From www.bloodjournal.org by guest on February 2, 2015. For personal use only. In Vivo Effect of Human Granulocyte-Macrophage Colony-Stimulating Factor on Megakaryocytopoiesis By Massimo Aglietta, Clara Monzeglio, Fiorella Sanavio, Franco Apra; Silvia Morelli, Alessandra Stacchini, Wanda Piacibello, Federico Bussolino, GianPaolo Bagnara, Giorgio Zauli, Angelika C. Stern, and F. Gavosto The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on megakaryocytopoiesis and platelet production was investigated in patientswith normal hematopoiesis. Three findings indicated that GM-CSF plays a role in megakaryocytopoiesis. During treatment with GM-CSF (recombinant mammalian, glycosylated; SandozIScheringPlough, 5.5 p,g proteinlkgld, subcutaneously for 3 days) the percentage of megakaryocyte progenitors (megakaryocyte colony forming unit [CFU-Mk]) in S phase (evaluated by the suicide technique with high JH-Tdr doses) increased from 31% & 16% to 88% f 11%; and the maturation profile of megakaryocytes was modified, with a relative increase in more immature stage 1-111 forms. Moreover, by autoradiogra- phy (after incubation of marrow cells with 'Bl-labeled GMCSF) specific GM-CSF receptors were detectable on megakaryocytes. Nevertheless, the proliferative stimulus induced on the progenitorswas not accompanied by enhanced platelet production (by contrast with the marked granulomonocytosis). It may be suggested that other cytokines are involved in the regulation of the intermediate and terminal stages of megakaryocytopoiesis in vivo and that their intervention is an essential prerequisite to turn the GM-CSFinduced proliferativestimulus into enhanced platelet production. o 1991 by The American Society of Hematology. H (including differential) and platelet counts, and normal hemoglobin (Hb)levels. Liver and kidney functions were normal, and no signs of abnormalities were present. No concomitant treatments with corticosteroids, sulphonamides, H, antagonists, nonsteroid anti-inflammatory drugs, or lithium were administered. GM-CSF. Recombinant GM-CSF (mammalian, glycosylated; Sandoz/Schering Plough) in purified lyophilized form was obtained by the recombinant DNA technique from a mammalian cell system. Study design. GM-CSF was administered to six patients by subcutaneous route (2.8 pg of p r o t e i a g every 12 hours for 3 days). Before and during the course of the study, the patients were monitored daily by recording of vital signs, physical examination, and determination of the complete blood counts with differential. Bone marrow morphology. Megakalyocyte maturation was detined according to Williams and Levine.l6 finetic investigation of marrow-committed progenitors. The fraction of CFU-GM, BFU-E, and CFU-Mk in DNA synthesis (S phase) was assessed, as previously described, by the suicide technique after exposure to high-specific-activity tritiated thymidine (3H-Tdr) before culturing in semisolid media! The marrow light-density fraction was collected after Ficoll Hypaque (FH)separation (Lymphoprep; Nyegaard, Oslo, Norway), washed three times and resuspended in 0.5 mL of Iscove's modified Dulbecco's medium (IMDM, Flow Laboratories, Imine, UK) + 10% fetal calf serum (FCS; Flow Laboratories) at a concentration of 2 x lo6 cells/mL. Five tubes were prepared and UMAN GRANULOCYTE-macrophage colony-stimulating factor (GM-CSF) is a cytokine active on hematopoietic cells. In vitro, it stimulates the proliferation of all myeloid progenitors and, alone or in combination with other growth factors, supports the formation of colonies composed of mature cells.'-5 In vivo, in both normal subjects and patients with hematopoietic disorders, GM-CSF stimulates the production of granulocytes and monocytes and activates mature cells."" This finding confirms in vitro observations that the molecule acts at all maturation levels of the granulomonopoietic differentiation pathway. Its effects on the other myeloid lineages are less clear: in situations of impaired hematopoiesis, increased production of erythrocytes and megakaryocytes has been reported incon~tantly.'*-'~ In a previous paper: we provided data to explain the variable effects of GM-CSF on erythropoiesis: it increases the proliferative activity of erythroid progenitors (erythroid burst forming unit [BFU-E]), whereas it does not affect that of erythroblasts, presumably as a consequence of a progressive loss of GM-CSF receptors during erythroid maturation. These data suggested that the endogenous levels of other cytokines such as erythropoietin were crucial in order that the proliferative stimulus induced on BFU-E resulted in an increased production of erythrocytes. We now extend our analysis to megakaryocytopoiesis to demonstrate that, in subjects with normal hematopoiesis, GM-CSF treatment increases the proliferative activity of megakaryocyte progenitors (megakaryocyte colony forming unit [CFU-Mk]) and modifies the maturation stages of megakaryocytes. Moreover, we show that megakaryocytes have receptors for GM-CSF. Accordingly, these findings suggest that GM-CSF has an in vivo effect on megakaryocytopoiesis. MATERIALS AND METHODS Patients. Patients with histologicallyproven neoplasia not involving the myeloid system and with normal bone marrow participated in the study. Each patient gave written informed consent, according to the Helsinki declaration. All patients had normal leukocyte 8/ood,Vol77,No6(March15),1991: pp1191-1194 From the Clinica Medica A, Dipadmento di Scienze Biomediche ed Oncologia Umana, Universita di Torino; Istituto Di Istologia ed Embriologia Generale, Univemith di Bologna; Divisione Ginecologia C, Ospedale S. Anna, Torino; Dipaiiimento di Genetica, Biologia e Chimica Medica, Universitri di Torino, Italy; and Clinical Research, Sandoz Phanna Ltd, Basel, Switzerland. Submitted May 23,1990; accepted November 8, 1990. Suppoiied by grants from the Italian Association for Cancer Research and from MPI 60%. Address reprint requests to Massimo Aglietta, MD, Clinica Medica A, via Genova 3,10126 Torino, Italy. 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. section 1734 solely to indicate this fact. 0 I 9 9 1 by The American Society of Hematology. 0006-4971/91/7706-ooO2$3.00/0 1191 From www.bloodjournal.org by guest on February 2, 2015. For personal use only. 1192 AGLIETTA ET AL incubated with: medium (in duplicate), 0.5 mg/mL cold thymidine (Sigma Laboratories, St Louis, MO), 'H-Tdr (100 p,Ci/mL, specific activity: 20 Ci/mmol/L; Amersham International, Buckinghamshire, UK, in duplicate). After 30 minutes of incubation at 3 7 T , the reaction was stopped by the addition of 5 mL ice-cold Hank's balance salt solution (HBSS; Flow Laboratories) containing 0.5 mg/mL unlabeled thymidine. After three washes, the cells were resuspended in IMDM: CFU-GM, BFU-E, and CFU-Mk cultures were prepared with previously described procedures6." by seeding 1 X 105 cells/dish (for CFU-GM and BFU-E) and 3 x 10' cells/dish (for CFU-Mk) of the original cell suspension. The following medium and growth factors were used: 10% conditioned medium of the 5637 cell line for CFU-GM; 10% human cord blood endothelium supernatant and 1.5 IU erythropoietin (Toyobo, Osaka, Japan) for BFU-E; and 10% conditioned medium of the Mo cell line for CFU-Mk. After 7 and 14 days of incubation, the number of clones was evaluated by two independent investigators. CFU-GM and BFU-E were identified by their morphology, CFU-Mk identification was performed by means of 515 (CD4lw), a monoclonal antibody (MoAb) directed against the glycoprotein (GP) IIb-IIIa complex: binding was shown by fluorescinated goat antimouse IgG (Ortho-Diagnostic System, Raritan, NJ). The percentage of progenitors in the S phase of the cell cycle (Ns) was determined by applying the following formula: Ns = Nc Nt/Nc, where Nc is the number of colonies or bursts in the controls, and Nt is the number of colonies or bursts in the samples treated with high-dose 'H-Tdr. Receptor studies. Marrow cells from normal donors were obtained with two separation techniques. In two cases (samples 1 and 2), buf€y coat cells were obtained by centrifugation for 10 minutes at 80%. In one case (sample 3), enriched marrow megakaryocyte populations were obtained by separation on Percoll gradient (density 1.050). Ten million cells per milliliter were incubated for 2 hours at room temperature in a rotating system, with 1,000 pmol/L Y-GMCSF with and without a 1,000-fold excess of unlabeled GM-CSF. They were then layered on a solution at 75% FCS. Cells were centrifuged for 10 minutes at 4 Q , suspended in phosphatebuffered saline (PBS), and cytocentrifuge slides were prepared. Slides were fixed for 30 seconds at room temperature in phosphate buffer with 8% formaldehyde and 65% acetone, covered with Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY),and incubated in the dark. After 4 weeks, slides were developed, fixed, and cells were counterstained with May-Grunwald-Giemsa. The degree of megakaryocyte labeling was evaluated under the light microscope by counting the number of grains per cell and subtracting the background labeling. Specific labeling is equal to the difference between the number of grains in specimens incubated with '"I-GM-CSF only and those incubated with unlabeled GMCSF in excess. Table 2. Effect of GM-CSF Treatment on the Percentage of S Phase Marrow Progenitors ~~ Day of Study Day 7 CFU-GM Day 14 CFU-GM BFU-E CFU-Mk 6.7 4.5 0.16 0.34 11.7 291 1.5 1.0 + 0.08 0.20 + 0.4 ? 49 ? + + 36 ? 8 40 f 15 39 14 31 16 59 L 5 t 58 L 6' 71 + 1 8 t 88 ? 14t + + RESULTS Subcutaneous administration of GM-CSF resulted in a marked granulo-monocytosis, whereas lymphocyte, erythrocyte, and platelet numbers were not affected (Table 1). These results are similar to those previously observed after continuous intravenous infusion in a similar group of patients6 Table 2 shows that the percentage of myelopoietic progenitors in S phase increased during treatment. A particularly interesting new finding was the increment in the proliferative activity of megakaryocyte progenitors (the percentage of S phase CFU-Mk increased from 31% ? 16% to 88% ? 14%). Moreover, the number of CFU-Mk per milliliter of marrow increased from 299 ? 269 to 842 2 599. Despite this proliferative stimulus induced by GM-CSF on CFU-Mk, the number of circulating platelets was unchanged, suggesting that either megakaryocyte development or platelet release was not affected by the treatment. To analyze the effect of GM-CSF treatment on morphologically recognizable megakaryocytes,two approaches were used. First, a morphologic analysis was performed to investigate whether GM-CSF treatment could modify the maturation profile of megakaryocytes (Table 3). A significant decline in the percentage of mature (stage IV) megakaryocytes with a relative accumulation of the immature forms occurred during treatment. Second, a search for specific GM-CSF receptors on megakaryocytes was made by incubating normal marrow cells with '"I-GM-CSF and subsequent analysis of labeled Table 3. Modifications in the Maturation Stages of Megakaryocytes Induced by GM-CSF Treatment Day of Study Leukocytes (xlOg/L) Neutrophils (xlOg/L) Eosinophils (x109/L) Monocytes (x109/L) Hb (9%) Platelets (x109/L) 3 Results are expressed as means f SD of the values obtained from six patients (CFU-Mk data refer to four patients). Absolute growth in controls (range of colony number per dish): day 7 CFU-GM: 40-273; day 14 CFU-GM: 21-104; BFU-E: 38-372; CFU-Mk: 20-131. *P < .05 compared with data at day 0. t P < .01 compared with data at day 0. Table 1. Modificationsof Peripheral Blood Parameters Occurring After Three Days of Treatment With GM-CSF 0 0 Day of Study 3 17.0 f 8.4* 13.0 f 7.0* 0.93 f 0.63' 0.78 + 0.20 11.4 f 0.9 301 76 + Results are means ? SD of the values obtained from six patients. * P < .01 compared with data at day 0. Stage 1 Stage 2 Stage 3 Stage 4 0 3 7?3 21 + 7 5 3 + 15 19 5 7+4 30 9 58 + 13 5 + 3* * + ~ Results are expressed as means f SD of the values obtained from six patients. At least 100 megakaryocytes were evaluated per each patient. *P < .01 compared with data at day 0. From www.bloodjournal.org by guest on February 2, 2015. For personal use only. GM-CSF AND MEGAKARYOCYTOPOIESIS 1193 Table 4. Binding of "'OM-CSF (expressed as mean number of grains per cells 2 SEI to Marrow MegakaryocytesFrom Normal Donors Not Undergoing GM-CSF Treatment Sample 1 Sample 2 Sample 3 Total GM-CSF Bound GM-CSF Binding Specific Binding 11 f 0.9 19 f 2.4 75 f 5.1 4.9 2 0.8 9.0 2 1.4 4425 56% 53% 40% Nonspecific Megakaryocytes were obtained from marrow buffy coat (samples 1 and 2) or after Percoll separation (sample 3). At least 50 megakaryocytes were counted for each point in samples 1 and 2 andmore than 150 in sample 3. Fig 1. Autoradiographic preparation showing a megakaryocyte labeled with '"I-GM-CSF. A megakaryocyte-enriched populationwas obtained by Percoll separation of normal marrow cells. cells. Megakaryocytes showed a significant labeling (Fig 1) that was partially decreased by incubating marrow cells with an excess of unlabeled GM-CSF (Table 4). This displacement of labeled GM-CSF, similar to that observed by Fraser et a1," who studied the presence of erythropoietin receptors on marrow megakaryocytes, strongly suggests the presence of specific GM-CSF receptors on megakaryocytes. DISCUSSION These findings in subjects with normal hematopoiesis throw some light on the effect of GM-CSF in thrombocytopoiesis in vivo. Three observations suggest that GM-CSF plays a role in megakaryocytopoiesis: (1) There is a highly significant increase in the proliferative activity of CFU-Mk during subcutaneous GM-CSF administration. Based also on in vitro evidence^'^^" showing that GM-CSF alone supports the formation of megakaryocyte colonies, we suggest that GM-CSF stimulates CFU-Mk by directly acting on progenitors. However, the possibility of an indirect effect, caused by enhanced release of other cytokines (ie, interleukin-6 [IG6], etc) can not be ruled OUt.7,21-ZS (2) GM-CSF treatment modifies the maturation profile of megakaryocytes, inducing a relative increase in the more immature forms. (3) Incubation with labeled GM-CSF and autoradiography shows the presence of specific GM-CSF receptors on megakaryocytes. However, despite these observations, treatment with GM-CSF did not alter the number of circulating platelets. This finding contrasts with the marked proliferative stimulus induced by GM-CSF through the granulocyte-monocyte line, resulting in a modulation in marrow composition with a rapid and marked granulo-monocytosis (Table 1)?(' Seeking an explanation for this difference in effect, it must be remembered that many cytokines promote (eg, thrombopoietin, IL-6) or limit (eg, transforming growth factor p) platelet p r o d ~ c t i o n . ' ~ "In~ subjects with normal hematopoiesis, changes in marrow levels of these cytokines could offset the proliferative stimulation of megakaryocyte progenitors induced by pharmacologic doses of GM-CSF. This hypothesis may also explain why GM-CSF has an inconstant effect on the restoration of platelet production when hematopoiesis has been affected (ie, by chemotherapy, marrow transplantation). In these situations the final outcome of the proliferative stimulus of GM-CSF on early phases of megakaryocytopoiesis is less predictable because it depends on the number of residual progenitors and on endogenous cytokine levels. In conclusion, our data show that GM-CSF action is restricted to early phases of megakaryocytopoiesis and does not influence platelet production in subjects with normal hematopoiesis. This result is presumably because of the fact that other stimulating or inhibiting factors are of decisive importance in the regulation of the intermediate and final stages of platelet production in vivo. In perspective, a sequential treatment with GM-CSF followed by cytokines acting at a late stage (presumably thrombopoietin or IL-6) might prove to stimulate platelet production in vivo. ACKNOWLEDGMENT We are indebted to M. Roland0 for secretarialassistance. REFERENCES 1. 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Ishibashi T, Miller SL, Burstein SA: Type-beta transforming growth factor is a potent inhibitor of murine megakaryocytopoiesis in vitro. Blood 69:1737, 1987 27. Kishimoto T: The biology of interleukin 6. Blood 74:1, 1989 28. Lotem J, Shabo Y, Sachs L Regulation of megakaryocyte development by interleukin 6. Blood 74:1545,1989 29. Renninck D, Jackson J, Yang G, Wideman J, Lee F, Hudak S: Interleukin-6 interacts with interleukin-4 and other hematopoietic growth factors to selectively enhance the growth of megakaryocytic, erythroid, myeloid, and multipotential progenitor cells. Blood 73:1828,1989 30. Mitaivila MT, Vinci G, Villeval JL, Kieffer N, Henri N, Testa U, Breton Gorius J, Vainchenker W Human platelet alpha granules contain a non specific inhibitor of megakaryocyte colony formation: Its relationship to type beta transforming growth factor (TGF-beta). J Cell Physiol143:93,1988 From www.bloodjournal.org by guest on February 2, 2015. For personal use only. 1991 77: 1191-1194 In vivo effect of human granulocyte-macrophage colony-stimulating factor on megakaryocytopoiesis M Aglietta, C Monzeglio, F Sanavio, F Apra, S Morelli, A Stacchini, W Piacibello, F Bussolino, G Bagnara and G Zauli Updated information and services can be found at: http://www.bloodjournal.org/content/77/6/1191.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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