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From www.bloodjournal.org by guest on June 17, 2017. For personal use only. Thrombopoietin Stimulates Colony-Forming Unit-Megakaryocyte Proliferation and Megakaryocyte Maturation Independently of Cytokines That Signal Through the gp130 Receptor Subunit By Virginia C. Broudy, Nancy L. Lin, Norma Fox, Tetsuya Taga, Mikiyoshi Saito, and Kenneth Kaushansky Thrombopoietin (Tpo), the ligand for thec-Mpl receptor, is a major regulator of megakaryopoiesis. Treatment of mice with Tpo raises the platelet count fourfold within a few days. Conversely, c-mpl knock-outmice have platelet counts that are 15% that of normal. The subunit structure of the c-Mpl receptor is not fully understood. Some cytokines that stimulate megakaryopoiesis (IL-6, IL-11, leukemia inhibitory factor, and oncostatin M) bind t o receptors that use gp130 as a signal transduction subunit. For these reasons, we determined whethergp130 function wasrequired for Tpo-induced signal transduction. Murine marrow cells were cultured in semi-solid media in the presence of Tpo or IL-3, with or without a neutralizing anti-gpl30 monoclonalantibody (RX187) or a soluble form of c-Mpl receptor (soluble Mpl) that blocks Tpo bioactivity, and thenumbers of colony-forming unit-megakaryocyte (CFU-Meg) colonies were counted on day 5. Murine marrowcells were also cultured in suspen- sion under serum-free conditions for5 days, and megakaryocyte DNA content was measured by flow cytometry, as an index of nuclear maturation. The addition of RX187 did not block Tpo-induced CFU-Meg colony growth nor CFU-Meg nuclear maturation in suspension culture. However, IL-3induced CFU-Meg colony growth and megakaryocyte nuclear maturation decreased in the presence of RX187. Soluble Mpl completely ablated Tpo-induced CFU-Meg growth, andpartiallyblockedIL-3-stimulated CFU-Meg growth. Thus the effects of Tpo on megakaryopoiesis in vitro do not depend on cytokines that signal through gp130. Furthermore, it is unlikely that gp130 serves as a beta chain for the c-Mpl receptor, as Tpo signalling is unimpaired in the presence of RX187. In contrast, the effects of IL-3 on CFUMeg growthare mediated in part throughTpo and through gp130-signalling cytokines. 0 1996 b y The American Society of Hematology. P then initiation of signal transduction. Thus the gp130receptor subunit, which is ubiquitously expressed in vivo,'5 plays a critical role in the transmission of signals of the IL-6family of cytokines across the cell membrane. Thrombopoietin, also known as Mpl ligand, megapoietin. or megakaryocyte growth and development factor, is a key regulator of megakaryopoiesis.'" In vitro, Tpo synergizes with erythropoietin, SCF, and IL-I 1 to promote growth of CFU-Meg colonies, and is a potent stimulant of megakaryocyte nuclear endoredupli~ation.~"'~~'~ Treatment of mice with Tpo increases the platelet count fourfold," while mice engineered to eliminate c-Mpl receptor expression exhibit platelet counts that are 15% that of normal levels.I9 Tpo is likely to be used clinically to speed platelet recovery after myelosuppressive chemotherapy, so an understanding of its mechanism of action is important. Thec-Mpl receptorisamember of the hematopoietic growth factor receptor as are receptors the for the IL-6 family of cytokines. Members of this receptor superfamily share structuralfeaturesincludingconserved cysteine residues and a characteristic WSXWS motif in the extracellular domain, and an absence of recognizable kinase domains in the intracellular portion of the receptor. Certain of the receptors in the superfamily homodimerize after ligand binding: others require association of a signal transducing P chain, which may be shared by several members of the family. The subunit structure of the functional c-Mpl receptor complex has not been fully defined. We investigated whether signal transduction via thegp 130 receptor subunit wasrequired forCFU-Meg proliferation and megakaryocyte nuclear maturation in response to Tpo or to IL-3. Our results demonstrate that the effects of Tpo on megakaryopoiesis in vitro do not requiregp130. Conversely,theeffects of IL-3 on CFU-Meg proliferation irz r'itro are dependent in part on Tpo andoncytokines that signal through the gp 130 receptor subunit. LATELET PRODUCTION is anorderly process in which committed megakaryocyte progenitor cells (colony-forming unit-megakaryocyte [CFU-Meg]) proliferate and differentiate into immature megakaryocytes,which then undergo cytoplasmic maturation and nuclear endoreduplication to generate mature,polyploid megakaryocytes. The mature megakaryocytes release platelets.',' These processes are governed by a flotillaof cytokines. Someof these hematopoieticgrowthfactors (including IL-3 and stemcell factor [SCF]) predominantly affect CFU-Meg proliferation, while others (including IL-6, IL- 1 l , leukemia inhibitoryfactor [LIF], and oncostatin M) mainlyinfluence megakaryocyte maturation.'.' Thrombopoietin enhances both CFU-Meg growth and megakaryocyte Interleukin-6, IL- 1 1, LIF, oncostatin M, and ciliary neurotropic factor have also been called neuropoietic cytokines,' I because of their diverse biological effects on hematopoiesis, B lymphocyte differentiation, induction of acute phase proteins, and survival and differentiation of neurons. The neuropoietic cytokines. together with cardiotrophin-I , l 2 use gpl30 asa component of the cell surface receptor complex.".'4 Ligand binding triggers association of gp 130, and formation of gp 130 homodimers or gp 130-LIFreceptor heterodimers, From the University of Washington. Seattle. WA; and Osaka University, Osaka, Japan. Submitted December 21. 1995; accepted May 17, 1996. Supported by National Institutes rgHealtlz Grunts No.DK 44194, CA 31615, and DK 49855 and an American Cancer Society Faculo Research Award to Virginia C. Broudy. Address reprint requests to Virginiu C. Broudy, MD, University of Washington, Division of Hematology. Bo.x 357710, Seattle, WA 98195-7710. The publication costs of this article were defrayed in part by page churge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 17.34 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8806-0031$3.00/0 2026 MATERIALSANDMETHODS Megakaryocyte colony assays. BhD2FI mice (JacksonLaboratories, Bar Harbor. ME), 8 to 10 weeks old, were killed by rapid Blood, Vol 88, No 6 (September 15). 1996:pp 2026-2032 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. TPOACTSINDEPENDENTLYOF 2027 ~ ~ 1 3 0 Table 1. Effect of Blocking gp130 Activity on CFU-Meg cervical dislocation, andmarrow cells were obtained by flushing Colony Growth the femurs with Iscove’s Modified Dulbecco’s Medium (IMDM, GIBCO, Grand Island, NY) supplemented with 2% fetal calf serum CFU-Meg Colonies/ Experiment (FCS; Hyclone, Logan, UT). The marrow cells were cultured in 2 x 10’ Cells Addition No. IMDM supplemented with 15% horse serum (Hyclone) and 5 X 0 1 0 IO-’ molar p mercaptoethanol (Sigma Chemical CO,St Louis, MO) 35.0 2 4.0 TPO in 0.285% agar (DIFCO, Detroit, MI) in triplicate plates as pre37.7 2 2.4 Tpo + RX187 viously described.8 Megakaryocyte colonies were counted after a 5Tpo + Sol Mpl 0 day incubation at 37°C in a humidified atmosphere containing 5% IL-3 22.7 2 0.3 COz. The growth factors used included recombinant murine Tpo 11-3 + RX187 12.7 2 0.3 and recombinant murine IL-3, both expressed in BHK cell^.'^^^^ Fifty 10.0 2 1.5 IL-3 + Sol Mpl units of Tpo or IL-3 activity were defined as that quantity that gave 0 2 0 one-half maximal stimulation in the BaF3Mpl proliferation assay, 41.0 2 2.2 TPO or in the murine marrow CFU-GM colony assay, respectively.” In 41.1 2 4.3 Tpo + RX187 some experiments, recombinant human IL-l1 (Genzyme, Boston, Tpo + Sol Mpl 0 MA) was also used. A soluble form of c-Mpl receptor (soluble Mpl) IL-3 33.0 2 1.4 was generated by truncation at the interface of the extracellular and IL-3 + RX187 18.0 t 1.2 and transmembrane domains, was purified as previously de~cribed?~ 15.3 2 2.6 IL-3 + Sol Mpl was provided by ZymoGenetics, Inc (Seattle, WA). Soluble Mpl is IL-3 + RX187 + Sol Mpl 18.3 2 3.9 a specific competitive antagonist of Tpo a~tivity.’~ Murine marrow cells were cultured in 0.285% agar in IMDM suppleThe RX187 rat antimouse monoclonal antibody (IgG2a) recogmol/L p mercaptoethanol mented with 15% horse serum and 5 x nizes the extracellular domain of gp130, and neutralizes the bioactivand Tpo (2,000 U/mL) or IL-3 (100 U/mL). The RX187 antibody was ity of IL-6 and IL-I I in vitro. To generate the M 1 8 7 antibody, a added at a concentration of 1.5 pg/mL, and soluble Mpl was added soluble form of the extracellular domain of murine gp130 was preat a concentration of 5 pg/mL. Thenumber of CFU-Meg-derived colopared by introducing a stop codon just above the transmembrane nies was counted on day 5. The data are presented as the mean domain. Soluble gp130 was used to immunize a rat, and the resultant 2 SEM of triplicate plates. Five additional experiments gave similar hybridomas were screened by flow cytometry for ability to recognize results. the native transmembrane form of murine gp130. The m 1 8 7 antibody was able to immunoprecipitate a 130 kD protein from methionine-labeled cells into which cDNA encoding murine gp130 had been introduced, but not from untransfected parental cells; congoat antirat IgG (Jackson Immunoresearch Labs, Inc, West Grove, trol rat IgG2a did not immunoprecipitate the 130 kD protein. The PA) for 1 hour at 4°C. The cells were then incubated with propidium M 1 8 7 antibody blocked the ability of the T1165 mouse plasmacyiodide (50 pg/mL; Molecular Probes, Inc. Eugene, OR) in a hypotoma cell line to proliferate in the presence of IL-6 or IL-11.25 Further tonic citrate solution, and analyzed in a Coulter Epics Elite flow evidence of the neutralizing activity of the m 1 8 7 antibody is procytometer.’ vided in the Results section. The control rat IgG2a antibody was obtained from PharMingen, San Diego, CA. RESULTS To determine if the horse serum used for the megakaryocyte colMegakaryocytic colony growth. We examined the ability ony assays contained Tpo, a Tpo-responsive murine cell line, DAof the neutralizing anti-gpl30 monoclonal antibody W 1 8 7 1-TpoZ6was cultured in the presence of horse serum with or without to block growth of CFU-Meg colonies in the presence of soluble Mpl, and cell proliferation was quantitated. Parallel cultures Tpo or IL-3 (Table 1). In seven independent experiments, of the DA-l-Tpo cells contained known quantities of Tpo. These experiments showed that the quantity of horse serum used for the the addition of RX187 antibody did not affect the number CFU-Meg colony assays would contribute approximately 2 U/mL of CFU-Meg colonies found in the presence of 2,000 U/mL. of Tpo activity to the culture. Megakaryocyte colony assays conof Tpo. In contrast to the results with Tpo, the addition of taining 15% horse serum but no added growth factor had no CFUM 1 8 7 antibody blocked IL-3-stimulated CFU-Meg colony Meg colonies, demonstrating that this quantity of Tpo alone was growth by approximately 40% to 45%. The addition of coninsufficient to support CFU-Meg colony growth. trol IgG2a had no effect on CFU-Meg colony growth in the Analysis of megakaryocyte ploidy. Murine marrow cells were ~ ~ ~ ~ of Tpo or of IL-3 (data not shown). Soluble Mpl, cultured in serum-free suspension as previously d e s ~ r i b e d . ~ ~ ’presence which competes with cell surface c-Mpl for binding Tpo, Briefly, the marrow cells were cultured at a concentration of 1 X completely inhibited Tpo-stimulated megakaryocyte colony lo6cells/mL in IMDM supplemented with 1% Nutridoma-SP (Boehm o m p mercapringer Mannheim COT, Indianapolis, IN), 5 X growth, and inhibited XL-3-stimulated megakaryocyte coltoethanol, and growth factors with or without M 1 8 7 antibody as ony growth by approximately 50% (Table 1). The horse described above. The cells were incubated at 37°C for 5 days. The serum used in the CFU-Meg colony assay contributed apDNA content of the megakaryocytes was analyzed by flowcytometry proximately 2 U/mL of Tpo activity to the culture; this may after labeling with the 4A5 monoclonal antibody and withpropidium account in part for the ability of soluble Mpl to inhibit CFUiodide as previously des~ribed.~,~’ The 4A5 monoclonal antibody Meg colony growth in cultures containing IL-3. recognizes a 74 kD glycoprotein present on murine megakaryocytes To determine whether R X I 87 antibody would inhibit and and was generously provided by Dr Sam Burstein CFU-Meg colony growth in the presence of lower concentra(University of Oklahoma, Oklahoma City, OK). Briefly,the cells tions of Tpo, we cultured murine marrow cells with Tpo (0, were labeled with the 4A5 monoclonal antibody (15 &mL) or with 15, 60,350, 1,OOO, or 2,000 U/mL) with or without RX187 control rat IgG2a antibody in 0.15 moVL NaCl (pH 7.4) containing antibody (Fig 1). The addition of RX187 antibody did not 0.5% bovine serum albumin (Sigma) and0.05% sodium azide (Sigma) for 30 minutes at 4”C, then incubated with FITC-conjugated block Tpo-induced CFU-Meg colony growth in the presence ’%- From www.bloodjournal.org by guest on June 17, 2017. For personal use only. BROUDYET AL 2028 45 T 40 Y) 3 35 r' g 30 0 -1J1 0 2._ 25 c 20 I I 0 350 0 15 l000 U. 04 0 3 10 D Concentrationof IL-l1 (@mi) 1 - rm 2000 60 Concentrationof Tpo (Ulml) Fig 1. Effect of neutralizing gp130 activity on growthof CFU-Meg colonies in the presence of Tpo. Murine marrow cells (2 x 105/mL) were culturedin semi-solid media in the presence of various concentrations of Tpo (0 t o 2,000 UlmL) without (0)or with (M) RX187 antibody (1.5 pglmL) for 5 days. The data represent the number of CFU-Megcolonies (mean f SEM of triplicate plates)from one of two similar experiments. of lower concentrations of Tpo (Fig l ) . The combination of Tpo plus IL-3 is a very potent stimulant of megakaryocytic colony growth (Fig 2). The addition of RX 187 antibody did not diminish CFU-Meg colony growth in thepresence of Tpo plus TL-3 (Fig 2). suggesting that signal transduction via gp130 is not required for megakaryocytic colony growth under these conditions. Although IL-l1 exerts its predominant effects on megakaryocyte maturation rather than on CFU-Meg proliferation, IL-l I can synergize with Tpo to augment the growth of megakaryocytic colonies.x The addition of IL- I 1 ( I O to 100 ng/mL) to a submaximal concentration of Tpo ( I O 0 U/mL) T Fig 3. Effect of neutralizing gp130 activity on growthof CFU-Meg colonies. Murine marrow cells were cultured as in Fig 1 in the presence of Tpo (100 UlmL) plusvarious concentrations of IL-l1 ( 0 t o 100 nglmL) withor without RX187 antibody (1.5 pg/mLI for 5 days. The data represent the number of CFU-Meg colonies (mean f SEM of triplicate plates) from one of two similar experiments. enhanced CFU-Meg colony growth (Fig 3 ) . The RX I87 antibody was able to ablate the incremental effect of IL-l 1 on growth of CFU-Meg colonies (Fig 3). directly demonstrating the neutralizing activity of this antibody. Consistent with the data in Table 1 andFig I , the RX187 antibody did not diminish CFU-Meg colony growth below that seen withTpo alone. To further define the neutralizing activity of RXI 87 antibody, murine marrow cells were cultured in the presence of a range of concentrations of IL-l I , with or without RX187 antibody or control IgG2a antibody (Fig 4).The addition of RX I X7 antibody blocked the effects of IL-l1 on CFU-Meg colony growth. At high concentrations of IL-l I , a modest number of CFU-Meg colonies was found even in the presence of RX I87 antibody (Fig 4).This suggests that IL-1 1 and RXI X7 antibody compete for binding tothe gp130 subunit of T 11 1 15 20( 3 100 Concentrationof IL-3 (Ulml) 20$8.') 0 1 5 Concentration of IL-l1 (ng/ml) Fig 2. Effect of neutralizing gp130 activity on growth ofCFU-Meg colonies in the presence of Tpo plus 11-3. Murine marrow cells were cultured as in Fig 1 in thepresence of various concentrations of IL-3 (0). 11-3 plus Tpo l100 UlmL, O), or 11-3 plus Tpo plus RX187 (1.5 p g l mL, M) for 5 days. The data represent the number of CFU-Meg colonies (mean f SEM of triplicate plates) from one of two similar experiments. Fig 4. Effect of neutralizinggp130 activity on growth of CFU-Meg colonies in the presence of IL-11. Murine marrowcells were cultured as in Fig 1 in the presence of various concentrations of IL-l1 ( 0 t o 80 nglmL) in the presence of control ratlgG2a 11.5 pglmL, 0 )or RX187 antibody (1.5 pg/mL, M) for 5 days. The data represent the mean f SEM of triplicate plates from one experiment. ) From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 2029 TPO ACTS INDEPENDENTLY OF ~ ~ 1 3 0 the receptor, and also demonstrates that the effects of m 1 8 7 antibody are not due to nonspecific toxicity. To ensure that appropriate concentrations of M 1 8 7 antibody were used for these experiments, an additional experiment examined the effects of varying amounts of the M 1 8 7 antibody on CFUMeg colony growth in the presence of Tpo plus IL-11 (Table 2). The RX187 antibody at a concentration of 0.5 to 1.5 pg/ mL completely blocked the incremental effect of IL-l1 on CFU-Meg colony growth. The control IgG2a antibody had no effect (Table 2). Megakulyocytenuclearendoreduplication. We examined the DNA content of the megakaryocytes generated in serum-free suspension cultures in the presence of Tpo or IL3, with or without RX187 antibody, to assess the effects of these cytokines on nuclear endoreduplication. Some megakaryocytes were produced even in the absence of exogenous growth factor, and the modal ploidy of these megakaryocytes was 16N (Fig 5). When m 1 8 7 antibody was added to the cultures containing no exogenous growth factor, megakaryocyte ploidy decreased (data not shown), suggesting that endogenous production of cytokines that require gp130 as a signal transduction subunit is contributing to megakaryocyte endoreduplication in these cultures. IL-6 and IL-11, which are produced by stromal ~ e l l s , ~are ~ . ~likely ' candidate cytokines; IL-6 can also be produced by megakaryocyte^.^',^^ The addition of Tpo profoundly increased megakaryocyte nuclear endoreduplication over that seen withno added growth factor (Fig 5). In the presence of Tpo, the majority of megakaryocytes were of 32-64N ploidy (Fig 5). The addition of RX187 did not diminish the proportion of 32N, 64N, and 128N megakaryocytes produced in the presence of Tpo (Fig 5). A relatively small proportion of the megakaryocytes generated in the presence of IL-3 exhibited 32N, 64N, or 128N DNA content (Fig 6), in contrast to megakaryocytes produced in the presence of Tpo. Neutralizing gp130 activity decreased megakaryocyte nuclear endoreduplication in the presence of IL-3, and virtually eliminated the production of 64N megakaryocytes (Fig 6). We interpret these results to mean that the modest effect of IL-3 to stimulate megakaryocyte nuclear endoreduplication is dependent on the presence of endogenous IL-6 or IL-11 in these c u l t ~ r e s . ~ ~ - ~ * Table 2. Effect of RX187 Antibody on CFU-Meg Colony Growth Addition CFU-Meg Colonies1 2 X 10' Cells 0 (100 Tpo ng/mL) Tpo(25 IL-l1 IL-l1 Tpo Tpo + I L - l 1 L11 Tpo IL-l1 Tpo IL-l1 Tpo IL-l1 Tpo IL-l1 Tpo + + + + + + + + RX187 (0.05 pg/mL) + RX187 (0.15 pg/mL) + RX187 (0.5 Fg/mL) + RX187 (1.5 pg/mL) + IgG2a (0.15 pg/mL) + IgG2a (0.5pg/rnL) + lgG2a (1.5 pg/mL) 0 41.0 59.0 63.7 55.7 41.0 43.0 58.0 62.0 56.7 2 2.2 2 5.1 2 7.8 i 0.3 1.4 t 0.6 2 1.2 t 2.2 t 2.3 C Murine marrow cells were cultured as described in Table 1 in the presence or absence of RX187 antibody (0.05 t o 1.5 pg/mL). The data represent the mean 2 SEM of triplicate plates from one experiment. 2N TII DNA CONTENT Fig 5. DNA histogram ofmegakaryocytes generated in wspension culture. Murine marrow cells l1 x 106/mL) were cultured in IMDM supplementedwith 1% Nutridoma-SP with no added growth factor (A), Tpo (100 U l m l B), or Tpo plus RX187 IC). After 5 days of culture, the cells were labeled with the 4A5 antimouse megakaryocyte monoclonal antibody, stained with propidium iodide, and analyzed by flow cytometry. The results of one of four similar experiments are shown. DISCUSSION Hematopoiesis in vivo is intricately controlled to maintain thenumbers of circulating erythrocytes, leukocytes, and platelets with a narrow range under normal circumstances. Much is understood about the physiology of the key growth factors that regulate the terminal stages of erythropoiesis and myelopoiesis, and several of these cytokines have been in clinical use for approximately 10 years. In contrast, the cytokine that predominantly controls megakaryopoiesis had remained elusive. The cloning of the c-mpl proto-~ncogene,~~ and the subsequent use of c-Mpl receptor affinity chromatography to isolate its ligand 34,35 in parallel to a functional cloning strategy,36 and other purification appro ache^,^'.^' yielded Tpo. Contrary to predictions from experimental models of thrombocytopenia, and previous studies with partially purified molecules, recombinant Tpo was found to promote both the proliferation of CFU-Meg and the maturation of their progeny. The network of hematopoietic cytokines is characterized From www.bloodjournal.org by guest on June 17, 2017. For personal use only. BROUDY ET AL 2030 f I l 32N 2N I 4N 1 C 2N ..L I.. DNA CONTENT Fig 6. Effect of W187 antibody on nuclear endoreduplication of megakaryocytes generated in the presence of IL-3. Murine marrow cells were cultured as describedin Fig 2 in thepresence of no added growth factor (A), IL-3 (100 UlmL, B), or IL-3 plus RX187 (C). After 5 days of culture, the DNA content of the WNS was analyzed as described in Fig 2. The data from one of four similar experiments are shown. by functional redundancy. Previous work had shown that IL3 is a potent stimulant of murine CFU-Meg proliferation,"* and that the IL-6 family of cytokines including IL-6, IL-11, LIF, and oncostatin M stimulate megakaryocyte maturat i ~ n . ~ ,Some ~ , ' of the members of the hematopoietic growth factor receptor superfamily share a common p chain, which plays a critical role in signal transduction.** In this study, we used a neutralizing anti-gpl30 monoclonal antibody to determine if gp130 was a component of the c-Mpl receptor complex, and to explore the extent to which the IL-6 family of cytokines contribute to Tpo-stimulated and to IL-3-stimulated megakaryopoiesis in vitro. Ablation of signal transduction through gp130 did notalter the ability of Tpo to stimulate the cytoplasmic maturation of rnegakary~cytes~~ or the proliferation of CFU-Meg or the nuclear endoreduplication of megakaryocytes. These results demonstrate that gp130 is not a component of the functional receptor complex for Tpo. Expression of a chimeric receptor consisting of the extracellular and transmembrane domains of the IL-4 receptor with the cytoplasmic domain of c-mpl in a lymphoid cell line had shown that the cytoplasmic region of c-mpl is sufficient for signal transd~ction.~'Subsequent studies have dissected the regions of the c-mpl cytoplasmic domain that are required for activation of specific JAK and STAT protein~.~',~' However, because of the ubiquitous expression of gp13O,ls these studies do not rule out its participation in Tpo signal transduction. Furthermore, the present results with the anti-gp130 monoclonal antibody U 1 8 7 show that the activity of IL-11 is not required for the full spectrum of Tpo biological effects to be realized in vitro. These data confirm and extend a report that a polyclonal antibody to IL-11 did not impair the ability of Tpo to stimulate CFU-Meg growth." In contrast to the results with Tpo, the effects of IL-3 on megakaryopoiesis do require the cooperation of cytokines that signal through the gp130 receptor subunit. A monoclonal antibody that neutralizes the effects of IL-6 blocked the ability of IL-3 to enhance murine megakaryop~iesis.~~ IL-3 can also induce megakaryocytes to produce IL-6,"whichcan then stimulate megakaryocyte cytoplasmic maturationand nuclear endoreduplication via an autocrine loop mechanism. Studies with soluble Mpl have demonstrated that full megakaryocyte cytoplasmic maturation inthe presence of IL-3 also requires TPO.'~ Taken together, the presentresults establish that the effects of Tpo on CFU-Meg colony growth and on megakaryocyte nuclear endoreduplication arenot dependent on cytokines that signal through gp130, and it is unlikelythat gpl30 serves as a p chain for the c-Mpl receptor complex. However, the effects of IL-3 on CFU-Meg proliferation and on megakaryocyte nuclear maturation are mediated in part by Tpo and by the IL-6 family of cytokines. ACKNOWLEDGMENT The authorsthankDr C. Lofton-Day (ZymoGenetics, Inc) for providing soluble Mpl, Dr S . Burstein (University of Oklahoma) for providing the 4A5 monoclonal antibody, and Z . Sisk and A. Dimaunahan for manuscript preparation. REFERENCES l. Zucker-Franklin D, Petursson S: Thrombocytopoiesis-analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes. J Cell Biol 99:390, 1984 2. Choi ES, Nichol JL, Hokom MM, Hornkohl AC, HuntP: Platelets generated in vitro from proplatelet-displayinghuman megakaryocytes are functional. Blood 85:402, 1995 3. Segal GM, Stueve T, Adamson JW: Analysis of murine megakaryocyte colony size and ploidy: Effects of interleukin-3. J Cell Physiol 137:537,1988 4. Briddell RA, Bruno E, Cooper RJ,Brandt JE, Hoffman R: Effect of c-kit ligand on invitrohuman megakaryopoiesis. Blood 78:2854,1991 5. Ishibashi T, Kimura H, Uchida T, Kariyone S , Friese P, Burstein SA: Human interleukin 6 is a direct promoterof maturation of megakaryocytes in vitro. Proc Natl Acad Sci USA 865953, 1989 6 . Burstein SA, Mei R-L, Henthorn J, Friese P, Turner K: Leukemia inhibitory factor and interleukin-l 1 promote maturation of murineandhuman megakaryocytes in vitro. J Cell Physiol 153:305, 1992 7. Wallace PM, MacMaster JF, Rillema JR, Peng J, Burstein SA, From www.bloodjournal.org by guest on June 17, 2017. For personal use only. TPO ACTS INDEPENDENTLY OF ~ ~ 1 3 0 Shoyab M: Thrombocytopoietic properties of oncostatin M. Blood 86:1310, 1995 8. Broudy VC, Lin N L , Kaushansky K Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-l 1 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood 85:1719, 1995 9. Debili N, Wendling F, KatzA, Guichard J, Breton-Gorius J, Hunt P, Vainchenker W: The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 86:2516, 1995 IO. Nichol JL, Hokom MM, Hornkohl A, Sheridan W P , Ohashi H, Kat0 T, Li YS, Bartley TD, Choi E, Bogenberger J, Skrine JD, Knudten A, Chen J, Trail G, Sleeman L, Cole S, Grampp G, Hunt P: Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia. J Clin Invest 95:2973, 1995 11. 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For personal use only. 1996 88: 2026-2032 Thrombopoietin stimulates colony-forming unit-megakaryocyte proliferation and megakaryocyte maturation independently of cytokines that signal through the gp130 receptor subunit VC Broudy, NL Lin, N Fox, T Taga, M Saito and K Kaushansky Updated information and services can be found at: http://www.bloodjournal.org/content/88/6/2026.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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