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a First Department of Pathology, Kansai Medical University, Moriguchi City, Osaka, Japan;
b First Department of Surgery, North Tai-Ping Road Hospital, Beijing City, China;
c Department of Sales Skills Development, Human Competence Development Institute, Otsuka Pharmaceutical Inc., Tokushima, Japan
Key Words. Hepatocyte growth factor (HGF) • c-met • W/W mice • W/Wv mice • Sl/Sld mice • Hemopoiesis
Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570, Japan.
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Abstract |
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We next examine the stimulatory effects of hepatocyte growth factor (HGF) on hemopoiesis in W/W mice. HGF has a stimulatory effect on the colony formation (CFU-C) of W/W BM cells when cultured using either a methylcellulose assay (containing cytokines) or a long-term culture (LTC) assay. A similar stimulatory effect of HGF is observed in the other W or Sl locus-mutant mice (W/Wv and Sl/Sld mice), which show less severe anemia than W/W. The numbers of nonadherent cells and cobblestone colonies significantly increase in the LTCs using their BM cells. In addition, in vivo administration of HGF shows a transient increase in the CFU-C counts in BM cells and peripheral blood cells. RBC, WBC, and platelet counts also increased.
These results suggest that the SCF/c-kit system is not essential to hemopoiesis but that a compensatory system such as the HGF/c-met system functions in the SCF/c-kit system-deficient mice.
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Introduction |
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Stem cell factor (SCF) is an important hemopoietic regulatory factor for the growth and proliferation of primitive hemopoietic cells [20-22]. Mice harboring mutations at the dominant white spotting (W) or steel (Sl) locus express similar pleiotropic abnormalities [23]. They are characterized by a lack of pigmentation, gametogenesis, and a reduction in hemopoietic activity that leads to anemia and a deficiency of mast cells. Previous studies have shown that the Sl locus encodes SCF [24, 25], whereas the W locus encodes a receptor of SCF, a tyrosine kinase proto-oncogene (c-kit) [26], which is a tyrosine kinase-type receptor similar to c-met. The heterozygous W/Wv and Sl/Sld mice can survive to adulthood in spite of severe anemia [23]. However, W/W homozygotes, which completely lack the cell surface expression of the c-kit receptor, caused by a deletion of 234 nucleotides of the c-kit gene [27], die within two weeks of birth because of severe macrocytic anemia. A more severe anemia is observed on Sl/Sl homozygotes, which lack the SCF gene itself; they die on days 15-16 of gestation [28]. These facts strongly suggest that the SCF/c-kit system is essential to maintaining their life, and particularly to regulating immunohemopoiesis. In a recent report, however, it has been shown that the SCF/c-kit system is not essential, since the counts of both Thy-1low/Lin–/Sca-1+ hemopoietic stem cells (HSCs) and CFU-S increase in the Sl/Sl FL from day 13 to 14 of gestation [28]. It is conceivable that some unknown factor(s) or mechanism(s) may play a crucial role in the initiation of hemopoiesis. This finding encouraged us to investigate the possibility that the HGF/c-met system (a tyrosine kinase receptor system similar to the SCF/c-kit system) does function as a compensatory system in W or Sl mutant mice.
In the present study, we show that exogenous HGF has stimulatory effects on the proliferation and differentiation of HSCs in the BMCs obtained from W/W, W/Wv or Sl/Sld mice in vivo and in vitro. Moreover, we show that the differentiation of HSCs into all lineage cells is observed in W/W mice on days 3-5 after birth, although the total hematolymphoid cell number is greatly reduced. In addition, the functions of T and B cells obtained from their spleens is almost normal, in contrast to +/? (W/+ or +/+) littermates. These findings suggest the HGF/c-met system as at least one alternative mechanism in these mutant mice, although this compensatory system cannot completely substitute for the SCF/c-kit system.
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Materials and Methods |
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Monoclonal Antibodies (mAbs)
The following mAbs were used for cell surface molecule analyses: anti-CD4 (clone: GK1.5), anti-CD8 (53.6.7), anti-B220 (RA3-6B2), anti-Gr-1 (RB6-8C5), anti-Mac-1 (M1/70.5), anti-erythroid (TER119), anti-Thy-1.2 (30-H12), anti-Sca-1 (E13-161.7), and anti-c-kit (ACK-2 or 4). The mAbs were purchased from Pharmingen (San Diego, CA) or Caltag Laboratories (South San Francisco, CA). The mAbs were conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Anti-rhHGF antibody (Ab) was obtained from Otsuka Pharmaceutical Co., (Tokushime, Japan) and the anti-HGF Ab was conjugated with FITC using FITC labeling kit-K8081 (American Qualex; La Mirada, CA).
Fluorescence-Activated Cell Sorter (FACS) Analyses
Single cells (x 106) prepared from the FL, BM, spleen, or thymus were suspended in 50 µl of 2% fetal calf serum-phosphate buffered saline (FCS-PBS) with 0.1% NaN3 (staining buffer), mixed with optimal concentrations of the mAbs mentioned above, and incubated on ice for 30 min. Cells stained with the isotype controls were used as negative controls.
Some cells were stained with anti-HGF Ab-FITC, as follows: single cells (x 106) were preincubated at 10°C for 30 min with binding buffer (Hank's solution containing 20 µM Hepes and 2 mg/ml bovine serum albumin, pH 7.0). After spinning down, ice-cold binding buffer (100 µl) containing 200 ng/ml of rhHGF was added to the cells and the preparation was incubated at 10°C for 3 h. After washing, the cells were incubated with staining buffer (50 µl) containing 2 µg/ml of anti-HGF-FITC at 4°C for 30 min. Unstained cells or cells stained with anti-HGF-FITC alone were used as a control. The stained cells were analyzed using a FACScan (Becton Dickinson; San Jose, CA).
Mitogen Response and Mixed Lymphocyte Reaction (MLR)
Mitogen reactivity was assessed using spleen cells of three- to five-day-old W/W mice and their +/? littermates. Triplicate cultures (2 x 105/well) were set up in the presence of 25 µg/ml lipopolysaccharide (LPS) (Difco Lab.; Detroit, MI), 2.5 µg/ml concanavalin A (Con A) (Calchem-Behring Corp.; San Diego, CA) or 25 µg/ml phytohemagglutinin (PHA) (Difco Lab.) for three days in 96-well-flat bottom culture plates containing 10% FCS and 50 µM 2-mercaptoethanol (2-ME) in RPMI 1640. The cells were pulsed with 3H-TdR for the last 17 hours of the culture period. As a control, the spleen cells of eight-week-old adult +/+ mice and three- to five-day-old or eight-week-old normal B6 mice were also cultured in the same manner.
The spleen cells of W/W mice were also assessed for their capacity to react with allogeneic spleen cells. Irradiated (15 Gy) C3H spleen cells (4 x 105 /well) were added to culture medium containing the spleen cells of W/W mice (3 x 105/well). Three days later, 3H-TdR uptake was measured, as mentioned above.
Plaque-Forming Cell (PFC) Assay
The spleen cells were also assessed for their ability to differentiate into anti-sheep red blood cells (SRBC) PFCs. The spleen cells (5 x 106/ml) were cultured with or without SRBC (5 x 106/ml) as an antigen in 24-well plates, containing 10% FCS and 50 µM 2-ME in RPMI 1640. Five days later, the cultured spleen cells were collected, and the direct anti-SRBC PFC assay was performed.
Colony Forming Unit Culture (CFU-C) Assay
The fresh BMCs of three- to five-day-old W/W or +/? mice (5 x 103) were cultured using 12-well-culture plates in 1 ml of Methocult GF3434 (containing an optimal concentration of interleukin 3 (IL-3), IL-6, SCF, and erythropoietin from StemCell Technologies Inc.; Vancouver, BC, Canada) with or without rhHGF. To examine the direct effects of rhHGF on the BMCs of W/W mice, the cells were cultured for 12 days in Methocult GF3230 (hemopoietic growth factor-free, StemCell Technologies Inc.) with rhHGF alone. Colonies of 50 or more cells were scored as colonies under an inverted-phase microscope 12 days after culture.
BM-LTCs
BM-LTCs were carried out according to a modification of the Dexter's method [29]. Briefly, BMCs from W/Wv or Sl/Sld mice were cultured in 25 cm2 flasks containing 10% horse serum (Nikken Biological Medicine Laboratory; Kyoto, Japan, No. NDH-1128), 10–7M hydrocortisone, and 50 µM 2-ME in 
-medium. After the adherent cells reached subconfluence, each flask (in triplicate) was irradiated (20 Gy), then charged with fresh 2 x 105 BMCs of seven- to nine-week-old W/Wv or Sl/Sld mice, respectively, with or without 20 ng/ml rhHGF. At one-week intervals, the adherent foci or cobblestone colonies per flask were counted, and a half-culture medium containing nonadherent cells was then removed and replaced by fresh medium. The nonadherent cells per flask were counted, and some were examined for their ability to form hemopoietic colonies in methylcellulose culture in the presence of 10% medium conditioned by lung and abdominal wall tissues (containing mainly GM-CSF, M-CSF, G-CSF, IL-6, and SCF).
The BMCs from the W/W mice were cultured in the same manner as mentioned above, but with a BM stromal cell line (MS-5) [30] instead of W/W BM adherent cells.
Characterization of Mast Cells Derived from W/W BMCs
The mast-cell-like colonies derived from the methylcellulose culture using the BMCs of the W/W mice were picked up, washed with 2% FCS-PBS, then prepared for cytospin. The morphological characteristics of the cells were analyzed by staining with May-Giemsa, alcian blue or toluidine blue. The cells were also fixed with 2% glutaraldehyde containing 0.1 M phosphate buffer (pH 7.4), followed by post-osmification. After dehydration with graded ethanol, they were embedded in Epon (Oken Commercial Co, Ltd.; Tokyo, Japan). Ultrathin sections prepared by a Porter-Blum ultramicrotome (Sorval Inc.; Newtown, CT) were observed using a Hitachi H-600 electron microscope (Hitachi; Ibaragi, Japan) after double staining with uranyl acetate and lead citrate.
In Vivo Administration of rhHGF to W/Wv and Sl/Sld Mice
rhHGF was diluted in 2% FCS-PBS at a concentration of 20 µg/ml. W/Wv and Sl/Sld mice were injected i.v. with rhHGF at 200 µg/kg, or 2% FCS-PBS every two days for two weeks (total eight times). The first rhHGF injection was dated as day 0. Before or after rhHGF injection, each mouse was bled from the tail vein, and WBCs, RBCs, and platelets were counted.
Mononuclear cells (MNCs) in their blood were also collected using Lympholyte-M (
< 1.088, Cedarlane; Ontario, Canada). Thereafter, an HSC-enriched cell population (low-density fraction, < 1.077) was further purified using Lymphoprep (Nycomed Pharma AS; Oslo, Norway) from the MNCs. The purified cells were cultured in Methocult GF3434 for two weeks, and HPC (CFU-C) then counted. BMCs from the treated mice were also collected every week and assayed for CFU-C formation. Moreover, changes in cellularity of the BM cells were investigated every week by FACS analyses using mAbs (anti-B220, Thy 1.2, Gr-1, Mac-1, TER 119 and Sca-1 mAbs).
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Results |
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Expression of c-met in FLCs and BMCs of W/W, W/Wv and Sl/Sld Mice
Like other factors, HGF acts by binding to the cell surface receptor, c-met. The expression of c-met was examined on W/W FL cells and BMCs using a FACS. Since there is, at present, no mAb that binds to the extracellular domain of c-met, the cells were preincubated with rhHGF, then stained with anti-rhHGF Ab. The c-met expression was detected in the cells from the W/W mice, as seen in normal littermates (Fig. 5A). Moreover, in the FL, the number of c-met-positive cells increased in the low-density cell population containing HSCs and progenitor cells, in contrast to whole FLCs. A similar observation was obtained in the BMCs. To exclude the possibility that the Ab may react with cell surface molecules having a similar epitope to rhHGF, the Ab was incubated with FLCs or BMCs that had not been preincubated with rhHGF. No positive staining was observed in this condition (data not shown), indicating that the Ab shows a specific binding to rhHGF.
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Effects of Exogenous HGF on Colony Formation of BMCs from W/W Mice
To examine the effects of HGF on the colony formation of fresh BMCs from W/W mice, methylcellulose culture assays were performed using Methocult GF3434 containing optimal concentrations of hemopoietic growth factors. As shown in Figure 6, when 20, 50, or 100 ng/ml of HGF was added to the culture, increases in the number of colony-forming unit-cell (CFU-C) counts were noted in a dose-dependent manner in both W/W and +/? mice. An approximately 2.3- or 2.8-fold increase in the CFU-C counts was observed in the W/W mice at the concentration of 50 or 100 ng/ml HGF, whereas there was no significant increase after the addition of HGF in the +/? mice. The colonies included colony-forming unit-mast cells (CFU-Masts), CFU-Ms, CFU-GMs, and CFU-GMMs determined by cytospin. No burst-forming units-erythroid (BFU-Es) or CFU-GEMMs were found in the methylcellulose culture, even if a high concentration of HGF (100 ng/ml) had been added to the culture. The differentiation pattern of these CFU-Cs was not affected by the addition of HGF. To examine the stimulatory effects of HGF alone on the BMCs of the W/W mice, HGF was added to the methylcellulose culture containing no cytokines (Methocult M3230) and cultured for 12 days. There were no CFU-C colonies; HGF alone had no stimulatory effect on CFU-C formation. Therefore, these results suggest that HGF has a synergistic effect with other growth factors (IL-3, IL-6, etc.) on colony formation of BMCs of W/W mice as well as those of normal littermates. This result is compatible with our previous data [19] using normal mice.
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The addition of 20 ng/ml HGF to the BM-LTCs significantly increased the number of cobblestone colonies and nonadherent cells at the seventh week of culture in the W/Wv mice (Fig. 8) and at three to six weeks of culture in the Sl/Sld mice (Fig. 9). Cytological analyses revealed that the nonadherent cells contained all lineage cells such as erythroblasts, macrophages, granulocytes, and megakaryocytes (data not shown). The nonadherent cells obtained from the BM-LTCs of the Sl/Sld mice were examined for their ability to form colonies in the methylcellulose culture in the presence of 10% medium conditioned by lung and abdominal wall tissues. A more than 15-fold increase in CFU-C counts was observed in the culture containing 20 ng/ml HGF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). When HGF was administered to Sl/Sld mice, significant increases in the WBC and platelet counts were observed (Table 2). In the W/Wv mice, the RBC and platelet counts increased significantly (Table 1). The WBC counts in the W/Wv mice and the RBC counts in the Sl/Sld mice increased slightly (not significantly in comparison with control mice). In both mutant mice, there was a transient increase in the level of HPCs (CFU-Cs) in the BM, and thereafter, a similar transient increase was also observed in their PB (Table 3). Such a short-term increase in the CFU-C level is probably because the CFU-C progenitors differentiate immediately into mature cells and return to normal levels. However, the possibility that antibodies against hHGF are produced in the treated mice and neutralize HGF cannot be ruled out. Taken together, these results indicate that HGF has a stimulatory effect on mutant mice both in vivo and in vitro.
The differentiation of HSCs into all the lineage cells was observed in perinatal W/W mice, although the total number of hematolymphoid cells significantly decreased (Fig. 1). Moreover, lymphocytes can function normally in contrast to perinatal +/? littermates and normal B6 mice (Figs. 2-4). These data raise the important question as to whether the SCF/c-kit system is indispensable in hematolymphopoiesis. Very recently, Rodewald et al. reported that all the stages of B cell development are phenotypically indistinguishable between W/W and +/+ mice, and that day 13 FL cells of W/W mice differentiate into mature B cells in immunodeficient RAG-2–/– mice as well as those of +/+ littermates [32]. It has also been reported that W/Wv BMCs can induce the same number of CFU-Ss in the spleen of irradiated hosts as their littermates, although the colony size is much smaller [23]. Based on these observations, we speculate that the SCF/c-kit system is essential for cell proliferation but not for the differentiation and maturation steps of HSCs. Recently, we have found that the phenotype of pluripotent HSCs with the long-term (>1.5 years) reconstituting activity is c-kit<low and H-2high [33, 34]. We have also found that the CD 34+/c-kit<low cells, purified from the human cord blood, express c-kit molecules (c-kit<low
c-kitlowc-kit+) after a short incubation together with cytokines, indicating that c-kit<low cells are the precursors of c-kit+ cells [35]. These findings strongly support our present data.
The results presented in this paper clearly indicate that rhHGF acts as a synergist in combination with other cytokines in W and Sl mutant mice. Moreover, the presence of c-met receptor, to which rhHGF can bind, was confirmed in these mutant mice; the level of expression was similar to that of normal mice (Fig. 5). However, there is the possibility that other cytokines such as M-CSF or flk2 (flt3), in which the receptors are closely related to the c-kit receptor, also have similar stimulatory effects to HGF on these mutant mice. Indeed, the addition of 10, 20, or 40 ng/ml of M-CSF to the methylcellulose culture (Methocult GF3434) of W/W BMCs induced an increase in the total CFU-C count, similar to that with HGF (data not shown). Previously, Dubreuil et al. [36] demonstrated that W/W mast cells, which are obtained from the culture of their FLCs with IL-3 and express c-fms (a receptor of M-CSF) ectopically by infection with a c-fms retrovirus, can survive in the presence of M-CSF. It is known that mast cells normally do not express c-fms but express c-kit and utilize it for their survival and proliferation. W/W mast cells lack the extracellular domain of c-kit, but the intracellular transduction pathways are considered to be normal. Therefore, they speculated that c-fms shares common downstream effector elements with c-kit signal transduction. This finding supports the concept that the tyrosine kinase receptor family has overlapping or identical signal transduction pathways, and these can complement each other in the normal or unphysiological state.
Rodewald et al. [32] reported that no T cell differentiation was observed in RAG-2–/– mice that received W/W FLCs, although B cells were reconstituted similarly to +/+ littermates, as mentioned above. This result is not compatible with our present data, because functional T cells were observed, as shown in the T cell mitogen assay and mixed lymphocyte reaction using perinatal W/W spleen cells (Figs. 2 and 3). It is difficult to explain this discrepancy, since the cells examined and the experimental systems were different. We are now investigating in detail the pathway of T cell differentiation and the level of T cell development in W/W mice.
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Acknowledgments |
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References |
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