JOURNAL OF CELLULAR PHYSIOLOGY 153:305-312 (1992)

Leukemia Inhibitory Factor and Interleukin-I 1 Promote Maturation of Murine and Human Megakaryocytes In Vitro SAMUEL A. BURSTEIN,* R U I - L I A N MEI, JAMES H E N T H O R N , P A U L FRIESE, AND KATHERINE TURNER Department of Medicine and the Saint Francis Medical Research Institute, University of Oklahomd Health Scienccs Center, Oklahoma City, Oklahoma 73 J 90 (S.A. &., R.-L.M., I.H., P I . ) , and the Genetics Institute, Cambridge,, Massachusctts 02 140 (K.T.) The growth-promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin-I 1 (IL-1 I ) , a stromal cell-derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin-6 (IL-6), a known megakaryocytic maturation factor. Maximally stimulating concentrations of LIF (25 ngiml), IL-I 1 (1 0 ngiml), or IL-6 alone ( 1 0 ng/ml) promoted an 81, 157, and 1 5 3 % increase, respectively, in acetylcholine5terase (AchE) activity in murine serum-free cultures cornpared with controls (11= 5). In combination with 2.5 Uiml murine interleukin-3 (IL-3), LIF, 11.-6, and IL-I 1 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL-3 alone (n = 4). Flow cytometric analysis of 4-day-old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL-I 1 and IL-6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL-11 increased the numbers of Mks in comparison to LIF, IL-6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL-11 was used in combination with IL-3 (compared with either IL-3 alone of IL-3 IL-6). Flow cytometric ploidy analysis of 8-day-old human liquid marrow cultures showed that either LIF, IL-I 1, or IL-6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL-3. The data suggest that LIF and I L - I 1 promote murine and human M k maturation in vitro, although the effect of I L - l 1 exceeds that of LIF in mice. Despite the comparable influence of IL-11 and IL-6 on M k ploidy, IL-11 ha5 the additional characteristic of enhancing the number of Mks, particularly in combination with IL-3. CC? 1992 WiIcy-Liss. Inc.

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A number of growth factors has been shown to promote in vitro and in vivo megakaryocytopoiesis (Hoffman, 1989; Williams et al., 1989). IL-6, a cytokine with multiple biologic effects, has been shown to enhance the maturation of murine, canine, and human megakaryocytes in vitro, and murine, canine, and primate megakaryocytes in vivo (Bruno and Hoffman, 1989; Ishibashi et al., 1989a,b; Asano et al., 1990; Hill et al., 1990; Mayer et al., 1991; Stahl et al., 1991). Nevertheless, these effects of IL-6 do not preclude the notion that a variety of cytokines promote megakaryocytic maturation. Previous studies have shown that both leukemia inhibitory factor (LIF) and interleukin-1 1(IL-11) have a n influence on megakaryocytopoiesis (Metcalf et al., 1990, 1991; Bruno et al., 1991). In this report, we describe the influence of these factors on several aspects of murine and human megakaryocytic maturation in vitro. The data show that both LIF and IL-11 promote megakaryocyte maturation and suggest that the mechanisms that positively regulate later stages of megakaryocytopoiesis are complex and perhaps redundant. G 1992 WILEY-LISS, INC.

MATERIALS AND METHODS Animals All animals were housed according to the guidelines of the AAALAC in approved facilities either a t the University of Oklahoma Health Sciences Center or the Oklahoma Medical Research Foundation (OMRF). C,,B1/6 mice, 6-8 weeks old (Jackson Laboratories, Bar Harbor, ME), were used for all murine experiments. Marrow preparation Marrow was flushed from the femurs of C6,B1/6 mice with Iscove'e modification of Dulbecco's medium (IMDM) supplemented with 1%Nutridoma-SP (Boeh-

Received December 9,1991; accepted May 26,1992. *To whom reprint requestsicorrespondence should be addressed at Hoom 271, College of'Hea1t.h Building, P.O. Box 26901, Oklahoma City, OK 73190.

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ringer Mannheim, Indianapolis, IN), a serum-free medium supplement. For culture studies, a single cell suspension was made by repetitive expulsion through progressively smaller needles. I n some experiments, murine marrow cells were treated with 0.5 mM diisopropylfluorophosphate to inactivate endogenous AchE (a marker enzyme of megakaryocytes in murine marrow; Burstein et al., 1985). Human marrow was aspirated from the posterior iliac crest of normal volunteer donors with informed consent obtained according to the guidelines of the Human Subjects Committee of the University of Oklahoma Health Sciences Center.

Liquid cultures Murine liquid marrow cultures were performed in serum-depleted conditions a s described previously (Burstein, 1986). Nucleated nonadherent marrow cells (lo5) were set up i n 96-well culture plates in 0.2 ml IMDM supplemented with 1%Nutridoma in the presence of varying concentrations of growth factors. Control cultures contained bovine serum albumin (BSA), the diluent protein used for the cytokines. After incubation a t 37°C for 4-5 days, the numbers and size of megakaryocytes were assessed following histochemical staining for AchE (Ishibashi et al., 1989a). Human marrow (3 x 105inonadherent cellsiml) was cultured for 8-9 days according to a modification of a previously described method (Kimura et al., 1990) in IMDM supplemented with 2.5% human plasma and various concentrations of the tested growth factors. Control cultures contained 100 Uiml human IL-3 (in contrast with murine cells, human megakaryocytes required the addition of some exogenous cytokine to maintain viability in these culture conditions). Growth factors Murine and human recombinant IL-3 (1 x lo7 Uimg and 1 x lo8 Uimg, respectively) were purchased from Genzyme (Boston, MA). Human recombinant IL-6 (8 x 10' B9 Unitsimg) was purchased from R&D Systems (Minneapolis, MN). Purified LIF (1.7 x 10'Uimg) and IL-11 (2.4 x loGUimg) were provided by Genetics Institute (Cambridge, MA). AchE a s s a y AchE activity was measured a s previously described (Ishibashi et al., 1989a). To each well, 180 pl of a solution of 0.2%Triton X-100 in 1mM EDTA, 0.12 M NaC1, and 50 mM Hepes pH 7.5 was added, followed by the addition of 20 pl acetylthiocholine iodide (final concentration 0.56 mM). Following 3 h r incubation at room temperature, 20 pl of the reaction mixture from each well was transferred to the corresponding wells of a 96-well Microfluor "B" plate (Dynatech Laboratories, Alexandria, VA). Then, 20 pl of 0.4 mM coumarinphenylmaleimide (Molecular Probes, Junction City, OR) was added, followed by 160 p1 of diluent buffer consisting of 0.2% Triton X-100 in 1mM EDTA and 50 mM Na acetate pH 5.0. The fluorescence emission was determined on a fluorometer capable of reading 96-well plates (Multifluor; Dynatech). Measurement of megakaryocytic ploidy The relative DNA content of murine megakaryocytes was determined by flow cytometry (Mei and Burstein,

1991). Marrow cells or cultured cells were incubated a t 4°C for 30 min with 15 pg/ml of fluoresceinated 4A5, a r a t antimurine platelet monoclonal antibody specific for murine platelets and megakaryocytes (Mei and Burstein, 1991). Subsequently, a n equal volume of 0.1% sodium citrate solution containing 40 pgiml propidium iodide, 30 pgiml DNAse-free RNAse (Boehringer Mannheim, Indianapolis, IN), and 0.1% Triton X-100 was added and incubated at 37°C for 10 min. Cells were analyzed with a Coulter Epics V flow cytometer (Coulter Instruments, Hialeah, FL) or a FACScan (Becton-Dickinson; Mountain View, CA). Small megakaryocytes exhibiting low fluorescence were distinguished from nonspecifically stained cells and autofluorescence by setting a gate a t a level of fluorescence greater than that of cultured cells stained with a control antibody (fluoresceinated rat IgG). The ploidy distribution was determined by setting markers a t the nadirs between peaks using the 2N and 4N peaks of the cells a s internal standards. Ploidy analysis of cultured human megakaryocytes was performed as described using monoclonal antibody P4 (antiglycoprotein IIb/IIIa) to identify the megakaryocytes (Kimura et al., 1990). Induction of IL-6 To determine if LIF or IL-11 were capable of inducing IL-6 production in vitro, the factors were added at the outset of liquid culture. Aliquots of the culture supernatant were analyzed for IL-6 activity on a daily basis by the B9 bioassay. Bioassay for IL-6 IL-6 bioactivity was measured using the IL-6-responsive B9 cell line (provided by Dr. Lucien Aarden, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service; Brakenhoff et al., 1987). I39 cells were grown in IMDM containing 5% fetal calf serum (FCS), 50 U/ml penicillin G, 50 pgiml streptomycin, and 0.1 ngiml human recombinant IL-6. For assay purposes, B9 cells were harvested by centrifugation and washed three times in IL-6-free medium supplemented with 2.5% FCS. Twenty pl of sample to be tested were added to 5,000 B9 cells in a final volume of 200 p J IMDM supplemented with 15%FCS. Following incubation for 3 days, cell proliferation was determined by the MTT colorimetric assay (Hansen et al., 1989). Fifty pl of a 5 mgiml stock solution of MTT (3-[4,5-dimethylthiazol-2yl]-2, 5-diphenyltetrazolium bromide) were added to each culture well. After 2 h r incubation a t 37"C, the cells were centrifuged, the supernatant discarded, and 100 pl of a lysing buffer (20%sodium dodecyl sulfate, 50% dimethylformamide, pH 4.7) were added. After overnight at 37"C, the OD600 was measured with a Biomek 1000 Automatic Laboratory Work Station (Beckman Instruments, Palo Alto, CA). One unit (U) of activity is defined a s the amount of IL-6 required to attain half-maximal stimulation. The specificity of the assay for IL-6 was assessed using monoclonal antibody 6B4, a neutralizing rat antimurine IL-6 antibody (provided by Dr. Jacques Van Snick, Ludwig Institute, Brussels, Belgium). Isolation and culture of purified murine megakaryocytes To determine if the growth factors acted directly upon megakaryocytes o r indirectly via accessory mar-

LIF AND 1L-11 PROMOTE MEGAKARYOCYTE MATURATION LOO

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Fig. 1. Dose-response relationship bctween AchE production and IL-6 (-), IL-11 (k-4,and LIF ( . . ) concentration on 3-day-old liquid murine marrow cultures to ascertain maximally stimulating concentrations. For both IL-6 and IL-11,lO ngiml promoted maximum AchE production, with no significant difyerence observed between the 2 cytokines P > 0.5). Maximal AchE production was observed at 10 pgiml of LIF, but the percentage increase in AchE activity was significantly less than that of maximally-stimulating concentrations of cither IL-6 or IL-11 ( P < 0.01 a t 10 ngiml of each cytokine). The background control BSA level was 790 -t 71 arbitrary fluorescence units. In this and in subsequent figures, the error bars represent the mean 2 1 standard deviation

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Fig. 2. Influence of varying concentrations of cytokines on AchE production in short-term liquid murine marrow cultures. A marked augmentation of AchE production was noted with IL-3 and submaximally stimulating concentrations of IL-11 that significantly exceeded the response observed with 11,-3 plus IL-6 or 1,lP (P i0.01; 5 experiments). The absolute background BSA control activity was 215 34.

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Fig. 3. Effect of the tested cytokines on the number of megakaryocytes in culture. The combination of IL-3 plus IL-11, with or without 1L-6, promoted the maximal number of megakaryocytes detectable in liquid murine culture (3 experiments; 6 wellsiexperiment).

using a magnetic particle concentrator (MPC-6; Dynal). Following the final wash, the 4A5-beads were resuspended in 0.2% BSA-PBS. To enrich for megakaryocytes for this procedure, murine marrow cells were first cultured a t 2 x 106/ml for 3 days in 20% horse serum without cytokine supplements. The cells were removed, centrifuged at 100 x g for 7 min, and resuspended in 1 ml of PBS supplemented with 0.2% crystalline BSA. Next, the cells were transferred to a 15 ml round bottom polypropylene tube, followed by the addition of 50 p1 of 4A5-coated magnetic beads. The cells were incubated a t 4" for 40 min and gently swirled every 10 min. The cells were then placed in a magnetic particle concentrator for 5 min and the supernatant and nonbound cells were aspirated. Seven ml of PBS-O.2% BSA were added and the process repeated again. Finally, the cells were resuspended in 1.5ml IMDM supplemented with 0.1% BSA. Cell suspension (200 ~ 1was ) added to individual wells of a 96-well plate, followed by addition of growth factors. The cells were cultured for 2 days and their ploidy was analyzed by flow cytometry. The presence of the beads in the culture did not affect cell viability, cell identification, or ploidy analysis, but did interfere with flow cytometric measurement of cell size.

RESULTS AchE production in liquid culture. Both LIF and * IL-11 stimulate AchE production in murine liquid "serum-free" cultures. AchE content is a general measure of megakaryocyte mass, reflecting both the number row cells, the factors were tested on murine megakary- and the size of megakaryocytes (Burstein et al., 1985). ocytes purified by immunomagnetic beads (Leven and Figure 1represents three experiments showing that 10 Rodriguez, 1991). One mg (33.3 ~ 1of) sheep antirat IgG ngiml of IL-11 and 10 pgiml of LIF were maximally conjugated immunomagnetic beads (Dynabeads M-450; stimulating, although the biological effect of IL-11 exDynal, Great Neck, NY) was added to a 15 ml round ceeded that of LIF at optimal levels of each cytokine. bottom polypropylene tube containing 1 ml of 0.2% Figure 2 shows the influence of LIF, IL-11, and IL-6 on crystalline bovine serum albumin (BSA; Sigma) in AchE production in these cultures with or without IL-3. PBS. One pg of monoclonal antibody 4A5 (purified IgG) When used alone at maximally stimulating concentrawas added and the mixture was incubated overnight at tions, the factors promoted a n 81,157, and 155%incre4". The conjugate was subsequently washed four times ment, respectively, in AchE content compared to con-

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BURSTEIN ET AL.

P < 0.001). No additional increment was observed when 10 ngiml each of IL-6 and IL-11 was added to IL-3. LIF and IL-11 increase the diameter of megaE 30.karyocytes. Compared with BSA controls, all added 1 v growth factors augmented the size of cultured murine megakaryocytes. The mean diameter of megakaryo25.cytes is shown in Figure 4A. The size of these cells was U significantly augmented in the presence of IL-11 or x IL-6 compared with either BSA control (P < 0.002), LIF = 20.(P < 0.05) or IL-3 (P < 0.05). Figure 4B shows the effects of the tested cytokines on the number of large 15megakaryocytes > 30 pm in diameter. Similar numbers of megakaryocytes of this size were observed with either IL-3 or LIF. In contrast, a marked increase in the number of large megakaryocytes was observed with either IL-11 or IL-6. The addition of IL-3 to any of the other cytokines did not further augment the number of large megakaryocytes (data not shown). LIF and IL-11 increase the percentage of high ploidy megakaryocytes. In murine cultures, all added growth factors except IL-3 increased the percentI age of 32 and 64N megakaryocytes (Fig. 5A). A marked rightward shift in the ploidy distribution was observed using either IL-11 or IL-6. LIF enhanced the percentage of 32N cells, but the differences were not significant (P > 0.2). However, in human cultures, the influence of LIF was similar to that of IL-11 and IL-6, with these z 10 three cytokines all significantly augmenting the percentage of 32N cells compared with IL-3 (Fig. 5B; P < 0.01). BSA LIF IL3 IL11 The LIF and IL-11-related enhancement of IL6 (25V/m11 (lo"s/ml) (i*g/m4 megakaryocyte maturation is not mediated by induction of IL-6. Figure 6 shows the time course of Fig. 4. Influence of the tested cytokines on the size of megakaryoIL-6 accumulation in unfractionated liquid murine cytes in liquid culture. A. The mean diameter of megakaryocytes was marrow cultures. A significant elevation in IL-6 bioacsignificantly augmented with all tested cytokines compared to the BSA control (P < 0.002), but both IL-11 and IL-6 augmented size to a tivity was observed 2 h r following addition of IL-3, greater extent than either IL-3 or LIF ( P < 0.05). B. Both IL-11 and which increased to 772 Uiml by 48 hr. In contrast, neiIL-6 also increased the numbers of the largest detectable megakaryo- ther LIF nor IL-11 induced IL-6 production compared cytes (greater than 30 pni in diameter),campared with LIF (P 0.02), IL-3 (P 0.05) and to the BSA control ( P < 0.001). Three experi- with BSA controls. ments, 60 megakaryocytes analyzediexperiment for each condition. LIF and IL-11 augment the ploidy of purified isolated megakaryocytes. To determine if the cytokines could enhance the rate of megakaryocytic endoreduplication directly, they were added following isotrol marrow cells grown in the absence of exogenous lation of murine megakaryocytes from 2-day liquid cytokine. The effect of IL-11 was similar to that noted cultures. In this culture system, significant polywith IL-6 (10 ngiml). In combination with the multipoi- ploidization occurs in the absence of added cytokine for etin IL-3, a marked augmentation of AchE synthesis unknown reasons, and the differences among the was observed. The combination of IL-11 and IL-3 pro- growth factors are best observed in the 64N ploidy moted AchE accumulation in excess of the combination class. Figure 7 shows the appearance of the purified of IL-3 plus IL-6 or IL-3 plus LIF (6.9-fold vs. 3.7- and megakaryocytes 2 days following the addition of LIF 3.5-fold greater than BSA control, respectively). The (A), IL-11 (B), IL-6 (C), or no added cytokine (D). LIF, addition of IL-6 to the IL-1liIL-3 combination did not IL-11 and IL-6 all significantly augmented the percentsignificantly enhance AchE content (P > 0.2). age of 64N cells compared with BSA control (Fig. 8). LIF and IL-11 do not increase megakaryocyte When 6B4, a rat antimurine IL-6 neutralizing antibody number in the absence of IL-3. No differences were (provided by Dr. Jacques Van Snick, Ludwig Institute, observed in the mean number of megakaryocytesiwell Brussels, Belgium), was added to the cultures concuramong IL-6, LIF, IL-11, and BSA control, with 36-58 rently with LIF or IL-11, no abrogation of polyploidizamegakaryocyt.es noted (Fig. 3). When either IL-6 or LIF tion was observed (data not shown). was added together with IL-3 (25 Uiml), a n increase in DISCUSSION megakaryocytes to 131-167iwell was observed, with no LIF is a 20-60 kDa variably glycosylated protein that augmentation over IL-3 alone noted (P > 0.3). In contrast, 1ngiml IL-11 plus 25 Uiml IL-3 increased mega- exhibits no sequence homology with any known protein karyocyte number to 293iwel1, significantly greater (Metcalf, 1991). The factor was originally purified than the sum of the individual cytokines (185/well; based on its capacity to induce differentiation of the

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Fig. 8. Ploidy analysis of isolated purified murine rnegakaryocytes. Each of’the tested cytokines was capable of significantly augmenting

(Metcalf, 1991). LIF produces numerous biological effects on both hematopoietic and nonhematopoietic cells. Among these are differentiation of myelonionocytic cells, inhibition of commitment of embryonic stem cells, suppression of osteoblast proliferation, stimulation of

the hepatic acute phase response, and promotion of megakaryocytopoiesis (Metcalf, 1991). In vitro, LIF augments megakaryocyte colony formation in conjunc-

Fig. 7. Photomicrographs of isolated purified murine megakaryocytes 2 days following culture in the presence of (A) 25 ngiml LIF; (€5) 10 n g h l IL-11; (C) 10 ngiml IL-6; (D) no added cytokine.

LIF AND IL-11 PROMOTE MEGAKABYOCYTE MATURATION

tion with, but not in the absence of IL-3. The presence of LIF receptors on megakaryocytes was suggested by autoradiographic studies (Metcalf et al., 1991). After administration of LIF to mice, megakaryocytes and their progenitors increased in number in the marrow and spleen, followed by a n almost twofold increment in the platelet count (Metcalf et al., 1990). IL-11 is a recently described 20 kDa cytokine derived from a transformed primate stromal cell line and was cloned on the basis of its capacity to stimulate the IL-6dependent T1165 cell line (Paul et al., 1990). IL-11 also has a variety of biological effects, including promotion of B-cell formation, macrophage proliferation, augmentation of IL-3-dependent proliferation of murine multipotent progenitors, synergism with IL-4 to stimulate early murine progenitors, and inhibition of adipogenesis (Paul et al., 1990; Kawashima et al., 1991; Musashi, 1991; Musashi et al., 1991). IL-11 synergizes with IL-3 in the promotion of proliferation of murine and early and late human megakaryocyte progenitors (Paul et al., 1990; Bruno e t al., 1991). The present study was undertaken to examine the effect of these cytokines on two aspects of megakaryocytic maturation-size and DNA content. Both factors were capable of enhancing size and ploidy in serum-free murine cultures. Moreover, each was able to stimulate polyploidization of human megakaryocytes. To determine if the stimulatory effects of the cytokines required accessory marrow cells, murine megakaryocytes were purified to homogeneity, and the ploidy was measured by flow cytometry 2 days following addition of the factors. An augmentation of 64N cells was observed. The changes in these maturation parameters are thus likely related to cytokine binding to receptors on megakaryocytes. In the case of LIF, the presence of receptors has been shown directly by in situ binding studies (Metcalf et al., 1991). The potential role of IL-6 to serve as a n intermediary in the maturation process in unfractionated marrow was assessed by measuring IL-6 levels following addition of various growth factors. Only IL-3 proved capable of augmenting endogenous IL-6 production. In cultures of purified murine megakaryocytes, addition of antiIL-6 did not prevent the LIF or IL-11 induced augmentation of ploidy. This does not preclude the possibility that either of these cytokines may enhance endogenous nonsecreted IL-6 or IL-6 receptor (Navarro et al., 1991). In this regard, it is of additional interest that culture of purified murine megakaryocytes in close proximity resulted in a greater percentage of megakaryocytes of higher ploidy classes (a16N) even in the absence of exogenous cytokine, than is usually observed in liquid culture (Fig. 81, suggesting the possibility that megakaryocytes may be producing growth-enhancing cytokines other than IL-6. Despite the wide variety of biological effects of IL-6, LIF, and IL-11, all appear to promote both the hepatic acute phase response and megakaryocytopoiesis. Among the hepatic acute phase proteins are C4 binding protein, a n inhibitor of the anticoagulant Protein S (Dahlback, 1983; Matsuguchi et al., 1991); and fibrinogen, elevated concentration of which may accelerate the velocity of platelet aggregation (Landolfi et al., 1991). Thus concurrent augmentation of the hepatic

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acute phase response and thrombocytopoiesis by several disparate cytokines may not be unrelated, but rather may represent a redundant mechanism to augment hemostasis in response to injury. Although the effects of IL-11 on size and ploidy seemed more potent than those of LIF, it cannot be extrapolated from these in vitro data that IL-11 would prove to be more potent in vivo. The marked augmentation of megakaryocyte number observed in combination with IL-3 is of interest, in that it exceeded the IL-6 or LIF combinations. The basis for that increment is unclear but may represent either recruitment of uncommitted progenitor cells into the megakaryocytic pathway, o r insertion of additional mitoses of proliferating early megakaryocytes by shortening the cell cycle prior to entering the endoreduplicative pathway. The availability of three unrelated cytokines that promote megakaryocytopoiesis provides new opportunities to: (1)investigate the mechanisms of megakaryocytic maturation, ( 2 ) analyze the potential physiologic roles of megakaryocyte growth factors, and ( 3 ) ascertain their pharmacologic effects in augmenting the platelet count in pathologic conditions.

ACKNOWLEDGMENTS This work was supported in part by NIH grant HL29037, the Saint Francis Medical Research Institute, and a grant from the Oklahoma Center for the Advancement of Science and Technology. LITERATURE CITED Asano, S., Okano, A., Ozawa, K., Nakahata, T., Ishibashi, T., Koike, H., Kimura, H., Tanioka, A., Shibuya, T., Hirano, T., Kishinioto, T., Takaku, F., and Akiyama, Y. (19901 In vivo effects of recombinant human interleukin-6 in primates: Stimulated production of platelets. Blood, 75t1602-1605. Brakenhoff, J.P.J., Tlegroot, E.R., Evers, R.F., Pannekoek, H., and Aarden, L.A. (1987) Molecular cloning and expression of hybridoma growth factor in Escherichia Coli. J. Immunol., 139;41164121. Bruno, E., and Hoffman, R. (1989) Effect of interleukin 6 on in vitro human megakaryocytopoiesis: its interaction with other cytokines. Exp. Hematol., 17:1038-1043. Bruno, E., Briddell, R.A., Cooper, R.J., and Hoffman, R. (1991)Effects of recombinant interleukin 11on human megakaryocyte progenitor cells. Exp. Hematol., 19;37%381. Burstein, S.A. (1986) Interleukin-3 promotes maturation of murine megakaryocytes in vitro. Blood Cells, IIt469479. Burstein, S.A., Boyd, C.N., and Dale, G.L. (1985) Quantitation of megakaryocytopoiesis in vitro by enzymatic determination of acetylcholinesterase. J. Cell. Physiol., 122,159-165. Dahlback, €3. (1983) Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S. Biochem. J., 209:847-856. Hansen, M.B., Nielsen, S.E., and Berg, K. (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growthicell kill. J. Immunol. Methods, 119;203-210. Hill, R.J., Warren, M.K., and Levin, J. (1990) Stimulation of thrombopoiesis in mice by human recombinant interleukin-6. J . Clin. Invest., 85t1242-1247. Hoffman, R. (1989) Regulation of megakaryocytopoiesis. Blood, 74:1196-1212. Ishibashi, T., Kimura, H., Uchida, T., Kariyone, S., Friese, P., and Burstein, S.A. (1989a) Human interleukin 6 is a direct promoter of maturation of megakaryocytes in vitro. Proc. Natl. Acad. Sci. USA, 865953-5957. Ishibashi, T., Kimura, H., Shikama, Y . , Uchida, T., Kariyone, S., Hirano, T., Kishimoto, T., Takatsuki, F., and Akiyama, Y. (1989133 Interleukin-6 is a potent thrombopoietic factor in vivo in mice. Blood, 74t1241-1244. Kawashima, I., Ohsumi, J . , Mita-Honjo, K., Shimoda-Takann, K., Ishikawa, H., Sakakibara, S.,Miyadai, K., andTakiguchi, Y. (1991)

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