabilized before further assays, and then cultured in the presence or absence of PNA antisense indicated as described in Materials and Methods for 6 days and subsequently stimulated with ␥ IP-10 (100 ng/ml) for different time intervals as indicated. As described in Fig. 1, the cells were then lysed, immunoprecipitated, and subjected to kinase reactions (KA) or immunoblotted (IB). The migration (B) and adhesion (C) of CD34⫹ hemopoietic progenitors toward ␥ IP-10 (filled bars), Mig (gray bars), or SDF-1␣ (open bars). The cells were cultured with 10 ng/ml GMCSF for 36 h and subsequently stimulated with Syk PNA antisense for 6 days before chemotaxis and adhesion assays. All results were determined as described in Materials and Methods and expressed as migration or adhesion of percentage of input cells (mean ⫾ SD) (n ⫽ 3), and based on triplicate determinations of chemotaxis or adhesion on each concentration of chemokines as indicated. ⴱ, Significant difference from controls (medium).
4410 the PNA Syk antisense blocked the ␥ IP-10-induced activation of Syk kinase in CD34⫹ hemopoietic progenitors, even though CXCR3 were expressed on these cells. As expected, ␥ IP-10 induced Syk protein kinase phosphorylation in non-Syk-blocked CD34⫹ hemopoietic progenitors. We observed the same phenomenon where Mig was used as a stimulus (data not shown). This experiment verified the blocking effect of PNA Syk antisense. We next examined the abilities of ␥ IP-10 and Mig to induce chemotaxis and adhesion in these cells. Surprisingly, ␥ IP-10 can induce chemotaxis (41.1 ⫾ 6.5% of input cells at 100 ng/ml; Fig. 6B) and adhesion (47.5 ⫾ 7.3% of input cells at 100 ng/ml; Fig. 6C) in Syk-blocked GM-CSF-stimulated (CXCR3 expressing) CD34⫹ hemopoietic progenitors. Mig also, surprisingly, induces chemotaxis (42.3 ⫾ 9.1% of input cells at 100 ng/ml; Fig. 6B) and adhesion (46.7 ⫾ 8.1% of input cells at 100 ng/ml; Fig. 6C) in these cells. SDF-1␣ has been used as positive control where it induces chemotaxis (43.7 ⫾ 4.4% of input cells at 100 ng/ml; Fig. 6B) and adhesion (52.1 ⫾ 5.5% of input cells at 10 ng/ml; Fig. 6C) in these cells. All enhanced migrations of these cells were due to chemotaxis rather than chemokinesis (data not shown). Thus, Syk-blocking seems not to interfere with the chemotactic and adhesive function of CD34⫹ hemopoietic progenitors in terms of responsiveness to CXCR3 ligands ␥ IP-10 and Mig, if these cells already expressed CXCR3. As shown in Fig. 7A, the PNA Cbl antisense blocked the ␥ IP-10-induced Cbl phosphorylation in CD34⫹ cells, even though CXCR3 were expressed on these cells (data not shown). As expected, ␥ IP-10 induced Cbl phosphorylation in non-Cbl-blocked CD34⫹ hemopoietic progenitors. We observed the same phenomenon where Mig was used as a stimulus (data not shown). This experiment verified the blocking effect of PNA Cbl antisense. We next examined the abilities of ␥ IP-10 and Mig to induce chemotaxis and adhesion in these cells. ␥ IP-10 can induce chemotaxis (43.6 ⫾ 4.7% of input cells at 10 ng/ml; Fig. 7B) and adhesion (58.9 ⫾ 10.2% of input cells at 10 ng/ml; Fig. 7C) in Cbl-blocking GM-CSF-stimulated (CXCR3 expressing) CD34⫹ hemopoietic progenitors. Surprisingly, Mig can also induce chemotaxis (42.8 ⫾ 8.5% of input cells at 10 ng/ml; Fig. 7B) and adhesion (61.8 ⫾ 12.1% of input cells at 10 ng/ml; Fig. 7C) in these cells. SDF-1␣ has been used as control where it induces chemotaxis (31.4 ⫾ 5.4% of input cells at 10 ng/ml; Fig. 7B) and adhesion (34.2 ⫾ 6.2% of input cells at 10 ng/ml; Fig. 7C) in these cells. All enhanced migrations of these cells were due to chemotaxis rather than chemokinesis (data not shown). Thus, Cbl blocking seems only to be marginally modulating the chemotactic and adhesive function of CD34⫹ hemopoietic progenitors in terms of responsiveness to CXCR3 ligands ␥ IP-10 and Mig, e.g., increasing the sensitivity of the cells in the chemotaxis and adhesion assays (optimal concentration was 10 ng/ml instead of 100 ng/ml). As shown in Fig. 8A, the PNA Cbl-b antisense blocked the ␥ IP-10-induced Cbl-b phosphorylation in CD34⫹ cells, even though CXCR3 was expressed on these cells (data not shown). As expected, ␥ IP-10 induced Cbl-b phosphorylation in Cbl-b nonblocking CD34⫹ hemopoietic progenitors. We observed the same phenomenon where Mig was used as a stimulus (data not shown). This experiment verified the blocking effect of PNA Cbl-b antisense. We next examined the abilities of ␥ IP-10 and Mig to induce chemotaxis and adhesion of these cells. As shown in Fig. 8, B and C, ␥ IP-10 can induce neither chemotaxis (9.8 ⫾ 2.4% of input cells at 100 ng/ml; Fig. 8B) nor adhesion (11.7 ⫾ 1.8% of input cells at 100 ng/ml; Fig. 8C) in Cbl-b-blocked GM-CSF-stimulated (CXCR3 expressing) CD34⫹ hemopoietic progenitors. Mig also, surprisingly, induces neither chemotaxis (12.1 ⫾ 1.7% of input cells at 100 ng/ml; Fig. 8B) nor adhesion (11.5 ⫾ 1.9% of input
SIGNALING AND CXCR3 IN CD34⫹ STEM CELLS
FIGURE 7. A, Blocking effects of PNA Cbl antisenses on the activation of Cbl kinase in CB CD34⫹ hemopoietic progenitors. The cells were freshly isolated from CB or stimulated with GM-CSF (10 ng/ml) for 36 h, then permeabilized before further assays, and then cultured in the presence or absence of PNA antisense indicated as described in Materials and Methods for 6 days and subsequently stimulated with ␥ IP-10 (100 ng/ml) for different time intervals as indicated. As described in Fig. 1, the cells were then lysed, immunoprecipitated, and subjected to kinase reactions (KA) or immunoblotted (IB). The figure shows the migration (B) and adhesion (C) of CD34⫹ hemopoietic progenitors toward ␥ IP-10 (filled bars), Mig (gray bars), or SDF-1␣ (open bars). The cells were cultured with 10 ng/ml GMCSF for 36 h and subsequently stimulated with Cbl PNA antisense for 6 days before chemotaxis and adhesion assays. All results were determined as described in Materials and Methods and expressed as migration or adhesion of percentage of input cells (mean ⫾ SD) (n ⫽ 3), and based on triplicate determinations of chemotaxis or adhesion on each concentration of chemokines as indicated. ⴱ, Significant difference from controls (medium).
cells at 10 ng/ml; Fig. 8C) in these cells. SDF-1␣ has been used as control where it induces chemotaxis (39.8 ⫾ 9.7% of input cells at 100 ng/ml; Fig. 8B) and adhesion (43.1 ⫾ 12.4% of input cells at 100 ng/ml; Fig. 8C) in these cells. All enhanced migrations of these cells were due to chemotaxis rather than chemokinesis (data not shown). To be sure of the effects of Cbl-b PNA antisense on chemotaxis and adhesion of CD34⫹ hemopoietic progenitors, we used Cbl-b DNA oligo antisense as a control. The results showed
The Journal of Immunology
4411
Discussion
FIGURE 8. A, Blocking effects of PNA Cbl-b antisenses on the activation of Cbl-b kinase in CB CD34⫹ hemopoietic progenitors. The cells were freshly isolated from CB or stimulated with GM-CSF (10 ng/ml) for 36 h, then permeabilized before further assays, and cultured in the presence or absence of PNA antisense indicated as described in Materials and Methods for 6 days and subsequently stimulated with ␥ IP-10 (100 ng/ml) for different time intervals as indicated. As described in Fig. 1, the cells were then lysed, immunoprecipitated, and subjected to kinase reactions (KA) or immunoblotted (IB). The figure shows the migration (B) and adhesion (C) of CD34⫹ hemopoietic progenitors toward ␥ IP-10 (filled bars), Mig (gray bars), or SDF-1␣ (open bars). The cells were cultured with 10 ng/ml GMCSF for 36 h and subsequently stimulated with Cbl-b PNA antisense for 6 days before chemotaxis and adhesion assays. All results were determined as described in Materials and Methods and expressed as migration or adhesion of percentage of input cells (mean ⫾ SD) (n ⫽ 3), and based on triplicate determinations of chemotaxis or adhesion on each concentration of chemokines as indicated. ⴱ, Significant difference from controls (medium).
that Cbl-b DNA antisense had not affected the chemotaxis and adhesion of CD34⫹ hemopoietic progenitors induced by ␥ IP-10 and Mig (data not shown). Thus, Cbl-b-blocking seems to completely abolish the chemotactic and adhesive function of CD34⫹ hemopoietic progenitors in terms of responsiveness to CXCR3 ligands ␥ IP-10 and Mig, indicating that the Cbl-b signaling pathway is essential for the CD34⫹ cells in ␥ IP-10- and Mig-induced chemotaxis and adhesion by means of CXCR3.
It is still unclear what mechanisms and specific molecules are involved in 1) the mobilization of hemopoietic progenitor cells from the hemopoietic organs into peripheral blood, or the homing of hemopoietic progenitor cells; and 2) their trafficking through the hemopoietic organs during their maturation. Recently, we have described that CXCR3 is expressed on GM-CSF-stimulated, but not freshly isolated, CD34⫹ hemopoietic progenitors from human CB at both mRNA and protein levels. By means of CXCR3, ␥ IP-10 and Mig induce GM-CSF-stimulated CD34⫹ progenitor chemotaxis and adhesion (8), indicating that CXCR3-␥ IP-10 and -Mig receptor ligand pairs as well as the effects of GM-CSF on them may be especially important in cytokine/chemokine environment for the physiological and pathophysiological events of differentiation of CD34⫹ hemopoietic progenitors into lymphoid and myeloid stem cells, and subsequently to immune/inflammatory cells. What is the molecular mechanism behind these phenomena? In the present study, we further report that phosphorylation of tyrosine kinase Syk is a crucial step in the processes of GM-CSFinduced CXCR3 expression on CD34⫹ progenitors. ␥ IP-10 and Mig induce Syk protein kinase, Cbl, and Cbl-b phosphorylation in CD34⫹ cells by means of CXCR3, and Cbl-b phosphorylation is essential for ␥ IP-10 and Mig-induced chemotaxis and adhesion in CD34⫹ progenitors. In this study, there is no evidence that phosphorylation of Sky and Cbl is required for these events, noting the important difference between involved phosphorylations in CXCR3 induction and cellular activation by means of CXCR3. The Syk and -associated protein-70 (ZAP-70) protein tyrosine kinases form a family of signal-transducing molecules required for normal hemopoietic development (20). By virtue of their tandem Src homology 2 domains, Syk and ZAP-70 associate with tyrosinephosphorylated immunoreceptor tyrosine-based activation motifs contained within the cytoplasmic domains of activating cell surface receptors, including the B cell Ag receptors, TCRs, and the FcRs for IgG and IgE (20, 21). The modulation of Syk/ZAP-70 activity may result in the formation of different intracellular adapter protein complexes and thereby offer a mechanism to regulate biological responses. The Syk protein tyrosine kinase is essential for B, but not T or NK cell development (20). B cells are strictly dependent on Syk to transduce signals through the IgR (22). In the absence of Syk, B cell development is partially blocked at the pro-B cell stage and completely blocked at the pre-B cell stage (22). In a recent report, the role of Syk in T cell development in hemopoietic chimeras generated by using Syk-deficient fetal liver hemopoietic stem cells was investigated. It was found that Syk⫺/⫺ chimeras developed intestinal ␥␦T cells as well as other T cell subsets with reduction in number, indicating that Syk intervenes in early T cell development, but is not essential for the intestinal ␥␦T cell lineage to develop (23). In our study, we have found that GM-CSF by means of CD116 directly causes phosphorylation of tyrosine kinase Syk in CD34⫹ cells, whereas neither Cbl nor Cbl-b can be activated into phosphorylation by GM-CSF (Fig. 1). Syk protein kinase phosphorylation induced by GM-CSF was totally abolished in specific Syk-blocked CD34⫹ hemopoietic progenitors by PNA antisense (Fig. 2), resulting in totally blocked CXCR3 expression both at protein (Fig. 3A) and mRNA (Fig. 3B) levels. Thus, phosphorylation of Syk tyrosine kinases is a key step during the induction of CXCR3 by GM-CSF. It should be pointed out that in some of our PNA blocking assays, the direct target proteins are not significantly inhibited or totally abolished. Nevertheless, the target mRNA level has always been significantly inhibited (data not shown), and the function of phosphorylation has always been significantly inhibited or totally abolished. To our
4412 knowledge, the observations on phosphorylation of tyrosine kinases Syk induced by GM-CSF and on its vital relation with CXCR3 expression are novel. It indicates that Syk kinase at a very early stage of hemopoiesis plays a vital role in terms of CXCR3 expression on CD34⫹ hemopoietic progenitors. Expression of chemokine receptors (such as CXCR3) as well as the functions of their ligands (such as ␥ IP-10 and Mig) are widely considered as important events in various stages of hemopoiesis, such as maturation of hemopoietic progenitors, homing of immune cells to the extravascular compartment (24), optimal recruiting of hemopoietic progenitor cells to the bone marrow (25, 26), selectively entering into different hemopoietic organs, nesting on sites, differentiating, and further transmigrating into functional destinations, establishing T cell-dependent immunity, migration to or from the bone marrow during their development, and mutual direction mobilization between the bone marrow and the peripheral blood during immune reaction. Our results indicate that Syk protein kinase phosphorylation is involved in these processes. The questions still remain. Is it real directly that GM-CSF activates Syk protein tyrosine kinase? If not, by whom and how is phosphorylation of Syk protein tyrosine kinase mediated upstream and downstream? It will be very interesting to subject these questions to investigation. In general, signal transduction induced by chemokine receptors leads to the activation of G proteins and phospholipase C, and the elevation of cytosolic free calcium. A number of works have shown that stimulation of chemokine receptors results in the transient activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)-2 (27–30). Activation of ERK-2 is Ras-dependent, and prolonged activation causes its nuclear translocation and activation of transcription (31). Chemokines also stimulate phosphatidylinositol 3-kinase, leading to the formation of phosphatidyl 3,4,5,-triphosphate (32, 33) and the activation of protein kinase B (34). Phosphatidylinositol 3-kinase activity is necessary and sufficient to stimulate protein kinase B (35). Binding of its pleckstrin homology domain to 3-phosphoinositides and the phosphorylation of two critical residues accomplish activation of protein kinase B by phosphoinositide-dependent kinase(s) (35). At least four Cbl family members have been described: Cbl (c-Cbl), Cbl-b, Sli-1, and D-Cbl (36, 37). Cbl was originally noted for its negative regulation of signaling downstream of v-ErbB, the oncogenic form of the epidermal growth factor receptor (38). In the immune system, Cbl profoundly inhibits the function of T, B, and mast cells by down-regulating kinases such as ZAP-70 and Syk (39 – 42). The studies on Cbl-b-deficient mice reveal a provocative connection between Cbl-b-mediated protein degradation and the regulation of the threshold of T and B cell signaling leading to the development of autoimmunity (11, 12). Additionally, Cbl-b is widely expressed in many tissues and cells including hemopoietic cells (43, 44). An overexpression of Cbl-b in T cells induces the constitutive activation of NFAT (45), suggesting that Cbl-b plays a positive role in T cell signaling, most likely via a direct interaction with the upstream kinase ZAP-70. It should be stressed that Syk protein expression is apparently only partially blocked by the PNA treatment (Fig. 6A). Could that account for the absence of an effect of the Syk PNA treatment on CXCR3-dependent chemotaxis and adhesion? The consideration could be positive because Syk protein kinase phosphorylations induced by ␥ IP-10 and Mig totally vanished after Syk PNA treatment and that CXCR3-dependent chemotaxis and adhesion were still unaffected. In the present study, we have first demonstrated that ␥ IP-10 and Mig induce Syk, Cbl, and Cbl-b activation in CD34⫹ cells by means of CXCR3, and that Cbl-b activation, but not Syk and Cbl, is necessary for ␥ IP-10- and Mig-induced chemotaxis and adhesion in CD34⫹ cells. There are at least two ways
SIGNALING AND CXCR3 IN CD34⫹ STEM CELLS to interpret these results. First, the ligands by means of CXCR3 separately activate each kinase to induce chemotaxis and adhesion; Cbl-b activation is necessary and sufficient to cause biological functions. Second, the ligands by means of CXCR3 subsequently activate each kinase (first Syk kinase) to induce chemotaxis and adhesion; Cbl-b activation is in a key position to cause further biological cascades. The second pathway seems more likely. Therefore, further clarifying the pathways mentioned above is quite interesting in terms of understanding functions of chemokines and their receptors. It should be clear that Cbl and Cbl-b are not kinases. Their phosphorylation in pull-down immune complex kinase assays requires the preservation of associated kinases in the immunoprecipitates. It should also be mentioned that, in addition to Syk, there are other possible candidate kinases, for instance, Lyn and Src (46, 47). In summary, GM-CSF induces CXCR3 expression by means of Syk activation, and ␥ IP-10 and Mig induce chemotaxis and adhesion via Cbl-b activation in CD34⫹ progenitor cells, providing a useful insight into novel molecular mechanisms of the actions of CXCR3/␥ IP-10 and Mig, which may be especially important in cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.
Acknowledgments We thank Gitte Pedersen, Anne Corfitz, and Ulla Minuva for their excellent technical assistance.
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