* Department of Internal Medicine III, University Hospital, J. W. Goethe University of Frankfurt, Germany;
Institute of Transfusion Medicine, German Red Cross Blood Center, Frankfurt, Germany;
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
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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
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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.
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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.
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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.
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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 (-chain of VLA-4), CD11a (-chain of LFA-1), and CD49e (-chain of VLA-5) are shown. The profiles are representative for data obtained from three different donors.
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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.
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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).
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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 Gi 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.
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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 4 and 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 Gi 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 Gi-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.
Received December 13, 2001; revised May 18, 2002; accepted June 6, 2002.
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