Jun 28, 1995 - *Walter and Eliza Hall Institute of Medical Research, tThe Cooperative Research ... binding of GM-CSF was abolished in 3c-/- bone marrow.
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9565-9569, October 1995 Medical Sciences
Hematopoietic and lung abnormalities in mice with a null mutation of the common f3 subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5 (gene targeting/clearance of granulocyte-macrophage colony-stimulating factor)
LORRAINE ROBB*t, CATHERINE C. DRINKWATER*tt, DONALD METCALF*t, RUILI LI*, FRANK K6NTGEN*, Nicos A. NICOLA*t, AND C. GLENN BEGLEY*t§¶ *Walter and Eliza Hall Institute of Medical Research, tThe Cooperative Research Centre for Cellular Growth Factors, and §Rotary Bone Marrow Research Institute, Post Office, The Royal Melbourne Hospital, Victoria, 3050, Australia
Contributed by Donald Metcalf, June 28, 1995
IL-3Ra (6). However, 3tL3 does not form a high-affinity receptor with murine GM-CSFRa or IL-5Ra (7). To further understand the complex interplay of growth factors and their receptors in the regulation of hematopoiesis, we generated and characterized mice with a null mutation of the ,Bc receptor.
ABSTRACT Gene targeting was used to create mice with a null mutation of the gene encoding the common 13 subunit (,8c) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (,Bc-/- mice). High-affinity binding of GM-CSF was abolished in 3c-/- bone marrow cells, while cells from heterozygous animals (I3c+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3specific 13 chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of ,Bc-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of 13c-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. .Bc-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between Pc-/and wild-type littermates. The data establish that j8c is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.
MATERIALS AND METHODS 13c Gene Targeting and Generation of t8c Null Mice. The targeting construct was created from a 5-kb Sac I-EcoRV 129-derived genomic fragment of the ,Bc locus containing exons 5-9. This fragment was subcloned into pBluescript (Stratagene), and the blunt-ended EcoRI-HindIII insert of pKJl (8) was cloned in reverse transcriptional orientation into a BamHI site in exon 7 (Fig. 1A). The construct was linearized and electroporated into W9.5 embryonic stem cells (9), which were selected in the presence of 175 Ag of G418 for 10 days. Surviving clones were screened as described (10) except that Southern analysis was performed with DNA from single clones. To detect homologous recombinants, BamHI-digested DNA from resistant clones was hybridized to probe A (Fig. IA), and the mutation was confirmed by Southern analysis with a probe 3' of the construct and a neomycin phosphotransferase (neo) probe (not shown). Of 250 clones screened, 9 had targeted mutations of the 13c locus, and 3 of these were used to derive chimeric mice (10), which were mated with C57BL/6 mice. Offspring bearing the f3c mutation were interbred, and wild-type (13c+/+), heterozygous (3c+/-), and homozygous (3C-/-) pups were obtained in the expected Mendelian ratio (Fig. 1B). All experiments were performed using 6- to 12week-old F2 mice derived from two independent cell lines. Binding Assays and Clearance Studies. Radioiodination of murine GM-CSF and IL-3 and binding assays were carried out as described (11). For clearance studies, 4 x 106 cpm of 125I-labeled GM-CSF (125I-GM-CSF) in Dulbecco's modified Eagle's medium were injected into a tail vein of 8-week-old female ,3c+/+ and 3c-/- mice. Blood and organs were then collected and processed as described (12). Hematological Analysis and Histology. Mice were anesthetized, and orbital plexus blood was collected for white blood cell, hematocrit, and platelet estimations. Peritoneal cavity cells were collected by using 2 ml of 5% (vol/vol) fetal calf
Hematopoiesis is regulated by the interaction of a variety of growth factors with their cognate receptors. Granulocytemacrophage colony-stimulating factor (GM-CSF) and multiCSF [interleukin 3 (IL-3)] act on a wide range of hematopoietic cells to stimulate their proliferation and differentiation (1). Interleukin 5 (IL-5) is a more lineage-restricted cytokine that acts mainly on eosinophils but also on basophils and, in the mouse, on some B cells (2). The receptors for these cytokines (GM-CSFR, IL-3R, and IL-5R) are composed of at least two subunits, both of which are members of the hematopoietin receptor superfamily. The a subunits (GM-CSFRa, IL-3Ra, and IL-5Ra) are specific for each cytokine and bind their respective ligands with low affinity. In the human, a common 1B subunit (,Bc) forms a high-affinity receptor with all three a subunits but does not detectably bind the cytokines alone (3-5). In the murine receptor system there is, in addition to these subunits, a second P subunit known as ,IL3, which is highly homologous with ,c, binds murine IL-3 with low affinity, and forms a high-affinity IL-3-binding complex with
Abbreviations: G-CSF, GM-CSF, and M-CSF, granulocyte, granulocyte-macrophage, and macrophage colony-stimulating factors; SCF, stem cell factor; PAS, periodic acid/Schiff reagent; IL-3, IL-5, and IL-6, interleukins 3,5, and 6; GM-CSFRa, IL-3Ra, IL-5Ra, ,3 subunits of receptors for GM-CSF, IL-3, IL-5; Pc, common ,B subunit. *Present address: Amrad Operations, Burnley, Victoria, 3121, Australia. 1To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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FIG. 1. Targeting the ,Bc locus. (A) Partial map of the 83c genomic locus with the targeting construct and the resulting targeted loci. E, EcoRI; B, BamHI; S, Sac I; V, EcoRV. (B) Southern blot of tail DNA from offspring of a heterozygous intercross, hybridized with probe A. Sizes of the germ-line (5.0 kb) and targeted (5.6 kb) alleles are indicated, as is the band due to the 83 gene. +/+, Wild type; +/-, heterozygote; -/-, homozygote.
serum in normal saline. Total femur cell counts were performed, and cytocentrifuge preparations were prepared and stained with May-Grunwald Giemsa. Bone marrow progenitor cells were assayed in semisolid agar cultures of 50,000 bone marrow or spleen cells, and colonies were scored, fixed, and typed after 7 days of incubation (13). Colony formation was stimulated by purified recombinant bacterially synthesized growth factors at the following fmal concentrations: murine GM-CSF, 10 ng/ml; murine IL-3, 10 ng/ml; murine stem cell factor (SCF), 10 ng/ml; murine IL-6, 500 ng/ml; human granulocyte-CSF (G-CSF), 10 ng/ml; murine macrophageCSF (M-CSF), 10 ng/ml; and unfractionated baculovirusderived IL-5, 103 units/ml. Organs were fixed in Bouin's solution, sectioned, and stained with hematoxylin and eosin, Luxol fast blue, or periodic acid/Schiff (PAS) reagent. Flow Cytometry. Cell suspensions were stained with monoclonal antibodies that had been prepared and conjugated to fluorochromes as described (14) and were analyzed on a fluorescence-activated cell sorter (FACScan; Becton Dickinson). The following monoclonal antibodies were employed: CD4 and CD8 (Becton Dickinson), M1/70 (Mac-1), RB6-8C5 (Gr-1), F4/80, Terll9, 5.1 (IgM), RA3-6B2 (B220), 30H12 (Thy-1.2), H57-697.1 [T-cell antigen receptor (TCR)a/,B], GL3-1A (TCR-y/8) (see references in refs. 14 and 15).
RESULTS Confirmation of the t8c Null Mutation. The targeting construct introduced a neo expression cassette into exon 7 of the pc locus, creating stop codons in all three reading frames. Binding studies were performed to verify that this targeting event resulted in a null mutation. Bone marrow cells were incubated with increasing concentrations of 125I-GM-CSF or 125-I-labeled IL-3 (125I-IL-3) with or without an excess of
unlabeled competitor, and specific equilibrium binding data were obtained. Scatchard transformation of the binding data is shown in Figs. 2 and 3. GM-CSF binding to fc+/+ bone marrow cells demonstrated an apparent single class of highaffinity binding (Kd = 250 pM, n = 430 receptors per cell). An intermediate pattern was observed with c+/- cells, with both high-affinity (Kd = 86 pM, n = 170 receptors per cell) and low-affmity (Kd = 1.7 nM, n = 290 receptors per cell) binding being observed. Cells from 3c-/- mice bound GM-CSF only with low affinity (Kd = 4.6 nM, n = 450 receptors per cell), thus confirming the null mutation. IL-3 binding was of the highaffinity type (Kd 300 pM) in all three groups with, if anything, slightly higher affinity binding to j3c-/- cells (Fig. 3). Phenotype and Histological Analysis of 1lc Null Mice. The 3c-/- mice survived normally during a 6-month observation period and were phenotypically indistinguishable from their
littermates. At 6 and 14 weeks of age, histological abnormalities were confined to the lungs of Oc-/- mice. Focal peribronchovascular infiltrates of lymphoid cells and intraalveolar eosinophilic material and foamy macrophages were present. In older Bc-/- mice, nongranular PAS-positive material was observed in a small percentage of the alveoli (Fig. 4). Hematological Studies. Hematocrit, total white cell counts, and platelet counts were normal in f3c-/- mice. However, there was a consistent 95% reduction in eosinophils (3 ± 10/1l) compared with ,Bc+/+ and 3c+/- mice (90 ± 80 and 100 ± 80/,ul, respectively). Bone marrow and spleen cellularity did not differ between 3c+/+ and 13c-/- mice, but a reduction in the percentage of eosinophils was seen in 3c-/- bone marrow (4 ± 3% in ,Bc+/+ versus 0.7 ± 1.2% in Bc-/- mice). Luxol fast blue-positive eosinophils were observed in 3c-/- mice in those tissues that normally contain eosinophils, such as spleen, intestinal villi, and uterine wall, although numbers were reduced compared with those in ,Bc+/+ mice. Peritoneal cell numbers and composition were similar in 13c+/+ and c-/mice. Flow cytometric analysis was performed on cells from thymus, spleen, bone marrow, and lymph node using a panel
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of monoclonal antibodies against myeloid and lymphoid antigens. No alterations were found in the immunophenotypic composition of these organs (data not shown). Responsiveness to Growth Factors. In clonal cultures of marrow cells, stimulated by up to 1 jig of GM-CSF per ml, c-/- cells failed to exhibit any clonal proliferation. In contrast, the responsiveness of ,Bc+/+ and 3c+/- cells was identical (Fig. 5). When stimulated by IL-3, all three types of marrow cells showed identical quantitative responsiveness, colony size also being similar. Overall, the frequency of clonogenic progenitor cells responding to IL-3, G-CSF, M-CSF, SCF, or IL-6 was slightly higher in Bc-/- cells than in the ,Bc+/+ or ,c+/marrow population (Table 1), and the same situation was observed in spleen cell cultures (data not shown). With these latter stimuli, the only consistent anomaly shown with 3c-cells was a slightly elevated frequency of IL-3-stimulated megakaryocytic progenitor cells. In cultures stimulated by IL-5, ,Bc+/+ marrow cells produced an average of 26 eosinophil colonies per 50,000 marrow cells, whereas in cultures of 3c-/cells no eosinophil colonies or smaller clones developed. However, normal numbers of eosinophil progenitor cells were detected in cultures of jSc-/- bone marrow cells stimulated by IL-3 (Table 1). In Bc+/+ or Bc-/- marrow cultures initiated without stimulation and then stimulated by the delayed addition of IL-3, progenitor cells died with a half-life of 22-26 hr. Incubation of such cultures with a final concentration of up to 1 ,ug of
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FIG. 5. Proliferation of bone marrow cells from 3c null mice and littermates. Bone marrow cells (50,000) from wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice were stimulated in agar cultures by GM-CSF (Upper) or IL-3 (Lower) and colony growth was scored at day 7. Mean data from five separate experiments are shown.
GM-CSF per ml did not increase the survival of 13c-/progenitor cells, unlike the situation with gc+/+ cells, where 85% of clonogenic cells survived over a 4-day incubation period with GM-CSF before the addition of IL-3. As described (16), culture of 3c+/+ cells with M-CSF plus GM-CSF led to marked enhancement of colony formation. In contrast, in cultures of Bc-/- cells, no enhancement of M-CSF stimulation was observed, even with the addition of up to 1 ,ug of GM-CSF per ml (final concentration). Systemic Clearance of Radiolabeled GM-CSF. Clearance of intravenously injected 125I-GM-CSF was essentially identical in P3c+/+ and c-/- mice. In both there was an initial rapid fall in the level of radiolabeled GM-CSF (as assessed by CC13COOHprecipitable 125I in blood) with a half-life of 2.5 min. This was followed by a slower second phase with an estimated half-life of 157 min (Fig. 6). Six hours after injection, high levels of radioactivity were detected in kidney, salivary gland, lung, and
FIG. 4. Lung histopathology of t3c null mice. (A and B) Lung from 14-week-old k+/+ (wild-type) mouse (A) and a j6c-/- (homozygous) littermate (B). The arrow indicates a peribronchial lymphoid aggregate. (PAS; x100.) (C) PAS-positive homogeneous material in lung from the same mouse as in B. (PAS; X400.)
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Table 1. Bone marrow progenitor cells in I3c null and wild-type mice
Colonies, no. M Eo Meg Blast G GM Total Stimulus 9±2 1±1 2± 1 23 ± 7 GM-CSF 96 ± 11 46 ± 6 15 ± 8 17 ± 6 13 ± 8 29 ± 3 11 ± 6 55 ± 17 27 ± 9 IL-3 152 ± 27 0 0 0 0 3 2 0 0 29 ± 8 2±2 G-CSF 34 ± 11 0 0 0 0 0 0 83 5 110 ± 7 11 ± 6 16 ± 6 M-CSF 0.2 0.5 19 6 89 ± 11 5 4 0 0 62 ± 10 3±4 SCF 3± 1 3 4 0 0 34 ± 11 4 3 0 0 25 ± 4 IL-6 0 0 0 0 0 0 0 0 0±0 0±0 0 0 GM-CSF -/-(n =5) 9 3 15 6 29 17 32 6 45 13 53 ± 11 183 ± 33 IL-3 0.2 ± 0.5 2 1 5 2 0 0 0±0 41 ± 8 34 ± 8 G-CSF 22 9 0 0 0±0 0± 0 16 ± 6 97 26 M-CSF 135 ± 33 0.4 ± 0.9 27 ± 4 6 2 0 0 SCF 106 ± 19 68 ± 21 5 3 0 0 1±1 0±0 32 ± 11 23 ± 13 0.5 0.7 7 4 IL-6 Cultures contained 50,000 bone marrow cells. Means and standard deviations are shown (±). n, Number of mice studied; G, granulocyte; GM, granulocyte/macrophage; M, macrophage; Eo, eosinophil; Meg, megakaryocyte; blast, blast colony. Mice +/+ (n = 4)
thyroid of both P3c+/+ and 13c-/- mice. There was no significant difference between the low levels of radioactivity in bone marrow from the two groups (data not shown). Accumulation of CCl3COOH-nonprecipitable 125I-labeled material in urine was high and similar in both groups, suggesting similar degradation pathways.
DISCUSSION The generation of mice with null mutation of the fc receptor enabled us to reexamine current models of the murine IL-3, IL-5, and GM-CSF receptor complexes and to study the role of GM-CSF and IL-5 in hematopoiesis. Our data are in general agreement with those of Nishinakamura et al. (17) and provide additional insights into the role of f3c in high-affinity binding of IL-3 and GM-CSF and in clearance of GM-CSF and into the role of the GM-CSFRa chain in cell survival and proliferation. High-affinity binding of GM-CSF on bone marrow cells was abolished by the ,c null mutation, while on Bc+/- cells high-affinity binding sites were reduced. This indicates that the number of P3c chains present on the surface of these cells is not in large excess relative to the number of a chains. The total number of GM-CSF binding sites (high and low), reflecting the number of a chains, was similar in B3c+/+ and 3c-/- mice, suggesting that the a chain was not up-regulated in response co0
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to the absence of Pc. As expected in cells with an intact PIL3 receptor, IL-3 binding to pc-/- cells was essentially unaltered. Bone marrow and spleen cells from 3c-/- mice failed to exhibit proliferation, enhanced survival in vitro, or synergistic interaction with M-CSF, even in extremely high concentrations (1 ,ug/ml) of GM-CSF, indicating an inability of the GMCSFRa chain alone to elicit any of these biological responses. 3c-/- cells also failed to proliferate in response to IL-5, supporting current models of IL-5 signaling via Pc in association with the IL-5Ra chain. Overall, hematopoietic alterations in 1c-/- mice were relatively minor. This suggests (i) that GM-CSF, despite its role in survival, proliferation, and differentiation of myeloid progenitors in vitro (13) and in perturbations of hematopoiesis when present in excess in the mouse (18), does not play a critical role in basal hematopoiesis or (ii) that other growth factors are able to substitute for this role. Similar observations have been made in GM-CSF null mice (19, 20). The most striking abnormality in the blood of 3c-/- mice was a reduction in eosinophil numbers with lesser reduction in bone marrow and tissues. Others have shown that 3c-/- mice fail to mount a normal eosinophilic response to parasitic infection (17). Intriguingly, however, we found the numbers of eosinophil progenitor cells in the marrow, as detected by IL-3 stimulation, did not differ from controls. Only GM-CSF, IL-5, and IL-3 are known to stimulate the proliferation of eosinophil precursors. As GM-CSF and IL-5 are unable to stimulate eosinophil formation by Oc-/- marrow, it is possible that IL-3 might be responsible for the production of eosinophils in fc-/- mice. However, IL-3 has not been documented to be produced in normal mice and an as yet undiscovered factor may be responsible for this. The pulmonary pathology, resembling alveolar proteinosis, seen in the 1c-/- mice has been observed by others and is similar to that seen in GM-CSF null mice (17, 19, 20). Its etiology is uncertain, although a local defect in GM-CSF-mediated macrophage function affecting surfactant clearance or infection control is possible, as GM-CSF is produced by several cell types within the lung and alveolar macrophages are GM-CSFresponsive (21). Clearance of a trace dose (-40 ng) of GM-CSF in c-/mice was compared with that in I3c+/+ mice in order to study the contribution of receptor-mediated internalization to this process. Previous pharmacokinetic studies have shown an inverse relationship between serum G-CSF levels and the level of neutrophils in the peripheral blood (22), and it has been postulated that neutrophil receptor-mediated endocytosis of G-CSF may act as an important clearance mechanism. A similar mechanism was proposed for M-CSF (23). Our results are in keeping with previous studies showing rapid clearance
Medical Sciences: Robb et al. of cytokines from the murine circulation on intravenous injection (12). However, the lack of alteration in the half-life of GM-CSF and its organ distribution and degradation in f3c / mice suggests that ,Bc plays only a minor role in the overall clearance mechanism for GM-CSF in the resting state. The role of the a chain in this process remains to be evaluated. We thank Dr. Andreas Strasser for his generous gift of monoclonal antibodies and Dr. Andrew Hapel (Australian National University) for mouse IL-5. We also thank Bette Papaevangeliou, Sandra Mifsud, Ladina di Rago, and Dale Cary for their excellent technical assistance and Jodie Stanley for animal husbandry. This work was supported by the Deutsche Forschungsgemeinschaft (to F.K.), the National Health and Medical Research Council of Australia, Anti-Cancer Council of Victoria, National Institutes of Health Grant CA22556, the Rotary Bone Marrow Research Centre Royal Melbourne Hospital, and the Cooperative Research Centres Grant of the Australian Government. 1. Metcalf, D. (1993) Blood 82, 3515-3523. 2. Takatsu, K., Takaki, S. & Hitoshi, Y. (1995) Adv. Immunol. 57, 145-190. 3. Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K., Yokota, T. & Miyajima, A. (1990) Proc. Natl. Acad. Sci. USA 87, 96559659. 4. Kitamura, T., Sato, N., Arai, K.-I. & Miyajima, A. (1991) Cell 66, 1165-1174. 5. Tavernier, J., Devos, R., Cornelis, S., Tuypens, T., Van der Heyden, J., Fiers, W. & Plaetinck, G. (1991) Cell 66, 1175-1184. 6. Itoh, N., Yonehara, S., Schreurs, J., Gorman, D. M., Maruyama, K., Ishii, A., Yahara, I. & Arai, K.-I. (1990) Science 247,324-327. 7. Hara, T. & Miyajima, A. (1992) EMBO J. 11, 1875-1884. 8. Tybulewicz, V. L. J., Crawford, C. E., Jackson, P. K., Bronson, R. T. & Mulligan, R. C. (1991) Cell 65, 1153-1163.
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9. Szabo, P. & Mann, J. R. (1994) Development (Cambridge, U.K) 120, 1651-1660. 10. Robb, L., Lyons, I., Li, R., Hartley, L., Kontgen, F., Harvey, R. P., Metcalf, D. & Begley, C. G. (1995) Proc. Natl. Acad. Sci. USA 92, 7075-7079. 11. Nicola, N. A. & Metcalf, D. (1986) J. Cell. Physiol. 128, 180-188. 12. Metcalf, D. & Nicola, N. A. (1988) Proc. Natl. Acad. Sci. USA 85, 3160-3164. 13. Metcalf, D. (1984) Clonal Culture of Hemopoietic Cells: Techniques and Applications (Elsevier, Amsterdam). 14. Strasser, A., Harris, A. W. & Cory, S. (1991) Cell 67, 889-899. 15. Elliott, M. J., Strasser, A. & Metcalf, D. (1991) J. Immunol. 147, 2957-2963. 16. Metcalf, D. & Nicola, N. A. (1992) Blood 79, 2861-2866. 17. Nishinakamura, R., Nakayama, N., Hirabayashi, Y., Inoue, T., Aud, D., McNeil, T., Azuma, S., Yoshida, S., Toyoda, Y., Arai, K.-I., Miyajima, A. & Murray, R. (1995) Immunity 2, 211-222. 18. Metcalf, D., Begley, C. G., Williamson, D. J., Nice, E. C., De Lamarter, J., Mermod, J. J., Thatcher, D. & Schmidt, A. (1987) Exp. Hematol. 15, 1-9. 19. Dranoff, G., Crawford, A. D., Sadelain, M., Ream, B., Rashid, A., Bronson, R. T., Dickersin, G. R., Bachurski, C. J., Mark, E. L., Whitsett, J. A. & Mulligan, R. C. (1994) Science 264, 713-716. 20. Stanley, E., Lieschke, G. J., Grail, D., Metcalf, D., Hodgson, G., Gall, J. A. M., Maher, D. W., Cebon, J., Sinickas, V. & Dunn,
A. R. (1994) Proc. Natl. Acad. Sci. USA 91, 5592-5596. 21. Bilyk, N. & Holt, P. (1993) J. Exp. Med. 177, 1773-1777. 22. Layton, J. E., Hockman, H., Sheridan, W. P. & Morstyn, G. (1989) Blood 74, 1303-1307. 23. Bartocci, A., Mastrogiannis, D. S., Migliorati, G., Stockert, R. J., Wolkoff, A. W. & Stanley, E. R. (1987) Proc. Natl. Acad. Sci. USA 84, 6179-6183.
