By Philip M. Wallace, John F. MacMaster, Jill R. Rillema, Jingpeng Peng, Samuel A. ..... the OM, Marcia Hanson for editorial assistance, Debby Baxter and.
Thrombocytopoietic Properties of Oncostatin M By Philip M. Wallace, John F. MacMaster, Jill R. Rillema, Jingpeng Peng, Samuel A. Burstein, and Mohammed Shoyab Oncostatin M (OM) is a 28-kD glycoprotein that exhibits a cell counts. The increment in counts exceededthat observed panoply of biologic effects. Based on histologic observationswith IL-6. The kinetics of the OM response suggested that of increased splenic megakaryocytes in nude miceimplanted maximal increases in plateletsoccurred 3 days afterthe ceswith an OM-secretingcell line, the thrombocytopoietic propsation of OM administration, irrespective of the duration of erties ofOM in mice were investigatedin culture and in vivo. administration. In irradiated mice, OM administrationaccelAlone, OM did not induce megakaryocytic colony formation, erated platelet recovery and prevented the decrease in red OM but incombination with murineinterleukin-3(IL-3). blood cells observedin irradiated control animals. The data markedly enhanced colony formation. Theeffects of OM on facshow that OM behaves as a megakaryocytic maturation colony formation were similar to those of IL-6. OM alone tor in vitroand augments platelet production in vivo. Based augmented acetylcholinesterase in short-term marrow culon these animal data, OM may have potential clinicalutility tures. In normal mice, the administrationof OM augmented as a thrombocytopoietic agent. platelet counts without increasing other circulating blood 0 1995 by The American Societyof Hematology.
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NCOSTATIN M (OM), a 28-kD glycoprotein,is a pleiotropic cytokine of human origin originally defined by its ability to inhibit growth of certain tumorcell lines but not normalfibroblasts.' OM produced by monocytes,macrophages, and activated T cells'" is a 196 amino acid protein that is synthesized as a 252 amino acid precursor encodedby an approximately 2-kb tran~cript.4.~ OM is a distinct member of a cytokine family whose members include interleukin-6 (IL-6), leukemia inhibitory factor (LIF), granulocyte colonystimulating factor (G-CSF), ciliary neurotrophic factor (CNTF), and myelomonocytic growth f a ~ t o r .OM ~ , ~ acts on a wide variety of cells and elicits a multitude of biologic responses, including growth modulation,l.63s"0 leukemia cell differentiation?" low-density lipoprotein receptor upregulation," stimulation of plasminogen a~tivator,'~,'~ induction of IL-6,I5 inductionof acute-phase proteins,I6 regulationof early gene expression," and induction of vasoactive intestinal peptide.I7 Specific receptors (molecular weight [Mr] -150,000) for OMhavebeencharacterizedon a widevariety of ce~ls.10,12.15.18 It has recently been shown that OM specifically binds gp130, the L 6 signal transdu~er,'~~'~ and that antibodies to gp130 block OM, LIF, IL-11, and CNTF-induced signaling.'@'' To elucidate the properties of this molecule in vivo, nude micewereimplantedwith a celllinetransfectedwiththe human OM gene and capable of producing high levelsof this protein. After subcutaneous injection of cells, these mice were examinedhistologically. A localizedvasodilationwasobserved around the tumor cells shortly after implantation and continued for the duration of implantation. Splenomegaly was From the Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA; and the W.K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK. Submitted July 20, 1994; accepted March 23, 1995. Address reprint requeststoPhilipM.Wallace,DPhil,BristolMyers Squibb, 3005 First Ave, Seattle, WA 98121. The publication costsof this article were defrayedin part by page charge payment. This article must therefore behereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. ~6-4971i95/8604-0032$3.00/0 1310
also noted. Histologic sections of the spleen showed extramedullary hematopoiesis anda marked increase in megakaryocytes. Therefore, we investigated the effects of human recombinant OM on murine megakaryocyte and platelet production, because platelets playa vital role in many normal and pathologic processes including homeostasis, wound healing, inflammation, and a number of vascular Although extensively characterized in vitro, noinvivo properties of this molecule have been described. In this report, we describe the OM-elicited alterations on murine thrombocytopoiesis in normal and irradiated animals and contrast the effects of OM with those of the known thrombocytopoietic cytokine IL-6. MATERIALSANDMETHODS Cytokines. Recombinant human OM (3 X IO6 Ulmg) was expressed in Chinese hamster ovary cellsz7and purified as described.' Human recombinant IL-6 (2 X 10' Ulmg) was produced in Escherichia coli and purifiedto homogeneity by reverse-phase chromatography." Endotoxin levels were measured by the limulus amebocyte lysate assay (Whittaker, Walkersville, MD) according to the manufacturer's instructions; for OM, endotoxin levels were less than 0.1 EUlmg, whereas for IL-6, endotoxin levels were less than 2 EUlmg. Recombinant murine IL-3 and polyclonal rabbit antimurine E - 6 were purchased from R&D Systems (Minneapolis, MN). Mice. Female C3WHeJ mice (8 to 10 weeks old) were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed according to theAmerican Association for the Accreditation of Laboratory Animal Care and institutional guidelines. Megakaryocyticcolonyassay. Marrow cells were prepared as described.*' The cells were enriched for progenitors on a 1.077 pgl mL Ficoll gradient, followed by 2 hours of plastic adherence. Megakaryocytic colony-forming cells (CFU-Mk) were assayed by culturing the enriched nonadherent marrow cells (2 X lo" celldml) in Iscove's modified Dulbecco's medium (IMDM) rendered semisolid with 0.9% methylcellulose and supplemented with 15% horse serum and I X lo-' m o m mercaptoethanol. Cytokines were added to various cultures at their initiation. Megakaryocytic colonies ( 2 3 cellskolony) were enumerated after 5 to 6 days of incubation at 37°C in a 95% air-5% COz tissue culture in~ubator.'~ Acetylcholinesterase(AchE)assay. Nucleated murine marrow cells (1 X 105; prepared as above) were treated with0.5 mmom diisopropylfluorophosphate to inactivate endogenous AchE (a marker enzyme of megakaryocytes in murine marrow). The cells were dispensed in a 96-well culture plate in 0.2 mL ofIh4DM containing 1% Nutridoma-SP (Boehringer Mannheim, Indianapolis, Blood, VOI 86, NO 4 (August 15), 1995: PP 1310-1315
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PRODUCTION OF PLATELETS BY ONCOSTATIN M Table 1. O M Effects on Megakaryocytic Colony Formation CFU-Mkt
Cytokine*
13.75
Control IL-3 IL-6 OM IL-3 + IL-6 IL-6
0.25
2
0.4
2
1.8
1.0 2 1.4 0.25 ? 0.4 21.25 2 1.1 1.25 2 1.5 24.25 2 3.5 31.5 -c 2.8
+ OM
IL-3 + OM IL-3 + IL-6 + OM
Cytokines used in methylcellulose cultures were as follows: IL-3, recombinant murine IL-3 at 10 ng/mL; IL-6, recombinant human IL-6 at 100 ng/mL: OM, recombinant human OM at 100 ng/mL. t Megakaryocytic colonies per 2 x 10‘ enriched marrow cells.
IN) in the presence of the indicated concentrations of OM. In some studies, polyclonal antimurine IL-6 was included throughout the culture period. After incubation at 37°C for 4 days, AchE activity was measured using a modification of a previously reported fluorometric method.30 One hundred eighty microliters of a solution of 0.2% Triton X-100, 1 mmovL EDTA, 0.12 m o a NaCl, and 50 mmol/L HEPES, pH 7.5, was added to each well, followed by the addition of 20 pL of acetylthiocholine iodide (final concentration, 0.56 mmoll L). After 3 hours of incubation at room temperature, 20 pL of the reaction mixture from each well was transferred to thecorresponding wells of a 96-well Microfluor “B” plate (Dynatech Laboratories, Alexandria, VA). Twenty microliters of 0.4 mmovL coumarin phenylmaleimide (Molecular Probes, Junction City, OR) was then added, followed by 160 pL of diluent buffer consisting of 0.2% Triton X-100, 1 mmom EDTA, and 50 mrnoVL Na acetate, pH 5.0. The fluorescence emission was determined on a fluorometer capable of reading 96-well plates (Multifluor; Dynatech). Effects of cytokines on circulating platelet levels. Mice (5 animals per treatment group) were injected intravenously via the tail vein with 100 pL of OM or IL-6 (dose range, 1.5 to 15 pg) twice daily for 7 days. The cytokines were diluted into 0.1 % bovine serum albumin (endotoxin
