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(Journal of Leukocyte Biology. 2002;72:837-845.)
© 2002 by Society for Leukocyte Biology

Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells

G. Bug*, T. Rossmanith*, R. Henschler{dagger}, L.A. Kunz-Schughart{ddagger}, B. Schröder§, M. Kampfmann*, M. Kreutz||, D. Hoelzer* and O. G. Ottmann*

* Department of Internal Medicine III, University Hospital, J. W. Goethe University of Frankfurt, Germany;
{dagger} Institute of Transfusion Medicine, German Red Cross Blood Center, Frankfurt, Germany;
{ddagger} Institute of Pathology, and
|| Department of Hematology/Oncology, University of Regensburg, Germany; and
§ MainGen Biotechnologie GmbH, Frankfurt, Germany

Correspondence: Dr. Gesine Bug, Medizinische Klinik, Abteilung für Hämatologie und Onkologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail: G.Bug{at}em.uni-frankfurt.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Seeding of hematopoietic progenitor cells (HPC) into the bone marrow requires a complex interaction between cell membrane and adhesion systems and cell signaling pathways. We established a multicellular, spheroid coculture model to study HPC migration in a three-dimensional stromal environment. Here, entry of primary CD34+ cells into stroma cell spheroids was independent of the integrins very late antigen (VLA)-4, VLA-5, lymphocyte function-associated antigen-1, and the chemokine receptor CXCR4. Experiments using a panel of bacterial toxins selectively targeting key regulators of cellular locomotion, the Rho family small GTPases Rho, Rac, and Cdc42, revealed a considerable reduction or even abrogation of TF-1 cell migration without an increase of apoptosis or impairment of proliferation. Pertussis toxin, an inhibitor of G{alpha}i proteins, showed a similar effect. In some in vitro invasion assays, phosphatidylinositol-3 kinase (PI-3K) was shown to mediate Rac- and Cdc42-induced cell motility and invasion. However, inhibition of the PI-3K pathway by LY294002 did not impair TF-1 cell migration in our three-dimensional model system.

Key Words: very late antigen • lymphocyte function-associated antigen-1 • phosphatidylinositol-3 kinase • pertussis toxin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell migration within tissues requires the integration of key events in signaling, cytoskeletal, membrane, and adhesion systems [1 2 3 ]. Small GTPases of the Rho family are central regulators of actin polymerization and motility in higher eukaryotes and include RhoA, RhoB, RhoC, RhoG, Cdc42, Rac1, and Rac2 in mammalian cells [4 ]. Upon activation, these heterodimeric GTP-binding proteins replace GDP by GTP catalyzed by guanine exchange factors [5 ].

Rac and Rho are considered candidate initiators and regulators of migration-driving membrane protrusions, such as ruffles, filopodia, and lamellipodia [4 , 6 ]. In T cell lines, dominant-negative Cdc42 abrogates chemokine-induced directional polarization and chemotaxis [7 ]. Recently, Rac2-deficient mice have been shown to display a reduced integrin-mediated adhesion of hematopoietic progenitor cells (HPC) accompanied by an increased proportion of HPC in the blood showing enhanced chemokine-induced migration and mobilization out of the marrow cavity [8 ]. This is in contrast to the decreased motility of their mature cell progeny (neutrophils, mast cells), suggesting that the consequences of Rac activation depend on cell type, maturation stage, and potential activation signals.

Targeting HPC to the bone marrow, e.g., in the clinical setting of stem cell transplantation, is a complex, multifaceted process in which adhesion molecules of the integrin family [very late antigen (VLA)-4, VLA-5, and lymphocyte function-associated antigen-1 (LFA-1); refs. 9 10 11 12 ] and the interaction of chemokines, such as the receptor CXCR4 with its ligand stromal cell-derived factor-1 (SDF-1) [13 14 15 ], are thought to be involved. However, the mechanisms of HPC locomotion, matrix interactions, and integrin dynamics in the bone marrow microenvironment are poorly defined, and the functional role of Rho family small GTPases in HPC migration is not well understood. Thus, the aim of the present study was to gain deeper insight into the impact of signaling through Rho, Rac, and Cdc42 on the mobility of human HPC in a stromal microenvironment. To closely mimic the in vivo situation of a three-dimensional, quiescent stroma, we have established a multicellular spheroid coculture model of murine stroma cell aggregates (cell line M2-10B4) and suspensions of umbilical cord blood (UCB)-derived CD34+ cells or the HPC line TF-1.

Among the strategies successfully used for the functional analysis of Rho GTPases, panels of bacterial toxins with selective inhibitory activity have proven to be particularly valuable experimental tools. The cell-permeable C3/C2 fusion toxin, consisting of the N-terminal part of Clostridium botulinum C2I and the full-length C. limosum adenosine 5'-diphosphate-ribosyltransferase C3, displays substrate specificity confined to the Rho family proteins RhoA, RhoB, and RhoC [16 ]. In contrast, lethal toxin produced by C. sordellii modifies Rac and Cdc42 from the Rho family and the Ras family members Ras, Ral, and Rap [17 18 19 ], and C. difficile toxin B was shown to monoglucosylate and thereby inactivate Rho, Rac, and Cdc42 [20 21 22 ]. We applied these three toxins (C3/C2 fusion toxin, lethal toxin, and toxin B) to differentially inhibit distinct GTPases and investigate signal transduction cascades involved in the regulation of HPC migration in a three-dimensional stromal environment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and reagents
The human leukemia cell line TF-1 (No. CRL-2003, American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640 (Gibco-Life Technologies, Paisley, UK) with 10% fetal calf serum (FCS; Hyclone, Greiner, Frickenhausen, Germany) in the presence of human interleukin (IL)-3 (R&D Systems, Wiesbaden, Germany) and granulocyte macrophage-colony stimulating factor (GM-CSF; 10 ng/ml each; Essex Pharma, Munich, Germany). The human bone marrow endothelial cell line (kindly provided by E. van der Schoot, CLB, Amsterdam, Netherlands) was cultured in gelatine-coated flasks in M199-medium (Gibco-Life Technologies) containing 10% human AB-plasma, 10% FCS, 1 ng/ml basic fibroblast growth factor (Cell Concepts, Umkirch, Germany), 5 U/ml heparin-sodium (Hoffmann-La Roche AG, Basel, Switzerland), 0.3 mg/ml glutamin, 100 U/ml penicillin/streptomycin, and 100 µg/ml geneticin (all from Gibco-Life Technologies). The murine bone marrow stroma cell line M2-10B4 (kindly provided by D. Hogge, Terry Fox Laboratory, Vancouver, Canada), modified to produce human IL-3 and GM-CSF, was maintained in RPMI 1640 (Gibco-Life Technologies) with 10% FCS, 0.06 mg/ml hygromycin B (Calbiochem, Bad Soden, Germany), 0.4 mg/ml geneticin, and 8 mM HEPES (both from Gibco-Life Technologies).

Collection of UCB samples and enrichment of CD34+ cells
UCB was collected immediately after delivery in a sterile tube containing 5000 I.U. heparin. Informed consent of the mother was obtained. Mononuclear cells (MNC) were isolated prior to processing of the cord blood sample over Ficoll/Hypaque density cushion (Biochrom, Berlin, Germany). The CD34+ selection was performed using the magnetic cell separation technology according to Miltenyi Biotec (Bergisch Gladbach, Germany). Briefly, UCB MNC were incubated for 15 min at 4°C with human immunoglobulin G (IgG) to block Fc receptors and with anti-CD34 antibodies (Ab) modified with a hapten (QBEND10, Miltenyi Biotec). Cells were washed with phosphate-buffered saline (PBS; Gibco-Life Technologies) containing 0.5% bovine serum albumin (BSA; Sigma Chemical Co., Deisenhofen, Germany) and 0.1% EDTA [Titriplex III, Merck, Darmstadt, Germany; referred to as magnetic cell sorter (MACS) buffer] and were incubated for 15 min at 4°C with a bead-conjugated antihapten mouse monoclonal Ab (mAb). Labeled cells were applied to an MS column (Miltenyi Biotec) placed in a permanent magnet; unbound cells were washed out with MACS buffer, and CD34+ cells were eluted from the column following removal from the magnet according to the manufacturer’s instructions. To improve the purity of the CD34+ cells, a second enrichment cycle was performed using a smaller column size (VS columns, Miltenyi Biotec).

Flow cytometry
For fluorescence-activated cell sorter (FACS) analysis, cells were washed in PBS containing 1% FCS and 0.1% NaN3 (Riedel de Haen, Seelze, Germany), and 5 µl phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated Ab was added, followed by incubation for 15 min at 4°C. After washing, cells were fixed in 300 µl PBS with 2% formaldehyde. FITC-conjugated Ab against CD34, CD45, and CD11a and PE-conjugated Ab against CD34, CXCR4, CD49d, and CD49e were applied. PE- and FITC-conjugated anti-human IgG1 were used as negative controls (all Ab from BD Bioscience, Heidelberg, Germany). Analysis was performed on a FACScan (BD Bioscience) using CellQuest and PC-Lysis software with 5000–10,000 collected events.

Determination of apoptotic cell death
Seven-amino-actinocycin (7-AAD; Sigma Chemical Co.) was diluted in PBS as a 200 µg/ml stock solution, which was stored at -20°C. A minimum of 2.5 x 105 cells was stained with FITC-conjugated mouse anti-human CD34 as described above. After washing, cells were incubated with 7-AAD in PBS at a final concentration of 20 µg/ml for 20 min in the dark. Cells were immediately analyzed by flow cytometry following incubation. At least 10,000 events were counted.

Spheroid assay
M2-10B4 spheroids were grown in 1% agarose-coated 96-well plates using the liquid overlay technique [23 ]. For initiation, 2.5 x 104 cells were inoculated per well in 200µl Iscove’s modified Dulbecco’s medium (IMDM; Biochrom) supplemented with 10% FCS. After 4 days, 1 x 104 CD34+ or TF-1 hematopoietic cells were added for cocultivation by replacing 100 µl of the medium. Spheroid cocultures were harvested 6–96 h later, washed with PBS, and dissociated by incubation with a 0.25% trypsin and 0.1% EDTA solution (1:3 in PBS; PAN Biotech, Aidenbach, Germany) for 5 min at 37°C and mechanic means (pipetting). Cell suspensions were filtered through a mesh (pore size 70 µm; Falcon, Becton Dickinson Labware, Le Pont de Claix, France) and incubated with FITC-conjugated anti-human CD45 Ab (or anti-human IgG1-Ab for control purposes) to determine the percentage of hematopoietic cells in the spheroids. Analysis was performed on a FACScan (BD Bioscience) using CellQuest and PC-Lysis software.

Immunohistological staining of multicellular spheroids (MCS)
For immunohistochemistry, spheroid cocultures and respective fibroblasts spheroid controls were shock-frozen in liquid N2 using Jung tissue-embedding medium (Leica, Nussloch, FRG) and stored at -80°C. Samples were serially sliced into 5–6 µm cryostat sections, mounted on SuperFrostPlus glass slides (Menzel-Glaeser, Braunschweig, Germany), and fixed in ice-cold acetone for 15–20 min. Sections were air-dried and eventually stored overnight at -20°C. A mouse anti-human CD45 mAb (1:250, working concentration 2 µg/ml; BD Pharmingen) was applied to demonstrate migration of hematopoietic cells into stroma MCS. Positive staining was visualized via the basic 3'-diaminobenzidine tetrahydrochloride (DAB) detection kit using the BenchMarkTM system (Ventana Medical Systems, Frankfurt, Germany). Sections were counter-stained with hematoxilin.

Blocking adhesion molecules and chemokine receptors
CD34+ UCB cells were stimulated overnight with 50 ng/ml stem cell factor (SCF), flt-3 ligand (FL), 20 ng/ml thrombopoietin (TPO; R&D Systems), and 10 ng/ml IL-3 in X-vivo10 medium (BioWhittaker, Walkerville, MD) with 1% BSA at 37°C in a humidified atmosphere. Before cocultivation, CD34+ cells were washed with PBS, and 5 x 105 cells/ml were incubated for 30 min at 4°C in X-vivo10 medium for 30 min at 4°C containing 5 µg/ml blocking mouse anti-human mAb: anti-VLA-4 mAb 2B4, anti-CXCR4 mAb 12G5 (both from R&D Systems), anti-VLA-5 mAb JBS5, and anti-LFA-1 mAb, Cat. No. MCA 1848XZ (both from Serotec, Eching, Germany). Mouse anti-human IgG1 (Immunotech, Marseille, France) was used as control. For cocultivation, cells were washed and resuspended in IMDM/10% FCS.

Cell adhesion assay
Fibronectin (FN; 5 µg/cm2), 1 µg/cm2 vascular cell-adhesion molecule 1 (VCAM-1), or 3% BSA diluted in PBS was adsorbed to wells of 24-well plates overnight at 4°C. Nonspecific bindings were blocked with PBS containing 0.1% BSA in PBS for the following 30 min at 37°C. TF-1 cells were washed once in PBS, resuspended in serum-free X-vivo10 with 1% BSA, and incubated with 5 µg/ml anti-IgG1, anti-VLA-5, or anti-VLA-4 antibodies for 30 min at 4°C. TF-1 cells were resuspended in IMDM containing 10% FCS and allowed to adhere to the coated plates for 1 h (VCAM-1) or 4 h (FN) at 37°C in a humidified atmosphere by 5% CO2. After a 1-h incubation, nonadherent cells were obtained by gentle agitation and were counted.

Inhibition of small GTPases and intracellular signaling pathways
HPC were incubated with 100 ng/ml toxin B or lethal toxin or with 1000 ng/ml C3/C2 fusion toxin and carrier in IMDM/10% FCS for 6 h at 37°C. (Toxins were kindly provided by H. Barth and K. Aktories, Institute of Pharmacology and Toxicology, University of Freiburg, Germany.) Before cocultivation, cells were washed with medium and subsequently resuspended in IMDM/10% FCS. The proportion of migrated cells was determined after 24 h.

To block intracellular signaling pathways, inhibitors were added to the M2-10B4/TF-1 spheroid cocultures with the following concentration: 100 ng/ml pertussis toxin (Calbiochem) and 10 µM LY294002 (Sigma Chemical Co.). To avoid deleterious cellular effects of these inhibitors, migrated cells were determined only after 12 h of coculture.

Western blot analysis
TF-1 cells (5x106) treated with or without 1 and 10 µM LY294002, respectively, were washed twice in PBS and lysed in 100 µl lysis buffer (20 mM Tris, pH 8, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM EDTA, 100 mM NaF; Sigma Chemical Co.) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 20 nM Na3VO4, 40 µM leupetin, and 100 U/ml aprotinin; Sigma Chemical Co.). After 30 min of incubation on ice, cell lysates were purified via centrifugation (13,000 rpm for 15 min at 4°C). Whole cell lysates (35 µg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was immunoblotted with the following polyclonal rabbit-anti-human Ab (New England Biolabs, Schwalbach, Germany): the phospho-specific AKT (Thr 308) Ab (1:1000) to determine the inhibition of the phosphatidylinositol-3-kinase (PI-3K) and an AKT Ab (1:1000) to verify that an equal amount of AKT protein was applied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HPC infiltrate spheroids of bone marrow stroma cells in a time- and dose-dependent manner
To analyze HPC migration into MCS, freshly isolated human UCB-derived CD34+ cells or the leukemic cell line TF-1 were cocultured with mouse bone marrow stroma MCS. HPC were identified by immunohistochemistry and FACS analysis using mAb against human CD45. This antigen is expressed abundantly on UCB-derived CD34+ and TF-1 cells and is negative on murine M2-10B4 cells (Fig. 1 ). Migration of HPC was detected as early as 6 h after initiation of cocultures and increased thereafter, reaching a maximum proportion of 53.3 ± 6.9% TF-1 cells after 72 h (Fig. 2A ) and 20.5 ± 2.6% UCB-derived CD34+ cells after 48 h (Fig. 2B) . Immunohistochemical staining of cryosections of spheroid cocultures demonstrated that HPC were located not only in the spheroid periphery but also in central areas (Fig. 3 ).



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  		Figure 1. Representative flow cytometric analysis of single-cell suspensions isolated from spheroid cocultures 12 h after addition of TF-1 cells (A, B, E, F) or 48 h after addition of UCB-derived CD34+ cells (C, D) to quantify migration of hematopoietic stem/precursor cells. The definition of regions and gates allowed the discrimination of stromal and hematopoietic cell populations. (A, C) R1 in the forward (linear)- versus side-scatter (log) dot plot was defined as cell population with intact size and granularity characteristics. (B, D) Labeling of the cell suspension with a FITC-conjugated mouse anti-human anti-CD45 mAb was performed to identify CD45-positive (B) TF-1 or (D) UCB-derived CD34+ cells (R2) and the highly autofluorescent M2-10B4 stromal cells in the FL-1 versus FL-2 fluorescence intensity dot plot. (E) TF-1 and (F) M2-10B4 cell controls stained with the FITC-conjugated anti-CD45 mAb.

 


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  		Figure 2. Time-dependent migration of (A) TF-1 cells (n=2–12) and (B) UCB-derived CD34+ progenitor cells (n=3; cells from different donors) into stroma cell spheroids. After defined time intervals, spheroid cocultures were harvested and trypsinized, and the proportion of HPC in the dissociated cell suspensions was determined by flow cytometric analysis according to Figure 1 . (C) Time-dependent migration of TF-1 cells into stroma cell spheroids (filled symbols). To distinguish migration processes from a proliferation-associated increase in the TF-1 cell population in spheroid cocultures, a subset of the MCS (open symbols) was separated from nonmigrated TF-1 cells by tranfer into fresh, agarose-coated 96-well plates. The figure shows two independent experiments.

 


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  		Figure 3. Immunohistochemical staining of (A) UCB-derived CD34+ progenitor cells and (B) TF-1 cells after 48 h in coculture with stroma MCS. Cryosections (5 µm) of spheroid cocultures were labeled with a mouse anti-human CD45 mAb and the Ventana basic DAB detection kit and were counter-stained with hematoxilin.

 
For establishing spheroid cocultures as a model system to study migration processes of hematopoietic stem cell populations, a subclone of the M2-10B4 murine stroma cell line was applied, as it is described to show a higher capacity to support long-term, in vitro survival of a very primitive population of HPC. This cell line was engineered to produce high levels of human G-CSF (190 ng/ml) and IL-3 (4 ng/ml) [24 ]. To distinguish proliferation and migration phenomena and exclude IL-3-dependent stimulation of TF-1-proliferative activity affecting cell numbers in spheroid cocultures, cocultures were removed from the nonmigrated TF-1 cell population after 24 h by transfer into a fresh 96-well plate (Fig. 2C) . The HPC content of TF-1 cell-depleted MCS slightly increased from 10.2 and 19.3% to 18.3 and 30.1% over another 48 h, and a proportion of 49.0 and 61.3% TF-1 cells was detected in cocultures that remained in the TF-1 cell suspension. These data indicate that the increasing number of TF-1 cells in stroma cell spheroids during the first three days of coculture primarily results from continuous infiltration, and the contribution of proliferation of TF-1 cells is rather negligible.

Migration of hematopoietic progenitor cells into stroma cell spheroids is not impaired by blocking ß1- and ß2-integrins or chemokine receptors
To investigate which adhesion molecules may be involved in the locomotion of HPC into MCS, we analyzed the integrin and chemokine receptor profile of UCB-derived CD34+ and TF-1 cells. As previously described, UCB-derived CD34+ cells express the major integrins LFA-1, VLA-4, and VLA-5 as well as the chemokine receptor CXCR4 on their cell surface (Fig. 4 ). CXCR4 receptors proved to be functional in transwell assays (data not shown). In contrast to primary CD34+ cells, the TF-1 cell line was negative for CXCR4 and for the ß2-integrin LFA-1.



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  		Figure 4. Surface expression of CXCR4 and integrins on viable UCB-derived CD34+ progenitor cells after prestimulation in the presence of SCF, TPO, FL, and IL-3 for 24 h. Isotype controls (mIgG1Ab) and expression profiles of CXCR4, CD49d ({alpha}-chain of VLA-4), CD11a ({alpha}-chain of LFA-1), and CD49e ({alpha}-chain of VLA-5) are shown. The profiles are representative for data obtained from three different donors.

 
Migration of UCB-derived CD34+ cells in spheroid cocultures was unaltered by incubation with mAb blocking VLA-4, VLA-5, and LFA-1, whereas blocking the chemokine receptor CXCR4 slightly reduced the number of migrating cells to 81 ± 12% (Fig. 5A ). The efficacy of the mAb used was determined in cell-adhesion assays. On plastic surfaces coated with VCAM-1 or FN, TF-1 cell adhesion was markedly reduced by anti-VLA-4 and anti-VLA-5 mAb, respectively (Fig. 5B and 5C) . As strong adhesion of HPC on intercellular adhesion molecule-1 was not achieved in our hands, blocking function of anti-LFA-1 mAb was proven in a cytotoxicity assay (data not shown).



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  		Figure 5. (A) Effect of blocking mAb against CXCR4, VLA-4, VLA-5, and LFA-1 on the migration of UCB-derived CD34+ progenitor cells after prestimulation in the presence of SCF, TPO, FL, and IL-3 for 24 h. To avoid rapid internalization, blocking mAb were permanently present in the supernatant throughout cocultivation. The graph shows the proportion of migrated cells in suspensions of dissociated MCS cocultures normalized for the respective isotype control for three independent experiments (mean±SEM). (B, C) Anti-VLA-4 and anti-VLA-5 mAb selectively reduce adhesion of TF-1 cells to plastic surfaces coated with (B) VCAM-1 (two independent experiments carried out in duplicates) or (C) FN (n=3) shown as mean ± SEM.

 
To avoid escape mechanisms via rapid internalization of mAb-integrin complexes and functional integrin re-expression, mAb were permanently present in the supernatants. To additionally exclude the contribution of constitutively produced growth factors by the M2-10B4 stroma cells (IL-3 and G-CSF), we examined TF-1 cell migration into stroma cell spheroids using media supplemented with excess concentrations of IL-3 or G-CSF added to the spheroid supernatant and performed experiments with M2-10B4-conditioned media. No alteration in the TF-1 migration was observed for both experimental designs (data not shown). Taken together, these results imply that HPC cells do not follow a gradient of human or M2-10B4-derived cytokines or chemotactic factors.

Inhibitors of Rho family small GTPases abrogate entry of hematopoietic progenitor cells into stroma cell spheroids
To investigate whether migration of HPC is differentially affected by individual members of the Rho family of small GTPases, we selectively inhibited small GTPases using a panel of bacterial toxins. To inactivate the small GTPases Rho, Rac, and Cdc42 [25 ], TF-1 cells were pretreated for 6 h with increasing concentrations of C. difficile toxin B (10, 50, and 100 ng/ml). In the presence of 100 ng/ml toxin B, the TF-1 content of 24-h cocultured MCS was reduced from 30.3 ± 1.2% to 4.2 ± 3.9% (Fig. 6A ). Likewise, entry of UCB-derived CD34+ cells into MCS decreased from 26.6 ± 0.5% to 8.8 ± 3.5% (Fig. 6B) . Additional flow cytometric analyses with 7-AAD imply that toxin B does not enhance TF-1 cell apoptosis during the time course of our experiments. Having demonstrated a significant blockade of TF-1 cell infiltration by toxin B, we attempted to further define the pathways involved in HPC migration by studying the influence of the more specific inhibitors C. limosum-derived C3/C2 fusion toxin and C. sordellii lethal toxin. Inhibition of the Rho family proteins Rho A, B, and C by C3/C2 fusion toxin and of Rac and Cdc42 by lethal toxin proved to be as effective as the more broadly acting toxin B in down-regulating TF-1 cell migration from 26.0 ± 8.5% to 5.0 ± 1.0% and 6.7 ± 3.8%, respectively (Fig. 6C) . This suggests that blockade of individual Rho family GTPases may be sufficient to significantly inhibit the migratory activity of HPC in vitro.



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  		Figure 6. Inhibitors of Rho family GTPases impair migration of TF-1 and UCB-derived CD34+ progenitor cells into murine M2-10B4 stroma cell spheroids. (A) C. difficile toxin B reduces entry of TF-1 cells into MCS in a dose-dependent manner. TF-1 cells were pretreated for 6 h with increasing concentrations of C. difficile toxin B (10, 50, and 100 ng/ml), washed, and cocultured with M2-10B4 spheroids for 24 h (n=3). (B) Bacterial toxins were added to UCB-derived CD34+ progenitor or (C) TF-1 cells 6 h prior to cocultivation with stroma spheroids. After 24 h, the proportion of HPC in spheroid cocultures was determined by flow cytometry according to Figure 1 (n=3, mean±SEM).

 
Effect of bacterial toxins is mimicked by pertussis toxin but not by PI-3K inhibitors
PI-3K is hypothesized to be involved in Rho family small GTPase-mediated effects. Compared with control MCS, exposition of TF-1 cells and stroma MCS to the PI-3K selective inhibitor LY294002 (10 µM) did not significantly alter cell migration into stroma MCS (15.8±5.8% vs. 13.2±5.2%; Fig. 7A ). To avoid deleterious effects of the inhibitor on TF-1 cells and MCS, this set of experiments was carried out within 12 h. In contrast, treatment of TF-1 cells with the G{alpha}i inhibitor pertussis toxin (100 ng/ml) resulted in a clear inhibition of TF-1 cell migration (4.1±1.4%), analogous to the results obtained for the inhibition of small GTPases by the bacterial toxins.



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  		Figure 7. Activated G{alpha}i proteins are required for TF-1 cell migration, whereas PI-3K seem to be dispensable. (A) Proportion of TF-1 cells in spheroid cocultures with murine M2-10B4 stroma cell spheroids incubated with the specific inhibitors pertussis toxin (100 ng/ml) and LY294002 (10 µM) for 12 h, beginning with the initiation of cocultivation (n=4). (B) Serum components or M2-10B4-derived cytokines do not compensate for the inhibitory effect of LY294002 on the PI-3K pathway. In the presence of 10 µM LY294002, PI-3K phosphorylation is blocked. Immunoblotting was performed 6 h after addition of LY294002. As control, TF-1 cells were incubated in serum containing medium without LY294002.

 
Supplementation of 10% FCS proved to be mandatory for coculture experiments, as serum-free medium completely abolished TF-1 cell infiltration of the MCS (data not shown). To investigate whether serum components or M2-10B4-derived human IL-3 may compensate the inhibitory effect of LY294002, immunoblotting was performed 6 h after addition of LY294002 (1 and 10 µM). As controls, cells were incubated in TF-1 medium alone. Total cell lysates were analyzed with Ab that specifically recognize Akt phosphorylated on threonine 308. The serine/threonine kinase Akt functions in a pathway involving PI-3K, and activation of Akt requires PI-3K [26 ]. To show that equal amounts of protein were loaded in each lane, the blot was restained with anti-Akt Ab. Phospho-Akt (Thr308) was clearly identified at 65 kDa in control cells; in the presence of LY294002 (10 µM), phosphorylation of Akt is blocked (Fig. 7B) . In summary, these experiments indicate that locomotion of TF-1 cells in stroma cell spheroids can be significantly inhibited by pertussis toxin but does not require PI-3K.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In healthy adults, primitive hematopoietic stem and progenitor cells are concentrated in the sinusoids of the bone marrow, where they are located in close proximity to arrested stromal elements. During hematopoietic development, HPC migrate in a sequential, temporo-spatially defined regime between different hematopoietic organs (yolk sac, spleen, liver, bone marrow). Likewise, HPC migrate to and home in the bone marrow microenvironment to re-establish the hematopoietic system following therapeutic stem cell transplantation. Although considerable progress was made in determining the role of chemokines and adhesion molecules in human stem cell engraftment, biochemical pathways controlling adhesion and migration of hematopoietic progenitor cells have rarely been studied [27 ].

Recently, Rho family small GTPases, which are considered key regulators of cell migration [4 ], have been implicated in HPC function [8 ]. In the present study, we demonstrate a significant impact of the Rho-related GTPases Rho, Rac, and Cdc42 on the in vitro, migratory activity of HPC in a three-dimensional network of bone marrow stroma cells. For these studies, we applied stroma cell spheroids of the murine stroma cell line M2-10B4, which is known to support long-term, in vitro cultures of a very primitive population of human HPC [24 ]. This model was adapted from a spheroid coculture system established to investigate reciprocal tumor-immune cell [28 29 30 ] and fibroblast-immune cell interactions [31 , 32 ]. Multicellular tumor spheroids serve as a well-established, three-dimensional in vitro model that more closely resembles the in vivo situation in tumors with regard to cell shape and cellular environment than monolayer cultures and thus better reflects the biological behavior of cells [33 , 34 ].

Our results indicate that the interaction between HPC and three-dimensional stroma cell spheroids is rather independent of the integrins that are, in general, considered essential for HPC migration; i.e., migratory activity of HPC was not inhibited by blocking mAb against {alpha}4 and {alpha}5 integrins, although the human progenitor cell line TF-1 was shown to express VLA-4 (CD49/CD29) and VLA-5 (CD49e/CD29) on the cell surface, and CD34+ UCB cells were positive for VLA-4, VLA-5, and LFA-1 (CD11a/CD18), which is in accordance with the expression pattern in human bone marrow-derived CD34+ cells [35 36 37 38 ].

Friedl et al. [39 ] studied T cell migration in three-dimensional collagen matrices and verified that interactions with collagen fibers with concomitant migration are unaffected by adhesion blocking anti-ß1 and anti-ß2 integrin antibodies, and adhesion and migration can efficiently be blocked by antagonistic antibodies in two-dimensional models [40 41 42 43 ]. From our observation that HPC do not require adhesion-mediating ß1 or ß2 integrins for locomotion in a three-dimensional, cellular microenvironment, we hypothesize that like T lymphocytes, HPC possess a broad spectrum of migration strategies with diverse, underlying, regulatory pathways. These strategies range from adhesive motility across two-dimensional surfaces to largely integrin-independent migration processes predominantly guided by alterations in shape and morphological flexibility as recorded in three-dimensional environments [44 ].

The chemokine SDF-1 was shown to regulate interactions between immature human CD34+ cells and the bone marrow microenvironment, i.e., stroma cells and extracellular matrix, by activating LFA-1, VLA-4, and VLA-5, which are thought to be crucial for stem cell engraftment [45 ]. However, two observations argue against a direct adhesion-mediating and/or migration-inducing role of SDF-1/CXCR4 in the migration of HPC into stroma cell spheroids: Blocking anti-CXCR4 mAb did not substantially reduce infiltration of UCB-derived CD34+ cells in the stroma spheroid model, although biological activity of the SDF-1/CXCR4 pathway was demonstrated in a transwell migration assay with an endothelial cell monolayer; and TF-1 cells migrate into stroma cell spheroids without expressing CXCR4 receptors. These observations are in accordance with a recent report by Soede et al. [46 ] who demonstrated in vivo dissemination of a murine myeloid leukemia cell line (MDAY-D2), which does not express CXCR4 and shows no chemotactic response to SDF-1 [46 ]. Thus, SDF-1 is unlikely to be involved in bone marrow colonization by MDAY-D2 or TF-1 cells. Infiltration of stroma cell spheroids by TF-1 cells was effectively inhibited by pertussis toxin, an inhibitor of signal transduction mediated by the G{alpha}i subunit, which was shown to reduce in vitro migration of human CD34+ cells toward a gradient of SDF-1 in the transwell system [13 ]. Regulation and function of large and small G proteins have long been studied as independent regulator molecules in signal tranduction. It is now evident that Rho and other small G proteins of the Rho family can be activated through stimulation of heterotrimeric G proteins [5 ]. Migration of HPC into the three-dimensional network of bone marrow stroma cells was significantly impaired by inhibitors of Rho or Rac and Cdc42. Various studies have suggested PI-3K as an upstream regulator of Rac in growth factor or integrin-induced cytoskeleton reorganization [4 , 47 48 49 ]. However, a contribution of the serine/threonine kinase Akt to the activation of Rac could not be verified in our studies. Previous investigations on Rac2 activation revealed that stimulation of human neutrophils with G{alpha}i-coupled receptor agonists leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate causes a slow but more sustained Rac2 activation in a PI-3K-independent manner [50 ].

In conclusion, we provide evidence for important regulatory functions of Rho family small GTPases in HPC migration processes using a novel stroma cell spheroid coculture model that is considered a valuable tool to further study the impact of Rho family members on HPC behavior in a three-dimensional stromal network. Recent in vivo data suggest that our in vitro model reflects the in vivo situation of HPC seeding into the bone marrow: Homing of the murine progenitor cell line FDCP-Mix was completely prevented by blocking Rho family GTPases, and inactivation of Rac and Cdc42 by lethal toxin led to a 50% reduction [51 ]. So far, we have no indication for integrins or chemokines to play an essential role in HPC infiltration of stroma spheroids. However, further experiments need to be performed to verify these observations and to identify additional pathways involved in the mediation of HPC locomotion in a three-dimensional stromal environment.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Knochenmarkspenderdatei, Germany. The authors acknowledge the excellent technical assistance of Frank van Rey. We gratefully thank Dr. Holges Barth and Prof. Dr. Klaus Aktories, Institute of Pharmacology and Toxicology, University of Freiburg, for the gift of the bacterial toxins and Dr. Hans-Jörg Grimmiger and the staff of midwives at the St. Vincenz- and Elisabeth-Hospital Mainz for the UCB collection.

Received December 13, 2001; revised May 18, 2002; accepted June 6, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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