,JOURNAL OF CELLULAK PHYSIOLOGY 153:362-372 (1992)

Growth Factors Produced by Human Embryonic Kidney Cells That-Influence Megakaryopoiesis Include Erythropoietin, Interleukin 6, and Transforming Growth Factor-Beta R A Y M O N D M. W I T H Y , L O R I F. RAFIELD, A N T O N K. BECK, H E N R Y HOPPE, NEIL WILLIAMS, AND JOHN M. M c P H E R S O N * Recombinant Protein Development and Molecular Biology Departmenb, (;enzyme Corporation, Framingham, Mdssachusetts 0 7 70 1 (R. M.W., L. F.K., A. K . B., t-1.H., 1.M.M.1; and University of Melbourne, Melbourne, Victoria, Australia (N. W.)

Partially purified protein preparations containing megakaryocyte growth factor activity were prepared from human embryonic kidney (HEK) cell conditioned medium using ammonium sulfate precipitation, Cibicron blue affinity chromatography, and wheatgerm lectin affinity chromatography. Treatment of these preparations with neutralizing antibodies directed against erythropoietin (EPO) and interleukin 6 (IL6) resulted in a dramatic reduction in their capacity to stimulate megakaryocyte maturation in vitro. The presence of EPO in these preparations was confirmed by both immunoblotting and use of a mouse spleen erythroid progenitor cell proliferation assay routinely used to quantitate EPO activity in vitro. Northern blot analysis of HEK cell-dcrivcd mRNA with IL6 DNA probes revealed the presence of an IL6 transcript with a molecular sizc of 1.3 kb. Analysis ofthe HEK cell-derived preparation by ELlSA confirmed the presence of imniunologically reactive IL6. In addition, it was shown that purified recombinant human EPO and IL6 stimulated megakaryocyte maturation in the in vitro assay used in this study. These data indicate that the activity in H E K cell conditioned medium that stimulates megakaryocyte maturation in vitro is predominantly due to the presence of IL6 and EPO. lmmunoneutralization studies of another HEK cell-derived preparation, which was inhibitory in the megakaryocyte maturation assay, demonstratcd that it contained transforming growth factor beta (TGFP), a potent inhibitor of megakaryocyte maturation. Taken together, these studies indicate that HEK cell conditioned mcdium, which has previously been reported to contain megakaryocytc growth factor activity, is comprised of a complex mixture of growth and differentiation factors, some of which promote and others that inhibit the process (c) 1992 WiIey-Lisi, tnr. of mcgakaryopoiesis.

A considerable body of evidence from both rodent and human studies indicates that megakaryopoiesis, the continuum of developmental cellular events that begin a t the bone marrow stem cell and end with the eventual appearance of platelets in the circulation, is under multilevel, temporal control (Hoffman, 1989). A reduction of the circulating platelet mass leads to a n increase in the number, size, and ploidy of identifiable megakaryocytes in the bone marrow within 48 hours, whereas the levels of clonable megakaryocyte progenitor cells are unaltered (Harker, 1968; Ode11 et al., 1976; Levin et al., 1980; Burstein et al., 1981). These observations have led to the hypothesis that megakaryopoiesis is under the control of two humoral factors: thrombopoietin (TPO), which stimulates the terminal nonmitotic maturation of megakaryocytes and whose levels are inversely regulated by the platelet mass, and megakaryocyte colony stimulating factor (Meg-CSF), which (0 1992 WILEY-LISS.

INC

primarily stimulates the proliferation of the megakaryocyte progenitor cell population, but whose levels are independent of platelet mass and are inversely regulated by the bone marrow megakaryocyte mass (Hoffman, 1989; Hill and Levin, 1989). Using a n in vivo bioassay for TPO in which the incorporation of radiolabel into newly released platelets is measured (Evatt and Levin, 1969), investigators have isolated TPO-like activities from a number of sources.

Received March 20,1992; accepted June 11,1992.

*To whom reprint requests correspondence should be addressed. Lori F. Rafield is now at COR Therapeutics, South San Francisco, California.

A.K. Beck is now at Sandoz Pharma Limited, Basel, Switzerland.

HUMAN EMBRYONIC KIDNEY CELLS PRODIJCE EPO, IL6, AND TGFp

TPO has been partially purified from the plasma of thrombocytopenic rabbits, and these preparations have been shown to increase the proportion of higher ploidy megakaryocytes in the bone marrow both in vitro and in vivo (Levin et al., 1982; Hill and Levin, 1986; Hill e t al., 1989). Human embryonic kidney cell conditioned medium has been reported to contain thrombopoiesis stimulating factor ITSF), which has similar biological properties to plasma TPO (McDonald et al., 1975). TSF has been purified to apparent homogeneity (McDonald et al., 1985,1989).A third factor, megakaryocyte stimulatory factor (MSF), has also been identified in the plasma from thrombocytopenic rabbits and has been purified to apparent homogeneity from HEK cells (Tayrien and Rosenberg, 1988). MSF was defined by its ability to stimulate the metabolic radiolabeling of platelet specific proteins by megakaryocytes in vitro (Greenberg et al., 1988). It has been suggested that MSF and TSF from HEK cells are similar if not identical molecules (McDonald, 1989). Discrepancies, however, exist in their in vitro biological functions. Purified MSF was found only to increase platelet factor 4 in megakaryocytes but did not increase megakaryocyte DNA content (Greenberg et al., 1988). This restricted activity profile is in contrast to the wide spectrum of activities exhibited by semipurified preparations of TSF from the same source (McDonald, 1989). Another in vitro approach to analyzing megakaryocyte development has been the growth of megakaryocytes in semisolid media either a s colonies from precursor cells or as single megakaryocytes (Metcalf et al., 1975; Long et al., 1982). Based on these assays, another protein factor has been identified that stimulates megakaryocyte growth but has no colony stimulating activity. This growth factor has been designated megakaryocyte potentiator (Williams et al., 1978,1982).Tissue sources of mouse megakaryocyte potentiator have included the bone marrow, lung, and peritoneal exudate, suggesting a n in vivo role for this activity (Williams et al., 1981). Megakaryocyte potentiator has been shown to be biologically similar to TSF, and elevated levels of this activity are found in the plasma from thrombocytopenic animals (Keller e t al., 1988). Based on the fact that HEK cells have been reported as a reliable source of megakaryocyte growth factors, we initiated a series of experiments aimed at purifying these factors, obtaining partial amino acid sequence data, and cloning the cDNAs corresponding to these factors. We report here that growth factors produced by

A bbreuiations AcChE

ELISA EPO HEK IL6 MSF PBS SDS-PAGE SEM TGFp TPO TSF

Acetylcholinesterase enzyme linked immuno-sorption assay erythropoietin human embryonic kidney interleukin 6 megakaryocyte stimulatory factor phosphate buffered saline sodium dodecyl sulfate-polyacrylamidc gel electrophoresis Standard error of the mean transforming growth factor beta thrombopoietin thrombopoiesis stimulating factor

363

HEK cells that influence megakaryopoiesis include erythropoietin (EPO), interleukin 6 (IL6), and transforming growth factor beta (TGFP).

MATERIALS AND METHODS Electrophoresis chemicals were purchased from Biorad (Richmond, CA). All other chemicals were of reagent grade. Tris acryl blue resin was obtained from LKB (Gaithersburg, MD). Wheatgerm Sepharose was obtained from Sigma (St. Louis, MO). Cell culture media and FBS were obtained from Gibco (Grand Island, NY), and HEK cells from Whittaker Bioproducts (Walkersville, MD). Recombinant IL6 (specific activity lo7unitsimg protein) was obtained from Genzyme (Boston, MA). Porcine TGFPl and 2 a s well as the antiTGFP antibody were obtained from R&D Systems (Minneapolis, MN). Confirmatory experiments were performed with TGFPl and two provided by Dr. Yasushi Ogawa, Celtrix Laboratories (Palo Alto, CAI. Recombinant human EPO (specific activity 155 UnitsiFg) came from C127 cells transfected with a BPV vector containing a n EPO cDNA. IL6 antibody was obtained from Genzyme. Oligonucleotide probes were synthesized using a n ABI 380B DNA synthesizer. Preparation of HEK conditioned m e d i u m fractions Serum-free HEK conditioned medium was prepared a t large scale as described by Tayrien and Rosenberg (1988). Initially, the media was fractionated by Cibicron Blue affinity chromatography as follows: 50 ml media were adjusted to pH 6 by the addition of 0.1 M phosphoric acid with stirring and then applied to 5 ml Tris acryl blue resin that had previously been equilibrated in 50 mM sodium phosphate, 150 mM sodium chloride pH 7.4 (PBS). The column was washed in PBS, and the adsorbed proteins were eluted from the column with 50 mM sodium phosphate, 2 M sodium chloride, pH 8. The eluate was recovered in a volume of 8 ml, which were then dialyzed against PBS. This preparation was designated Preparation I. Serum-free HEK conditioned medium (60 liters) was also concentrated about tenfold over YM-3 membrane (Amicon), the protein was precipitated with ammonium sulfate, and then fractionated by wheatgerm lectin affinity chromatography as described by Tayrien and Rosenberg (1988). Ammonium sulfate fractionated material was designated Preparation 11, and the preparation that did not bind t o the lectin column was designated Preparation 111. Biological a s s a y s Murine megakaryocyte maturation was measured by monitoring the growth of acetylcholinesterase (AcChE) positive single cells from i n vitro cultures of murine bone marrow cells (Long et al., 1982; Williams et al., 1990). Briefly, a single cell suspension of murine bone marrow cells was centrifuged in a Percoll gradient, and the 1.07-1.085 g/cm3fraction that was devoid of mature megakaryocytes was used. The cells were cultured a t 5 x l o 4 cells per ml in agar for 5 days a t 37°C in a humidified incubator in the presence of test substance and 5%' FBS in DMEM. The cultures were dried, stained for AcChE, and the number of single positive-

WITHY ET AL.

364

A 80 40 0

1

T

a-

ct 30

f

20

10

10

0

1

.1

10

100

1 -

.1 '

10

1

rhEP0 (Units/ml)

Preparation I (ul) Fig. 1. Dose-dependent stimulation of murine megakaryocyte maturation with Preparation I and rhEPO. A. The indicated volumes of Preparation I were incubated with assay culture media (U) or with 20 pg of affinity purified EPO antibodies in assay culture media (w) for 2 hours at 37°C in a 100 (~.1reaction volume. These conditions were sufficient to neutralize the activity of one unit of rhEPO in the mouse spleen erythroid progenitor cell proliferation assay (data not shown). Following incubation, the samples were added to a preparation of mouse bone marrow cells depleted of mature megakaryocytes and the degree of stimulation of megakaryocyte maturation determined by

quantitating the number of AcChE positive cells following 5 days of culture as described in Materials and Methods. Values are the average +SEM of triplicate determinations. "significantly different ( P 0.05) from the equivalent untreated sample. €3. Murine bone marrow cells depleted ofmature megakaryocytes were incubated with the indicated concentrations of rhEPO for 5 days in culture and megakaryocyte maturation was determined by quantitating the number of AcChE positive cells as described in Materials and Methods. Values presented are the average and standard error of the mean (SEM) of triplicate determinations.

TABLE 1 , Effect of normal rabbit IgG on megakaryocyte maturation in vitro

bator. Following incubation, 50 p1 of DMEM plus 10% serum containing 1pCi of 6-13Hl-thymidinewere added to each well and incubated a t 37°C for a n additional 12 hours. At this time the test solutions were removed from the wells by aspiration and the wells washed twice (15 minutes each) with 200 p1 of phosphate-buffered saline. The cell layer was then solubilized by the addition of 200 pl of 1 M NaOH followed by incubation a t 37°C for 4 hours. Aliquots were then removed and radioactivity quantified by scintillation counting. The assay provided a measurable range of TGF-p1 between concentrations of 0.05-0.5 ngiml with half-maximal inhibition observed at a TGF-P concentration of -0.1 ngi ml. Antibody neutralization experiments were carried out by preincubating active material with appropriate quantity of antibody prior to bioassay. The specific details of these experiments are described in corresponding figure legends. Statistical analysis of the data was performed with either the signed rank Wilcoxon text or the MannWhitney U test using the Statview program running on a Macintosh computer. Various samples analyzed in the megakaryocyte maturation assays were tested in triplicate wells and the results expressed as the mean i 1standard error of the mean (SEM) of the triplicate cultures.

0 36 75 150 300

Unstimulated value

Stimulated value

23 i 6 19 I 5 24 t 3 21 5 2 19 3

78 L 4 75 F 2 71 i 5 73 i 6 70 L 7

*

'IgG was purified from normal rabbit serum by a combination uf ammonium sulfate precipitation and DEAE chroniatography ufiing standard procedures. The indicaled amounts of thc purified antibody preparation were incubated for 2 hours a t 97°C with either 100 pl ofPreparation I (stimulatedl or 100 p.1 ofcnntrol medium (unstimulated) in a final reaction volume of 200 ~ 1The . various tcst samples were then incubated with mega.karyocyte-deplcted bunt. niarrow cells for 5 days and scored for maturation aclivity as described in Materials and Methods. Values presented are the mean and standard error ofthe mean of triplicate determinations.

staining cells greater than 20 pm in diameter were counted by light microscopy. EPO activity was measured by the incorporation of ['HI-thymidine into spleen erythroid progenitor cells (Krystal, 1983). TGFp activity was measured by the inhibition of 13Hl-thymidine incorporation into mink lung cells (Ranchalis et al., 1987). Briefly, 100 pl aliquots of trypsinized mink lung cells (ATCC, CCL64) suspended a t a concentration of 10,000 cellsiml, in DMEM supplemented with 10% fetal bovine serum, were introduced into 96-well tissue culture plates and allowed to attach for 3 hours. Following attachment, 50-pl aliquots of test samples or TGF-f3 standards, diluted in DMEM plus 10% fetal bovine serum, were introduced into individual wells and incubated for 48 hours a t 37°C in a tissue culture incu-

%
99%) latent form, which is characteristic of TGFp from platelets and numerous cellular sources (Pircher e t al., 1986). Quantitative analysis of the degree of activation of TGFp obtained from the conditioned medium of African green monkey kidney cells indicated that -20% of the fraction was biologically active, whereas the remainder was latent and required acidification or treatment with denaturing agents for expression of biologic activity (McPherson et al., 1989). The mechanism by which the human and monkey kidney epithelial cells activate TGF-f! in vitro is uncertain but may represent proteolytic activation of the factor as has been previously reported (Lyons et al., 1988). The results of other investigators have shown that TGF-p1 is a potent inhibitor of megakaryopoiesis in vitro (Ishibashi et al., 1987; Ottman and Pelus, 1988; Bruno and Hoffman, 1989). The results of our experiments confirm and extend these observations by showing that both TGF-pl and TGF-P2 inhibit megakaryocyte maturation in our in vitro assay system. This result is of particular interest since i t has been reported that TGF-61, but not TGF-PZ, is a potent inhibitor of the proliferative phases of hematopoietic and multilineage (i.e., granulocyte, macrophage, and megakaryocyte) cell development (Ohta et al., 1987). The simplest interpretation of these observations is that whereas both factors can inhibit the intermediate and latter stages of megakaryocyte differentiation, only TGF-p1 inhibits the proliferative phase of hematopoietic and megakaryocytic cell development. Another potentially important observation is that antibodies to TGF-P significantly increase endogenous megakaryocyte maturation in our assay system. This result suggests that TGF-p may normally play a n important role in controlling the process of megakaryopoiesis. Additional evidence to support this hypothesis has been provided by the results of experiments that demonstrate that a primary effect of intravenous administration of TGF-P in vivo is induction of thrombocytopenia (Carlino et al., 1990). Thus i t appears that megakaryopoiesis and platelet count may be stimulated not only by elevation of positive factors such as IL6, but also perhaps by the relief of inhibition imposed by TGF-P. Such a system could provide exquisite control and multiple pathways for different physiological signals to control platelet mass.

ACKNOWLEDGMENTS We are extremely grateful to Sara Yankelev, Louise Palmer, Bill Jacobson, J o e Kutzko, Nicholas Masiello, Karen Lee, Steven Reber, Dawn Ferrara, and Joe Windslow for their excellent technical assistance in the execution of these experiments. We acknowledge Dr.

HUMAN EMBRYONIC KIDNEY CELLS PRODUCE EPO, 1L6. AND TGFp

Susan Richards, who provided the anti-EPO antibody used in these studies. We also thank Dr. Cathy Oppenheimer and Dr. Alan Smith for their helpful suggestions during the preparation of this manuscript.

LITERATURE CITED Aviv, H., and Leder, P. (1972) Purification of biologically active messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA, 69t1408-1412. Banu. N., Fawcett, J., Williams, N., De Giorgio, T., and Withy, R. (1990) Tissue sources of murine megakaryocyte potentiator - biochemical and immunological studies. Br. J. Hematol., 7 5 3 1 3 3 1 8 . Berridge, M.V., Fraser, J.K., Carter, J.M., and Lin, F.K. (1988)Effects of recombinant erythropoietin on megakaryocytes and on platelet production in the rat. Blood, 72.970-977. Bruno, E., and Hoffman, R. (1989) Effect of interleukin 6 on in vitro human meeakarvocvtoDoiesis: Its interaction with other cvtokines. Exp. Hematol , 1?’~163~-1043. Bruno, E., Briddell, R.A., Cooper, R.J , and Hoffman, R. (1991) Effects of recombinant interleukin i 1 on human megakaryocyte progenitor cells. Exp. Hematol., 19t378-381. Burstein, S.A., Adamson, J.W., Erb, S.K. and Harker, L.A. (1981) Megakaryocytopoiesis in the mouse: Response to varying platelet demand. J . Cell. Physiol., 109r333-341. Carlino, J., Higley, H., Avis, P., Chu, S., Ogawa, Y., and Ellingsworth, L.E. (1990) Hematologic and hematopoietic changes induced by systemic administration of TGF-PI. Annals. NY Acad. Sci., 593:33& 333. Chomczynski, P., and Sacchi, N. t19S7) Single-step method of RNA isolation by acid guanidinium thiocynate-phenol-chloroform extraction. Anal. Biocheni., 162t156-159. Church, G., and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA, 81t1991-1995. Derynck, R., Jarrett, d.A., Chen, E.Y., Eaton, D.H., Bell, J.R., Assoian, R.K., Roberts, A.B., Sporn, M.B., and Goeddel, D.V. (1985)Human transforming growth factor-p complementary DNA sequence and expression in normal and transformed cells. Nature (Lond.), 316:701-705.

Evatt, B.L., and Levin, J. (1969) Measurement of thrombopoiesis in rabbits using 75Selenomethionine.J. Clin. Invest., 48: 1615-1626. Goldman, S., Hoysradt, J., McCarthy, K., Hayes, L., Neben, T., Stoudmire, J., and Schaub, R. 11991) Recombinant interleukin 11 stimulates megakaryocyte maturation and an increase in peripheral platelet count in vivo. J. Leukocyte Biol. (abstract)Suppl., 2:27. Greenberg, S.M.,Kuter, D.J., and Rosenberg, R.D. (19881 In vitro stimulation of megakaryocyte maturation by megakaryocyte stimulatory factor. J. Biol. Chem., 262:326%3277. Harker, L.A. (1968) Kinetics of thrombopoiesis. J. Clin. Invest., 47t458-465. Hill, R., and Levin, J . (1986) Partial purification of thrombopoietin using lectin chromatography. Exp. Hematol., 14.752-759. Hill: R.J., and Levin: J. (1989) Regulators of thrombopoiesis: Their biochemistry and physiology. Blood Cclls, 15:141-166. Hill, R.J., Leven, R.M., Levin, F.C., and Levin, J. (1989) The effect of partially purified thrombopoietin on guinea pig megakaryocyte ploidy in vitro. Exp. Hematol., 17r903. Hill, R.J., Warren, M.K., and Levin, J. (1990) Stimulation of thrombopoiesis in mice by recombinant interleukin 6. 6 . Clin. Invest., 85: 1242-1247. Hirano, T., Yasukawa, K., Harada, H., Taga, T., Watanabe, Y., Matsuda, T., Kashiwamura, s.,Nakajima, K., Koyama, K., et al. (1986) Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature (Lond.), 324t73-76. Hoffman, R. (1989) Regulation of megakaryopoiesis. Blood, 74:119& 1212. Ishibashi, T., Koziol, J.A., and Burstein, S.A. (1987a) Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro. J . Clin. Invest., 79:286289. Ishibashi, T., Miller, S.L., and Burstein, S.A. (198713) Type p transforming growth factor is a potent inhibitor of murine megakaryocytopoiesis in vitro. Blood, 69:1737-1741. Ishibashi, T., Kimura, H., Uchida, T., Kariyone, S., Friese, P., and Burstein, S.A. 11989a) Human interleukin 6 is a direct promoter of maturation of megakaryocytes in vitro. Proc. Natl. Acad. Sci. USA, 86.5953-5957.

371

Ishibashi, T., Kimura, H., Shikama, Y., Uchida, T., Kariyone, S., Hirano, T., Kishimoto, T., Takatsuki, F., and Akiyama, Y. (198913) Interleukin-6 is a potent thrombopoietic factor in vivo in mice. Blood, 74,1241-1244. Keller, K.L., Rolovic, Z., Evatt, B.L., Sewell, E.T., and Ramsey, R.B. (1988) The effects of thrombopoietic activity of rabbit plasma fractions o n megakaryocytopoiesis in agar cultures. Exp. Hematol., 16t262-267. Krystal, G. (1983) A simple microassay for erythropoietin based on 3H-thymidine incorporation into spleen cells from phenylhydrazine treated mice. Exp. Hernatol., 11r649-654. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nat,ure (Lond.),227r680685. Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. (1977) RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry, 16r4744-4751. Levin, J., Levin, F.C., and Metcalf, D. (1980) The effects of acute thrombocytopenia on megakaryocyte-CFC and granulocyte-macrophage-CFC in mice: studies of bone marrow and spleen. Blood, 56r274-283. Levin, J., Levin, F.C., Hull, D.F., and Penington, D.G. (1982) The effects of thrombopoietin on megakaryocyte-CFC, megakaryocytes, and thrombopoiesis: With studies of ploidy and platelet size. Blood, 60t989-998. Long, M.W., Williams, N., and McDonald, T.P. (1982) Immature megakaryocytes in the mouse: in vitro relationship t o megakaryocyte progenitor cells and mature megakaryocytes. J. Cell. Physiol., 122t339-344. Kishimoto, T. (1989)The biology of interleukin-6. Blood, 7411-16. Lyons, R.M., Keski-Oia, J., and Moses, H.L. (1988) Proteolytic activation of latent transforming growth factor-p from fibroblast conditioned medium. J. Cell Biol., 106t1659-1665. Massague, J. (19871 The TGF-p family of growth and differentiation factors. Cell, 49r437-438. McDonald, T.P. (1989) The re-plation of megakaryocyte and platelet production. Int. J. Cell Cloning, 7:139. McDonald, T.P., Clift, R., Lange, R.D., Nolan, C., Tribby, I.E., and Barlow, G.H. (1975) Thrombopoietin production by human embryonic kidney cells in culture. J. Lab. Clin. Med., 85:59-66. McDonald, T.P., Cottrell, M., Clift, R., Khouri, ?J.A.,and Long, M.D. (1985) Studies on the purification of thrombopoietin from kidney cell culture medium. J. Lab. Clin. Med., 106:162-174. McDonald, T.P., Cottrell, M.B., Clift, R.E., Cullen, W.C., andLin,F.K. (1987) High doses of recombinant erythropoietin stimulate platelet production in mice. Exp. Hematol., 15:719-721. McDonald, T.P., Clift, R.E., and Cottrell, M.B. (1989) A four-step procedure for the purification of thrombopoietin. Exp. Hematol., 17:865471. McDonald, T.P., Cottrell, M.B., Swearingen, C.J., and Clift, R.E. (1991) Comparative effects of thrombopoietin and interleukin-6 on murine megakaryocytopoiesis and platelet production. Blood, 77t735-740. McPherson, J.M., Sawamura, S.J., Ogawa, Y., Dineley, K., Carrillo, P., and Piez, K.A. (1989) The growth inhibitor of african green monkey (BSC-1) cells is transforming growth factors p l and p2. Biochemistry, 28t3442-3447. Mei, R.-L., and Burstein, S.A. (1991) Megakaryocyte maturation in murine long-term bone marrow culture: Role of interleukin 6. Blood, 78r1438-1447. Metcalf, D., MacDonald, H.R., Odartchenko, N., and Sordat, B. (1975) Growth of mouse megakaryocyte colonies in vitro. Proc. Natl. Acad. Sci. USA, 72t1744-1748. Metcalf, I)., Nicola, N.A., and Gearing, D.P. (1990) Effects of injected leukemia inhibitory factor on hematopoietic and other tissues in mice. Blood, 7650-56. Metcalf, D., Hilton, D., and Nicola, N.A. (1991) Leukemia inhibitory factor can potentiate murine megakaryocyte production in vitro. Blood, 77r2150-2153. Nagasawa, T., Neichi, T., Satoh, K., Nakazawa, M., and Abe, T. (1988) In vitro regulatory mechanisms for cytoplasmic maturation of murine megakaryocytes derived from colony-forming units megakaryocyte iCFU-M). Exp. Hematol., I6t667-673. Odell, T.T., Murphy, J.R., and Jackson, C.W. (1976) Stimulation of megakaryocytopoiesis by acute thrombocytopenia in rats. Blood, 4Hr765-775. Ohta, M., Greenberger, J.S., Anklesara, P., Bassols, A,, and Massague, J. (1987) Two forms of transforming growth factor-p distin-

372

WITHY ET AL.

guished by multipotential haematopoietic progenitor cells. Nature (Lond.), 329t539-541. Ottmann, O.G., and Pelus, L.M. (1988) Differential proliferative effects of transforming growth factor-p on human hematopoietic progenitor cells. J . Immunol., 140t2661-2665. Pircher, R., Jullien, P., and Lawerence, D.A. (1986) P-Transforming growth factor is stored in human blood platelets as a high molecular weight complex. Biochem. Biophys. Res. Commun., 13Gt3037. Ranchalis, J.E., Gentry, I,., Ogawa, Y., Seyedin, S., McPherson, J., Purchio, A,, and Twardzik, D.R. (1987) Bone-derived and recombinant transforming growth factor p’s are potent inhibitors of tumor cell growth. Biochem. Biophys. Res. Comm., 148:783-789. Roberts, A., and Sporn, M.B. (1988) Transforming growth factor-p. Adv. Cancer. Res., 51t107-145. Sporn, M.B., Roberts, A.B., Wakefield, T,.M., and DeCrombrugghe, B.J. (1987) Some recent advances in the chemistry and biology of transforming growth factor-beta. J. Cell. Biol., 105r1039-1045. Stahl, C.P., Zucker-Franklin, D., Evatt, B.L., and Winton, E.F. (1991) Effects of human interleukin 6 on megakaryocyte development and thrombocytopoiesisin primates. Blood, 78:1467-1475. Tayrien, G., and Rosenberg, R.D. (1988)Purification and properties of a megakaryocyte stimulatory factor present both in the serum-free conditioned medium of human embryonic kidney cells and in thrombocytopenic plasma. J. Biol. Chem., 2G2r3262-3268.

Teramura, M., Kobayashi, S., Hoshino, S., Oshimi, I