Key Words. Diamond-Blackfan anemia M-07e cell line Stem cell factor GM-CSF Bone marrow fibroblast Long-term bone marrow cultures Stromal cells

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1 M-07e Cell Bioassay Detects Stromal Cell Production of Granulocyte-Macrophage Colony Stimulating Factor and Stem Cell Factor in Normal and in Diamond-Blackfan Anemia Bone Marrow Laura Bonsi," Gian Paolo Bagnara,"Sb Pierluigi Strippoli," Francesca Bonifazi,a Lorenza Vitale? Massimiliano Bonaf2," M. Alessandra Santucci; Luciano Pinto? Pasquale Rosito," Andrea Pession," Maria Felice Brizzi,' Gian Carlo Avanzi,' Ugo Rarnenghi,g Vilma Gabutti,g Guido Paoluccibve %stitute of Histology and Embryology, bg. Prodi Interdepartmental Center for Cancer Research, 'Institute of Cancerology, University of Bologna, Bologna, Italy; ddepartment of Pediatrics I, 2nd University of Naples, Naples, Italy; cdepartment of Pediatrics 111, University of Bologna, Bologna, Italy; 'Department of Biomedical Sciences and Human Oncology, gdepartment of Pediatrics, University of Turin, Turin, Italy Key Words. Diamond-Blackfan anemia M-07e cell line Stem cell factor GM-CSF Bone marrow fibroblast Long-term bone marrow cultures Stromal cells Abstract. Ten healthy donors and four patients with Diamond-Blackfan anemia () have been investigated for granulocyte-macrophage colony stimulating factor (GM-CSF) and stem cell factor (SCF) production by bone marrow-enriched fibroblasts (BMEF) in a highly sensitive biological assay on growth factor-dependent M-07e cells. M-07e cells detected active soluble kit-ligand from bone marrow fibroblasts as well as from BMEF which produce constitutively significant amounts of SCF. Interleukin l p (IL-1p) induced a significant increase of soluble SCF from both and BMEF. GM-CSF was undetectable in unstimulated cultures, while its production by bone marrow microenvironmental cells was documented for both and patients after IL-lp stimulation in vitro. Introduction CD34' enriched bone marrow cells from Diamond-Blackfan anemia () patients are unable to form erythroid colonies when stimulated in vitro by granulocyte-macrophage Correspondence: Dr. Gian Paolo Bagnara, Istituto di Istologia ed Embriologia Generale, V. Belmeloro 8,40126 Bologna, Italy. Received March 24, 1993; accepted for publication March 24, OAlphaMed Press /93/$ colony-stimulating factor (GM-CSF), interleukin 3 (IL-3) and erythropoietin (Epo) with or without the simultaneous presence of IL-6. Stem cell factor (SCF) [ 1-31 may partially correct the in vitro impaired erythroid differentiation by inducing a significant increase of both number and size of erythroid burst forming unit (BFU-E) derived colonies [4-61. In a study on two patients with [7], SCF and c-kit were found to be structurally, while the number of SCF receptors on the total mononuclear bone marrow cell population appeared to be lower than in the adults. However, the role of SCF and its receptor in the pathogenesis of is still unclear, and the involvement in this disease of cellular sources of both SCF and cytokines with synergistic effects on hemopoietic stem cell differentiation cannot be excluded. In this study, growth factor-dependent M-07e cells were used as a highly sensitive bioassay to detect soluble SCF and GM-CSF produced by marrow stromal cells. In unstimulated cultures, active SCF, but not GM-CSF, is detectable by M-07e cell bioassay in both and patients. IL-lP induces an increase of soluble kit-ligand and makes GM- CSF detectable in both and bone marrow cell conditioned medium. STEM CELLS 1993;11(~~ppl2):

2 132 GM-CSF and SCF Production in Diamond-Blackfan Anemia Materials and Methods Patients Clinical data of the patients are summarized in Table I. After informed consent, bone marrow samples were obtained from four patients and from ten subjects or patients with non-hematological disease. Bone Marrow Stromal Cell Cultures Bone marrow long-term cultures (BMLTC) were performed as described elsewhere [8, 91. Briefly, 3-5 x lo7 buffy coat cells from bone marrow aspirates were inoculated in 25 cm2 tissue culture flasks, in 8 ml total volume of McCoy's modified medium (GIBCO, Grand Island, NY), supplemented with 12.5% pretested fetal calf serum (FCS; GIBCO), 12.5% pretested horse serum (GIBCO) and M hydrocortisone succinate (Sigma, St. Louis, MO). Cultures were incubated at 33 C in 3% COz in air. Between days 5 and 7, the nonadherent cell population was removed. The stromal cell cultures were fed twice weekly until 6-8 weeks. After at least 6 weeks, the medium was changed, and supernatants were harvested from subconfluent stimulated cultures or not by recombinant human (rh) IL-Ip (British Biotechnology, Oxon, England) 0.1 ng/ml for three days, and stored at -20 C for the bioassay. In this culture system, stromal marrow cells are represented by heterogeneous cell types including fibroblasts, macrophages, endothelial and reticular cells, and adypocytes. Immunocytochemical characterization of these cells is described elsewhere [9]. Bone Marrow Enriched Fibroblast Cultures (BMEF) The mononuclear bone marrow cells were separated by density centrifugation by Ficoll- Hystopaque (Sigma), and were incubated for h in IMDM (GIBCO), supplemented with 25% FCS (GIBCO). In order to obtain enriched fibroblast populations, after nonadherent cells had been removed, adherent cells were cultured in IMDM 10% FCS supplemented with rh basic fibroblast growth factor (b-fgf; GIBCO) 0.1 ng/ml until four cultured passages had been performed, by which time macrophagic, endothelial and reticular cells were virtually absent. Five days before the SCF and GM-CSF bioassay, cells were deprived of b-fgf and plated at a density of 1 x 104/ml in 24-well plates (final volume 1 ml) with or without rhil-1p (0.1 ng/d). Supernatants were harvested after 72 h and stored at -20 C. M-07e Cell Line Bioassay SCF and GM-CSF were evaluated by bioassay on M-07e cell line [ 101. Bone marrow stroma1 fibroblasts or BMLTC stromal cell supernatants were used as a source of SCF and GM-CSF. After 24 h of IL-3 deprivation, M-07e cells were washed twice and seeded in 200 pl microwells in IMDM with 5% FCS at a final concentration of 5 x lo4 cells/microwell in the presence of increasing concentrations of supernatants. After 24 h, triplicate wells were pulselabeled with 3H-TdR (1 pci/well) for 4 h. Alcohol-acid precipitated radioactivity was determined by liquid scintillation counting. The concentration of GM-CSF or SCF in the cultures' supernatants was determined by comparing the dose-response curves obtained with serial dilution of rhgm-csf (or rhscf) to that Table I. Clinical data of patients at the time of study Case AgelSex Congenital Treatment Hb Ret WBC Platelets Transfusion anomalies' (gm) (%) (~109~) (xi094 at study 1 2lF No Indb M No < DeP 3 251F Yes St Ind 4 21F No Indb Abbreviations: Ret, reticulocytes; St, steroid; Ind, independent; Dep, dependent. "Congenital anomalies: labiopalatoschisis patient 3. bpatients 1 and 4 were studied at diagnosis.

3 Bonsi et al. 133 obtained with the cultures supernatants. The specificity of cell proliferation in response to each factor was assessed by the neutralizing antibodies. GM-CSF and SCF levels were determined by subtraction of the number of counts per minute (cpm) obtained in the presence of anti-gm-csf monoclonal antibody (Sandoz, Vienna, Austria) or anti-scf polyclonal antibody (British Biotechnology), respectively, from the total cpm amount. Northern Blot Analysis In order to study SCF mrna expression, BMEF obtained from two patients and one subject, as described above, were cultured in the presence or absence of hydrocortisone (HC) (lo- M for 48 h). The adherent layers were then lysed on the flasks. Total RNA was extracted with guanidium thiocyanate and isolated by acid-phenol-chloroform extraction [ 111. Twenty pg RNA samples were fractionated on 6% formaldehyde-1.2% agarose gel and blotted onto nylon membrane (Hybond-N+, Amersham, Buckinghamshire, England). The DNA probe was 32P labeled by random priming, according to Sambrook et al [12]. The filter was hybridized and washed by the method of Church and Gilbert [ 131, and exposed to X-ray film for two to six days. The probe was a 900 bp BamH I-Hind 111 insert of pbluescript, containing the entire human mast cell growth factor (identical to SCF) coding region, kindly provided by Dr. s. Gillis (Immunex, Seattle, WA). Results In our experiments, BMEF from two patients expressed quantities of SCF mrna. HC, which is capable of improving the hematologic condition in vivo, does not appear to clearly augment the SCF mrna expression, either in or in fibroblasts (Fig. 1). We tested the bone marrow stromal cells and enriched fibroblasts from ten subjects and three patients on M-07e cells [lo]. These cells are SCF-dependent and consequently provide a useful tool to test the effective biological activity of SCF. Ten pg of anti-scf polyclonal antibody is able to neutralize five ng di rhscf (Fig. 2). Amounts of soluble SCF are spontaneously produced by both and BMEF SCF Fig. 1. Northern blot analysis of RNA from BMEF samples from two patients and one subject in the absence (lane 1, case no. 1; lane 3, case no. 4; lane 5, ) or in the presence (lanes 2,4,6 respectively) of HC lo- M (48-h exposure). SCF mrna size is 6 Kb. P-Actin probe hybridization was subsequentially performed as a control for the amounts of RNA loaded in each lane. (from 0.5 to 1.2 ng/ml) (Fig. 3A). IL-lp stimulated the BMEF from both the and two patients to produce increased amounts of SCF (Fig. 3B). Cultures from a third patient s bone marrow were performed by Dexter s technique, and IL-1p again induced a significant increase of SCF (Table 11). GM-CSF was undetectable in unstimulated cultures of both stromal cells and BMEF. IL-lp induced the release of detectable amounts of GM-CSF both in Dexter and in BMEF cultures (Figs. 4 and 5, respectively); similar amounts were found in subjects - and patients. hscf 3oooo - -t rhscf + anti-sc(10 pg/ml) m.- E ,Ol, loo lo00 rhscf ng/d Fig. 2. M-07e cell line growth response to different concentrations of rhscf (squares) and its corresponding neutralization by one fixed concentration (10 lg/ml) of anti-scf polyclonal antibody (rhombi).

4 134 GM-CSF and SCF Production in Diamond-Blackfan Anemia 2o 1 0 '1 A - \ C E M 300 a % y Fig. 4. GM-CSF production (M-07e bioassay) from IL- 1 p stimulated long-term bone marrow stromal cell cultures in 3 subjects and one patient (case no. 3). 0 '1' ' Fig. 3. SCF production (M-07e bioassay) from unstimulated (A) and IL-Ip stimulated (B) BMEF in four subjects and two patients (case nos. I, 2). Discussion Patients with [ 141 have macrocytic anemia with several hematological abities, similar to those of Wand Sl mutant mice [ 151. Studies on the in vitro growth of bone marrow cells from patients demonstrated that granulocytic and megakaryocytic progenitors are able to form colonies in cultures stimulated by several cytokines in a way similar to bone marrow cells. In contrast, erythroid progenitor proliferation and differentiation occurs only after addition of SCF in vitro, which is able to induce a dramatic increase of both number and size of erythroid burst colonies. SCF is produced by bone marrow stromal fibroblasts and plays an important role in hemopoietic cell differentiation through its synergistic activity with GM-CSF, IL-3, IL-7, leukemia inhibitory factor (LIF), macrophage inflammatory protein la (MIP-la) and other cytokines [16]. The role of these cytokines in hemopoietic cell differentiation needs to be taken into account, and our study focused on the production of SCF and GM-CSF by bone marrow stromal cells Table 11. SCF production (M-07e bioassay) in long-term bone marrow cultures in one patient (case no. 3) Stromal cells unstimulated IL- 1 p stimulated 0.5 ng/ml 12.0 nglml Fig. 5. GM-CSF production (M-07e bioassay) from IL-I p stimulated bone marrow enriched fibroblasts in seven subjects and two patients (case nos. 1, 2).

5 Bonsi et al. 135 SCF is constitutively produced by BMEF. Our results demonstrate that BMEF from two patients constitutively expressed SCF mrna and show that active soluble SCF protein is detectable in the conditioned medium of BMEF and of stromal cells in general. HC seems to be able to upregulate SCF expression by bone marrow stromal cells [16, 171. The in vitro effect of HC on BMEF SCF mrna expression recorded by us requires further investigation, particularly with regard to different HC concentrations and the time of HC exposure. In both subjects and patients, IL- 1 p induced increased secretion of SCF by BMEF and stromal cells and also triggered GM- CSF production by the same cells. These results suggest that GM-CSF production in the bone marrow microenvironment by patients is. Since amounts of IL-3 have been found to be produced by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells [5], these two cytokines, with which SCF synergizes, would not appear to be involved in erythroid impairment in. In, the selective requirement of SCF by erythroid progenitors in vitro may partially be explained by a relative depletion of c-kit receptors in these cells. In fact, molecular analysis of both c-kit and SCF in two patients failed to demonstrate structural abities in this cytokine or its receptor protein [7]. However, the structural analysis of SCF does not in itself reveal its biological activity on target cells in vivo. Our experiments provide evidence that SCF in the bone marrow microenvironment is biologically active on M- 07e cell line proliferation. SCF receptors are distributed among granulocytic, megakaryocytic, erythroid and lymphocytic cells. The lower number of c-kit receptors recently described in total mononuclear marrow cells in patients [7] requires confirmation in selected erythroid cells. Several biological aspects of SCF-producing cells and of c-kit receptor protein distribution remain unclear in. The rarity of this disease makes homogeneous experimental conditions difficult, but further investigations on a larger number of patients are needed in order to reach a correct approach to the study of pathogenesis. Acknowledgments This work was supported by the Italian Ministry of Scientific and Technological University Research, Italian National Research Council grant no PF39, and by the Italian Association for Cancer Research grants to G. P. Bagnara, G. Paolucci and L. Pinto. References 1 Zsebo KM, Wypych J, McNiece IK, Lu HS, Smith KA, Karkare SB, Sachdev RK, Yuschenkoff VN, Birkett NC, Williams LR, Satyagall VN, Tung W, Bosselman RA. Mendiaz EA, Langley KE. Identification, purification and biological characterization of hematopoietic stem cell factor from Buffalo rat liver-conditioned medium. Cell 1990;63: Anderson DM, Lyman SD, Baird A, Wignall JM, Eisenman J, Rauch C, March CJ, Boswell HS, Gimpel SD, Cosman D, Williams DE. Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 1990;63: Zsebo KM, Williams DE, Geissler EN, Broudy VC, Martin FH, Atkins HL, Husu R, Birkett NC, Okino KH, Murdock DC, Jacobsen FW, Langley KE, Smith KA, Takeishi T, Cattanach BM, Galli SJ, Suggs SV. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine-kinase receptor. Cell 1990;63: Abkowitz JL, Sabo KM, Nakamoto B, Blau CA, Martin H, Zsebo KM. Papayannopoulou T. Diamond-Blackfan anemia: in vitro response of erythroid progenitors to the ligand for c-kir. Blood 1991;78: Bagnara GP, Zauli G, Vitale L. Rosito P, Vecchi V, Paolucci G, Avanzi GC, Ramenghi U, Timeus F, Gabutti V. In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia. Blood 1991;78: Olivieri NF, Gmnberger T, Ban-David Y, Ng J, Williams DE, Lyman S, Anderson DM, Axelrad AA, Correa P, Bernstein A, Freedman MH. Diamond-Blackfan anemia: heterogeneous response of hematopoietic progenitor cells in vitro to the protein product of the Steel locus. Blood ;78:

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