kit receptor-specific antibody, we show that the c-kit/SF system contributes to the survival of lymphocyte prog- enitors and enhances the proliferative responses of ...
Development 115, 1133-1147 (1992) Printed in Great Britain © The Company of Biologists Limited 1992
1133
Developmentally regulated cell surface expression and function of c-kit receptor during lymphocyte ontogeny in the embryo and adult mice RONALD PALACIOS 1* and SHIN-ICHI NISHIKAWA2 1Basel
Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland of Pathology, Kumamoto University Medical School, Kumamoto, Japan
2Department
*Author for correspondence
Summary We have used a c-kit-specific monoclonal antibody, immuno-fluorescence staining and flow fluorocytometry or microscopy analysis to assess the cell surface expression of the c-kit receptor on a panel of non-transformed clones representing different stages of T- and Blymphocyte development, freshly isolated lymphoid cells from thymus, bone marrow and spleen of young adult C57BL/6 mice and cells from yolk sac, thymus and liver of developing C57BL/6 mouse embryos. Pro-T, Pro-B and Pre-B clones derived from thymus or liver of 14day embryos are c-kit+. Starting at day 8 to 8.5 in yolk sac, day-10 in fetal liver, and day 11 to 12 in fetal thymus, there are many c-kit+ cells. The number of ckit+ cells in liver and thymus increases up to day 15 and progressively decreases thereafter. Cell sorter purified c-kit+ day 14 fetal liver cells fully reconstitute the T and B cell compartments of immunodeficient Scid mice. Stromal cells or epithelial cells derived from fetal thymus or liver, which can support growth and differentiation of c-kit+ lymphocyte progenitor clones, synthesize mRNA for Steel Factor (SF), the ligand of c-kit. In the adult mouse, however, c-kit expression is restricted to very early stages of T- and B-lymphocyte development (multipotent progenitors, B-cell/myelocytic progenitors, Pro-T and Pro-B lymphocyte progenitors).
Most cells at the Pre-T, Pre-B and later stages of development do not bear detectable c-kit. Using Cos-1 cells tranfected with mouse SF-cDNA and an antagonistic ckit receptor-specific antibody, we show that the c-kit/SF system contributes to the survival of lymphocyte progenitors and enhances the proliferative responses of these cells to other growth factors (i.e. IL2, IL3, IL4, IL7). However, the c-kit receptor/SF ligand pair is neither sufficient nor necessary for the differentiation of lymphocyte progenitors into mature T- or B-lymphocytes. Finally, in stromal cell lines from fetal liver and adult bone marrow and thymic epithelial cell lines the level of steady state SF-RNA transcripts is inversely correlated with that of IL-7-mRNA. Moreover, IL7 inhibits the synthesis of SF-mRNA in stromal cells and rIL6 abrogates this inhibitory effect of rIL7. Thus, the expression of SF in stromal cells is subjected to complex regulation by other cytokines produced by the same stromal cells or by neighboring cells in a given microenvironment. The results strongly suggest that the c-kit/SF system plays an important role in the very early stages of development of lymphocytes in the mouse.
Introduction
vivo the membrane-bound form is probably the physiologically relevant ligand for c-kit (Flanagan et al., 1991). The expression and function of the c-kit receptor/SF ligand pair in the development of primordial germ cells, melanoblasts, erythrocytes and mast cells have been the subject of intensive study by several groups. In all these cell lineages, evidence has been obtained that this receptor/growth factor pair supports cell survival and facilitates the response to other growth factors (Matsui et al., 1990; Nishikawa et al., 1991; Ogawa et al., 1991; Migliaccio et al., 1991; Orr-Urtreger et al., 1990; McNiece et al., 1991a; Broxmeyer et al., 1991; Keshet et al., 1991; Dolci et al., 1991). Various cell types in the developing nervous system express RNA transcripts for the Steel and the W genes
Mice carrying mutations at the Steel and dominant white spotting (W) loci have abnormal development of primordial germ cells, neural-crest-derived melanoblasts, haemopoietic stem cells, and erythroid and mast-cell lineages (reviewed in Russel, 1979; Witte 1990). The W gene encodes a cell surface tyrosine kinase receptor, called c-kit (Chabot et al., 1988; Geissler et al., 1988) and the Steel gene encodes the ligand for c-kit, a polypeptide called here Steel Factor (SF) and also referred to as mast-cell growth factor or stem cell factor by others (Zsebo et al., 1990; Huang et al., 1990; Williams et al., 1990). SF can be expressed both in membrane-bound and soluble forms. In
Key words: lymphocyte development, growth factors, haemopoiesis, stromal cells.
1134
R. Palacios and S.-I. Nishikawa
(Keshet et al., 1991; Orr-Urtreger et al., 1990; Matsui et al., 1990), but the biological significance of this is unclear. Both Steel and W mouse mutants do not show gross abnormalities in the nervous system (Russel, 1979). Very little is known about the expression and potential functions of the c-kit/SF system in the development of lymphocytes in the embryo and adult (McNiece et al., 1991b; Palacios and Samaridis, 1992). The experiments reported here aimed to determine the cell surface expression of ckit protein on cells of the lymphocyte compartments both in the developing embryo and in young adult normal mice and to assess the effects of SF on growth and/or differentiation of T- and B-lymphocyte progenitors. We have used the c-kit-specific monoclonal antibody ACK2 (Nishikawa et al., 1991), immuno-fluorescence staining and flow fluorocytometry (FACS) or microscopy analysis to determine the cell surface expression of the c-kit protein on (1) a panel of non-transformed cell lines representing different stages of T- and B-lymphocyte development, (2) freshly isolated lymphoid cells from the thymus, bone marrow and spleen of young adult C57BL/6 mice, and (3) cells from yolk sac, liver and thymus of developing C57BL/6 mouse embryos. We then directly tested the contribution of c-kit/SF to growth and/or differentiation of lymphocyte progenitor clones by using Cos-1 cells stably expressing a mouse cDNA encoding the membrane form of the Steel gene product and a c-kit-receptor-specific antibody able to block the function of c-kit/SF system. Finally, we also studied the synthesis of SF-mRNA in stromal cell lines from fetal liver and bone marrow as well as in thymic epithelial cell lines, with known capacity to support growth/differentiation of Bor T-lymphocyte progenitors in vitro. The results of these experiments are the subject of the present communication. Materials and methods Mice C57BL/6, CBA/Thy1 a and C.B.17 Scid mice are bred and maintained in the animal barrier facility of the Basel Institute for Immunology, Basel. C.B.17 Scid mice (8- to 12-week-old female and male) without detectable serum Ig were used. The age of the embryos was determined by scoring the day of the appearance of vaginal plugs, which is taken as day 0. Further assessment of the age of gestation was based on the following criteria; 8 days, a well-developed ectoplacental cone and early membrane formation; 9 days, a well developed yolk sac, a conspicuous heart and no liver pigment; 10 days, early pigmentation in the liver; 11 days, a well-pigmented liver and the absence of a differentiated thymus; 13- to 17-days, the relative size and development of the fetus. As there were variations in the individual development of the embryos within a single pregnancy, the estimates in some instances may be in error by 1/2 to 1 day.
Cell lines The development and characterization of the bone marrow stromal cell lines RP.0.10 and RP.0.16, the fetal liver-derived stromal line FLS4.1 and the thymic epithelial cell lines ET and EA2, were described (Palacios et al., 1989a,b; Gutierrez and Palacios, 1991; Palacios and Samaridis, 1992). The growth factor-dependent clones: multipotent progenitors (PR-5, PR-8, PR-23) (Palacios, R. and Samaridis, J., submitted for publication), the Bcell/myelocytic progenitors (LyD9, LyB9, LyH7), the marrow Pro-B lymphocyte progenitors (Bc/Bm11, CB/Bm7), the fetal liver
Pro-B lymphocyte progenitors (FLB56, FLB99), the fetal liver Pre-B clones (FLB32, FLB41, FLB86), the marrow Pro-T lymphocyte progenitors (C4-77/3, C4-90/16), the fetal thymus Pro-T lymphocyte progenitors (FTH5, FTA2, FTF1), were described and have been propagated in culture as detailed before (Palacios and Samaridis, 1992; Palacios and Steinmetz, 1985; Palacios et al., 1987a,b; Pelkonen et al., 1987). The Pre-T cell line Degos was obtained from Balb/c adult thymus, requires IL2 + IL7 for continuous growth in culture, is CD4−8−3−TCRαβ−TCRγδ− and has rearranged both alleles of the TCR β and γ genes (R.P. unpublished results). The 97.2 macrophage line arose spontaneously from bone marrow of an X-ray-irradiated AKR/J mouse (R.P. unpublished results). The Cos-1 cells were provided by René Devos (Roche Research, Gent-Belgium). The mature T-cell lines CTLL (Gillis and Smith, 1977) and HT-2 (Watson, 1979) were also used.
Cytokines Supernatants from X63Ag8 myeloma cells transfected with cDNAs coding for IL2, IL3, IL4, IL5, IL6 (Karasuyama and Melchers, 1988) or from J558L/A2B2/44 myeloma cells transfected with mouse IL7-cDNA (Samaridis et al., 1991), tested for biological activity in proliferative assays as described (Palacios et al., 1987a; Samaridis et al., 1991), were used. The Cos-1/SF5 stable transfectant cells expressing mouse cDNA encoding the membrane form of SF were generated as follows: the XhoI-XhoI 0.85 kb fragment from the SF-cDNA clone, pCAMG, was subcloned in the XhoI-site of the expression vector pcDNAI/NEO (In Vitrogen, Lugano, Switzerland) and the resulting clone was designated pcDNAI/Neo SF. Linearized pcDNAI/NeoSF plasmid DNA was introduced into Cos-1 cells by Lipofection using Lipofectin Reagent (BRL, Uxbridge, England) as previously described in detail (Samaridis et al., 1991). Stable transfectants were then selected in culture medium (see below) supplemented with G418 (1 mg/ml). They have been propagated and expanded, if required, in the same selection medium indicated above. Cos-1 transfectant cells and their supernatants were tested for their ability to support proliferation of bone-marrow-derived granulocytes essentially as described by Flanagan et al. (1991) with the exception that we have used mitomycin-c-treated Cos-1 transfectant cells in the assays. Transfectant cells that scored positive in the biological assay were expanded and used to prepare total RNA to confirm the expression of the transduced SF-gene by northern blot analysis (see below). The Cos-1/SF5 stable transfectant cells produce both membrane-bound and soluble (low levels) SF and were used in the experiments described here.
Antibodies FITC-, PE- or biotin-conjugated antibodies against Thy1, Lyt2 (CD8), L3T4 (CD4), B-220 (hybridoma 6B2), TCRαβ (hybridoma 57-597), TCRγδ (hybridoma GL3), CD3 (hybridoma 145-2C11), H-2Kk were purchased from Pharmingen (San Diego, California). FITC- and biotin-labelled Mac-1-specific antibody were from Caltag (Zürich, Switzerland). FITC-labelled anti-mouse µ, kappa, lambda Ig chains and PE-streptavidin were from Southern Biotechnology Associates (Birmingham, Alabama). FITC-conjugated anti-rat IgG-specific antibody, mouse IgG and rat IgG were from Jackson Immunoresearch Laboratory. FITC-streptavidin was from Amersham (Zürich, Switzerland). The c-kit-specific antibody ACK2 (Nishikawa et al., 1991) and the Mac-1-specific antibody M1/70 (Springer et al., 1979) were purified by protein-G chromatography (Pharmacia, Uppsala, Sweden). Purified c-kit antibody was biotinylated as described before (Palacios and Leu, 1986).
Cell preparations Mononuclear cell suspensions from bone marrow, thymus, spleen
Role of c-kit/SF in lymphocyte development and fetal liver were prepared free of erythrocytes as described (Palacios et al., 1987a,b), yolk sac mononuclear cells from day 88.5 embryos were kindly provided by Beat Imhof (Basel Institute for Immunology). CD4–CD8– thymocytes were obtained by two cycles of antibody plus complement killing of total thymocytes as described (Palacios and von Boehmer, 1986). c-kit+ cells from 14day fetal liver of CBA/Thy1a embryos were isolated by cell sorter using biotin-conjugated c-kit-specific antibody and FITC-streptavidin in a FACStar plus instrument (Becton and Dickinson, Mountain View, California) as described in detail before (Palacios and Leu, 1986). Reanalysis of the cell sorter purified cells showed that >98% of the cells were c-kit+. The cells were washed and resuspended either in culture medium [Iscove’s modified Dulbecco medium supplemented with heat-inactivated fetal calf serum (7.5%), 2-mercaptoethanol (5×10−5 M), L-glutamine (2 mM) and gentamycin (50 µg/ml)] or FACS buffer (PBS pH 7.3, 0.2% BSA, 0.1% sodium azide) as required.
Isolation and analysis of nucleic acids DNA and total RNA preparation, restriction enzyme digestions, agarose gel electrophoresis, RNA blotting, probe preparations, hybridization procedures and autoradiography were performed as described (Palacios et al., 1989b; Pelkonen et al., 1987; Palacios and Samaridis, 1991; Gutierrez and Palacios, 1991).
DNA probes The probes used were: IL7 (450 bp SstI-HindIII fragment), SF (850 bp XhoI-XhoI fragment), β-actin (1.1 kb PstI-PstI fragment) - isolated DNA fragments and were labelled with 32P by using a random-primer DNA labeling kit (Boehringer Mannheim).
Functional assays Reconstitution of Scid mice Cell sorter purified c-kit+ fetal liver cells from day 14 CBA.Thy1a embryos (2 ×105 cells in 0.5 ml PBS) were injected in the lateral vein of the tail of C.B.17 Scid mice that were previously (4-6 hours before injection) exposed to 300 rads of γ-rays. Scid mice that received c-kit+ fetal liver or PBS only (control) were housed in sterile isolators with sterile water and food. Lymphoid repopulation of bone marrow and spleen of these mice was determined by single- and two-color FACS analysis twelve to sixteen weeks after transfer of the c-kit+ fetal liver cells.
Proliferative cell responses Cos-1/SF5 transfectant cells and wild-type Cos-1 cells were treated with mitomycin C (20 µg/ml at 37°C for 4 hours). Following four washes in 50 ml of PBS each, they were resuspended in culture medium, distributed in flat-bottomed microtiter wells (~104-2×104 cells/well) and left to adhere to plastic by incubation at 37°C for 2-4 hours. 104 lymphocyte progenitor cells (PR-23, FTH5, CB/Bm7 and FLB41) were added to the microplate wells in the presence or the absence of limited concentrations or rIL2 (3 units/ml), rIL3 (2 units/ml), rIL4 (5 units/ml), rIL7 (20 units/ml), in a final volume of 200 µl of culture medium per well. In some experiments, the c-kit-specific antibody ACK2 or the Mac-1-specific antibody M1/70 (isotype matched control antibody) were added (final concentrations of 1, 10 and 100 µg per ml) at the beginning of the cultures. Cell proliferation was measured by [3H]thymidine uptake (1 µCi/well) during the last 8 hours of a 48 hour culture period performed at 37°C. The results are expressed as mean counts/minute of triplicate samples per group where the s.e.m. was less than 10.4% of the mean.
In vitro differentiation of Pro-T and Pre-B clones These assays were carried out essentially as described before (Palacios et al., 1989a,b; Palacios and Samaridis, 1992) using ET
1135
cortical thymic epithelial cells to support T-cell differentiation of the Pro-T cells FTH5 and RP.0.10 bone marrow stromal cells, rIL7 (250 units/ml) and LPS (40 µg/ml) to induce differentiation of the Pre-B cells FLB41 into IgM+ B lymphocytes. FTH5 ProT and FLB41 Pre-B cells also were cocultured on monolayers of Cos-1/SF5 transfectant cells or wild-type Cos-1 cells to test whether SF would induce differentiation of c-kit+ lymphocyte precursor clones. All cultures were carried out in six-well costar plates in a final volume of 2 ml of culture medium per well and incubated at 37°C for 6 and 8-10 days. In some experiments, purified c-kit-specific antibody or control Mac-1 antibody were added (final concentrations 10 and 100 µg/ml) at the beginning of the cultures. At the end of the culture period, the haemopoietic cells were harvested, washed and the presence of TCRαβ/CD3+ T-cells and IgM + B lymphocytes, respectively, was determined by FACS analysis. In all cases, staining of the cells with PgP-1-specific antibody was also included and served as positive control. The results are expressed as percent TCRαβ/CD3+ T-cells or IgM+ B lymphocytes developed in the cultures.
Expression of SF- and IL7-RNA transcripts in stromal cell lines and thymic epithelial cell lines Total RNA was isolated from the fetal liver-derived FLS4.1 stromal cells, the bone-marrow-derived RP.0.10 stromal cells, and the thymic epithelial cell lines ET and EA2 as described before (Samaridis et al., 1991; Gutierrez and Palacios, 1991). In experiments addressing the regulation of SF-RNA expression in FLS4.1 stromal cells, the cells were cultured (~75% confluency) in the presence or the absence of rIL7, rIL4, rIL6 (final concentrations 10, 50, 100, 500 and 1000 units per ml) either single or in combinations at 37°C for 4 and 24 hours. Total RNA was isolated from each experimental group. RNA transcripts from the SF, IL7 and β-actin genes were determined by northern blot analysis as indicated above.
FACS analysis This was carried out as described in detail previously (Palacios et al. 1989b; Samaridis et al., 1991; Gutierrez and Palacios, 1991). Single- and two-color FACS analysis were performed using a FACScan instrument (Becton and Dickinson, Mountain View, CA). Bone marrow, spleen and thymocytes from CBA/j mice were used as positive controls as required and to set up electronically green and red compensations. Fluorescence emitted by single viable cells was measured with logarithmic amplification. Dead cells were excluded from analysis by forward and side scatter gating. Data collected from 5×103-4×104 cells were analyzed with consort 30 software and displayed in the form of fluorescence histograms (single color) or contour plots (two color).
Immunohistochemistry Embryo sections (4-6 µm) were prepared and acetone-fixed on glass slides and stored at −20°C until use. Immunofluorescence staining of the slides (four previously selected slides per embryo) was carried out using a previously determined optimal concentration of purified c-kit-specific mAb in staining buffer (PBS + 2% BSA + 0.1% sodium azide), slides were then incubated at 20°C for 1 hour. Following two washes with buffer, FITC antirat IgG-specific antibody (final dilution 1:200) was added and the slides were incubated at 4°C for 1 hour. Following three washes with buffer (each for 10 minutes), the slides were mounted under glass cover slips using 50 mM Tris (pH 8.6) containing Gelvatol and analyzed, both under phase-contrast and green fluorescence with an Axiophot Zeiss microscope. In all cases, the entire embryo was scanned for the presence of cells that specifically bound the c-kit antibody. c-kit+ cells usually displayed a ring-dotted pattern
1136
R. Palacios and S.-I. Nishikawa
of fluorescence, and sometimes a patchy pattern of fluorescence on one pole of the cells was also observed.
Results c-kit expression on lineage uncommitted and lineagerestricted lymphocyte precursor cell lines A panel of non-transformed cell lines isolated from bone marrow, liver and thymus of either embryos or young adult mice which represent different stages of lymphocyte development were tested by FACS for c-kit protein expression on the cell membrane. The results showed that the multipotent progenitor clones PR-5, PR-8 and PR-23 (able to give rise to T-lymphocytes, B-lymphocytes and myeloid cells) are c-kit+. The bipotent B-cell/myelocytic progenitor clones LyD9, LyH7 and LyB9 (able to generate B-lymphocytes and myeloid cells, but not T-lymphocytes), the monopotent Pro-B lymphocyte progenitor clones derived from either adult bone marrow (CB/Bm7, Bc/Bm11) or 14day fetal liver (FLB56, FLB99) and the Pre-B cell clones isolated from 14-day fetal liver (FLB32, FLB41, FLB86) were all c-kit positive. The monopotent Pro-T lymphocyte progenitor clones obtained from either 14-day fetal thymus (FTH5, FTF1, FTA2) or adult bone marrow (C4-77/3, C490/16) were c-kit+ while the late Pre-T cell line Degos isolated from adult thymus and the mature T-cell lines (CTLL, HT-2) were c-kit–. These results are illustrated in the form of fluorescence histograms in Fig. 1 and are summarized in Table 1. c-kit expression on lymphoid cell populations in the young adult mice Single-color FACS analysis of lymphoid cells in the bone marrow of young adult C57BL/6 mice showed that 2-5% of these cells express low levels of c-kit and analysis of the myeloid marrow population showed that 11-17.6% of them were c-kit+ (Fig. 2 and Table 1). Two-color FACS analysis revealed that most (94-97%) B-220+ B-cell precursors and all IgM+ mature B lymphocytes in the bone marrow did not carry detectable c-kit (Table 1). Single-color FACS analysis of total thymocytes showed that only 0.2-0.6% of these cells bound the c-kit-specific antibody; two-color FACS analysis revealed that all CD4+/CD8+ thymocytes were c-kit negative and about one seventh of the CD4– CD8– thymocytes (which comprise Tcell precursors) expressed low levels of c-kit (Fig. 2 and Table 1). Finally, all mature T-lymphocytes and B-lymphocytes in the spleen were c-kit negative (Fig. 2 and Table 1). c-kit expression on cells from yolk sac, liver and thymus in developing C57BL/6 mouse embryos FACS analysis of mononuclear cells from yolk sac of day 8-8.5 embryos (before fetal blood circulation has started) showed that 10-18% of these cells express low levels of ckit receptor and that 25-30% mononuclear cells from day14 fetal liver brightly stained with the c-kit specific antibody (Fig. 2). Next we searched for c-kit+ cells in the liver and thymus of embryos at different times of development
Table 1. c-kit surface expression on cell lines and cell populations representing different stages of lymphocyte development Cells
Origin
c-kit expression FACS analysis
PR-5 PR-8 PR-23
adult BM adult BM adult BM
+ + +
LyD9 LyH7 LyB9
adult BM adult BM adult BM
+ + +
Pro-B
CB/Bm7 Bc/Bm11 FLB 56 FLB 99
adult BM adult BM fetal liver fetal liver
+ + + +
pre-B
FLB 32 FLB 41 FLB 86
fetal liver fetal liver fetal liver
+ + +
Pro-T
C4-77/3 C4-90/16 FTH5 FTA2 FTF1
adult BM adult BM fetal thymus fetal thymus fetal thymus
+ + + + +
Degos CTLL HT-2
adult thymus adult spleen adult spleen
− − −
B-220+ IgM+ IgM+
adult BM adult BM adult spleen
2-3%*
