Mice lacking thrombopoietin (TPO), or its receptor c-Mpl, display defective megakaryocyte and platelet development and deficiencies in progenitor cells of multiple hematopoietic lineages. The contribution of alternative cytokines to thrombopoiesis in the absence of TPO signalling was examined in mpl−/− mice. Analysis of serum and organ-conditioned media showed no evidence of a compensatory overproduction of megakaryocytopoietic cytokines. However, consistent with a potential role in vivo, when injected intompl−/− mice, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) retained the capacity to elevate megakaryocytes and their progenitors in hematopoietic tissues and increase circulating platelet numbers. However, double mutant mice bred to carry genetic defects both in c-Mpl and IL-3 or the alpha chain of the IL-3 receptor, displayed no greater deficiencies in megakaryocytes or platelets than mpl-deficient animals, suggesting absence of a physiologic role for IL-3 in the residual megakaryocytopoiesis and platelet production in these mice.

THE PRODUCTION OF platelets, the small anuclear cells shed into the circulation by mature megakaryocytes, plays an important role in blood clotting and hemostasis. A number of hematopoietic growth factors have been implicated in megakaryocytopoiesis, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), and stem cell factor (SCF), as well as the cytokines that use the gp130 receptor signalling chain (IL-6, IL-11, leukemia inhibitory factor [LIF] and oncostatin-M [OSM]).1 However, recently it has become clear that thrombopoietin (TPO) is the major physiologic regulator of this process. Isolated and cloned on the basis of interaction with its receptor c-Mpl, a member of the hematopoietin receptor family, TPO is transcribed predominantly in the liver, kidney, and smooth muscle and is secreted as a glycoprotein, the circulating concentration of which is regulated by megakaryocyte and platelet mass.2 As a single agent, TPO is a specific inducer of the proliferation of progenitor cells committed to megakaryocyte production. In semisolid culture assays, TPO stimulates the formation of small colonies of mature megakaryocytes from bone marrow or spleen cells, and this activity is augmented by the addition of other factors particularly IL-3 and SCF.3-6 TPO also stimulates megakaryocyte maturation, promoting expansion of cell size, increased DNA ploidy, and the cytoplasmic reorganization that typically precedes platelet release.3,5-9 Indeed, culture systems have been devised in which TPO can support complete in vitro development of primitive CD34+ hematopoietic progenitor cells to mature, platelet-shedding megakaryocytes.8 

In vivo, administration of TPO elevates the number of circulating platelets up to 10-fold and stimulates production of mature megakaryocytes and their progenitors in hematopoietic tissues.6,10-14 Although other cytokines, particularly IL-3, IL-6, IL-11, and LIF, share these thrombopoietic properties,15-18 at maximal concentrations their effects are significantly less potent than those of TPO. The essential physiologic roles of TPO have been established in mice genetically manipulated to lack the cytokine or its receptor, c-Mpl. The hematopoietic organs of TPO−/− andmpl−/− mice produce only 5% to 10% of the normal number of megakaryocytes, most of which are relatively immature. Consequently, the mice are thrombocytopenic and display a platelet deficiency of similar magnitude.19-21 In addition to deficiencies in megakaryocyte progenitor cells, c-mpl−/− mice also display reduced numbers of immature cells committed to all other hematopoietic lineages.20 This phenotype is shared with TPO-deficient mice22 and suggests that signalling through c-Mpl may also play a critical role in regulation of the hematopoietic stem cell compartment.

Despite the severe thrombocytopenia that characterizes c-mpl–deficient animals, residual platelet production inmpl−/− mice is sufficient to prevent hemorrhage and allows ostensibly normal development and adult life. To examine the contribution of alternative stimuli to the residual megakaryocytopoiesis in mice lacking TPO signalling, we have examined cytokine production and activity in mpl-deficient animals. Although we found no evidence of elevation of non-TPO megakaryocytopoietic cytokines in mpl-deficient mice, injection of IL-6 or LIF stimulated megakaryocyte and platelet production inmpl−/− mice to a similar extent to that observed in normal animals, suggesting alternative megakaryocytopoietic stimuli can function in the absence of TPO signalling. However, IL-3 appeared not to contribute significantly to megakaryocytopoiesis because double mutant mice deficient in c-Mpl and IL-3 or its receptor alpha chain (IL-3Rα) displayed no greater deficiencies in platelets, megakaryocytes, or their progenitors than is evident inmpl−/− mice.

Mice.

NZB mice bearing mutant IL-3Rα genes (Il3ran),23 c-mpl-deficient mice,20 and mice lacking a functional IL-3 gene24 have been described previously. Mice genetically defective for both c-Mpl and IL-3Rα were generated by breedingmpl−/− and NZB mice to yield F1 offspring (mpl+/-Il3ra+/n), which were subsequently interbred. The peripheral blood platelet counts of 98 male F2 mice were measured at 6 weeks of age and 27 thrombocytopenic (mpl−/−) mice were identified and analyzed. The IL-3Rα genotype of these mice was then determined using the polymerase chain reaction method previously described.23 As mpl−/− mice were of mixed C57Bl/6 and 129/Sv genetic background, and IL-3−/− mice were a mixture of C57Bl/6, 129/Sv, and Balb/c, in the mpl−/−IL-3−/− intercross experiments, wild-type controls included a combination of data from mice of each of these strains. All mice were housed in a conventional animal facility and analyzed at between 2 and 4 months of age.

Cytokines.

Murine GM-CSF, IL-3, IL-5, and TPO were produced in purified recombinant form and kindly provided by Dr N. Nicola (The Walter and Eliza Hall Institute of Medical Research, Melbourne). Recombinant murine IL-6 was a kind gift of Dr R. Simpson (Joint Protein Structure Laboratory, The Walter and Eliza Hall Institute of Medical Research and The Ludwig Institute for Cancer Research, Melbourne). Recombinant human G-CSF and recombinant murine LIF were kindly provided by Amgen (Thousand Oaks, CA) and AMRAD (Boronia, Australia), respectively, and recombinant human IL-11 was purchased from R&D Systems (Minneapolis, MN).

Cytokine bioassays.

Organs from male mpl−/− mice or their wild-type littermates were collected, fragmented with scissors, and incubated in 2 mL of serum-free Dulbecco's modified Eagle's medium (DMEM) in a fully humidified atmosphere of 10% CO2 in air. Supernatants conditioned by each tissue were collected after 4 days, filter sterilized, and stored at 4°C for analysis. Cytokine concentrations were determined by bioassay as described25,26 using parental Ba/F3 cells for IL-3 (detection limit, 20 pg/mL) or Ba/F3 cells transfected with the specific receptors for IL-6 (detection limit, 100 pg/mL), IL-11 (detection limit, 100 pg/mL), LIF (detection limit, 200 pg/mL), G-CSF (detection limit, 400 pg/mL), or TPO (detection limit, 100 pg/mL). As no organ-conditioned medium contained IL-3, FDC-P1 cells, which respond to IL-3 or GM-CSF, provided a specific bioassay for GM-CSF (detection limit, 100 pg/mL). Microwell assays were performed in 60-well microtiter dishes (Lux, Nashville, TN) as described.25 Briefly, 200 cells in 10 μL DMEM containing 10% newborn calf serum (NCS) were added to duplicate 5 μL volumes of serially diluted organ conditioned medium. The numbers of viable cells in each well were determined by manual inspection under phase-contrast microscopy after incubation for 2 days at 37°C in a fully humidified atmosphere of 10% CO2 in air. Where cell counts exceeded 200 per well, the culture was scored as containing >200 cells. Each assay included a titration of a known concentration of the relevant purified recombinant cytokine, which was used as a standard for calculating the amount of that cytokine present in each conditioned medium. As previously described,25 the media conditioned by four organs, brain, salivary gland, kidney, and liver, contain components toxic to the assay cells. Because mixing experiments indicated that the toxic effects were eliminated at dilutions greater than 1:8, the limits of detection for cytokines in these conditioned media were eightfold higher than stated. Similarly, as serum samples were initially diluted fourfold, the limit of detection for cytokines in serum was fourfold higher than stated.

Cytokine injections.

Groups of weight-matched male mpl−/−mice or wild-type littermates were injected twice daily subcutaneously with 0.2 mL of cytokine solution or dilution medium (saline supplemented with 5% NCS). Mice injected with IL-6 (2 μg/day) and LIF (1 μg/day) were analyzed after 7 and 8 days, respectively.

Hematologic and progenitor cell analysis.

Peripheral blood was collected by retro-orbital bleeding and diluted into 3% acetic acid containing methylene blue (white cells) or 1% ammonium oxalate (platelets) for manual cell counts using hemocytometer chambers and standard microscopy. Megakaryocytes were enumerated by microscopic examination of hematoxylin and eosin-stained histologic sections of sternal bone marrow and spleen, which had been cut to standard thicknesses of 1 and 2 μm, respectively. The clonal culture of hematopoietic progenitor cells was performed in 1 mL cultures of 2.5 × 104 (bone marrow) or 105 (spleen) cells in 0.3% agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% fetal calf serum (FCS), 10 ng/mL murine IL-3, 100 ng/mL murine SCF, and 4 U/mL human EPO as previously described.18 Single stimulus cultures included cytokines at the following final concentrations: murine TPO, 100 ng/mL; IL-5, 103 U/mL; IL-6, 100 ng/mL; murine GM-CSF, 10 ng/mL; human G-CSF, 10 ng/mL; murine M-CSF 10 ng/mL. Agar cultures were fixed and sequentially stained for acetylcholinesterase, Luxol fast Blue, and hematoxylin, and the composition of each colony was determined at 100-fold to 400-fold magnifications.

Cytokine production in mpl−/− mice.

The concentration of cytokines in the serum or in media conditioned by the organs of adult mpl−/− mice was examined using factor-dependent cells ectopically expressing specific cytokine receptors (see Materials and Methods). Consistent with previous reports,19 the level of serum TPO, which was undetectable in wild-type mice, was elevated inmpl−/− animals (640 ± 0 pg/mL, n = 4). Recent studies suggest that lack of platelet-mediated clearance of TPO is the predominant mechanism accounting for the elevated TPO levels in mpl-deficient animals.27 We were unable to determine whether changes in production also contribute, as TPO production was not sufficient to be detected in conditioned medium from any organs of normal or mpl−/− mice. To investigate whether maintenance of the residual thrombopoiesis in mice lacking TPO signalling may be due to increased production of alternative cytokines with megakaryocytopoietic activities, we also examined production of IL-3, IL-6, IL-11, LIF, and GM-CSF inmpl−/− mice. None of these regulators was detected in the serum of wild-type or mpl-deficient animals (Table 1). Similarly, IL-3 was undetectable in all conditioned media examined from mice of both genotypes. As previously observed,25 LIF, IL-6, and GM-CSF were detected in a wide range of organ-conditioned media from normal mice, in particular that of the lungs and muscle, and no significant differences in the concentrations of these cytokines was observed in analysis ofmpl−/− animals (Table 1). Similarly, no difference in the organ distribution or level of IL-11 production, which was evident at lower concentrations and from a more restricted range of tissues, was observed between mpl-deficient and wild-type littermates (Table 1). Together, these data suggest that of the megakaryocytopoietic cytokines under study, dramatic elevation in production does not contribute to the mechanisms by whichmpl−/− mice maintain residual platelet levels.

Table 1.

Cytokine Production in mpl−/−Mice

Organ IL-3 (ng)LIF (ng) IL-6 (ng)IL-11 (ng) GM-CSF (ng)
mpl+/+mpl−/−mpl+/+mpl−/−mpl+/+mpl−/−mpl+/+mpl−/−mpl+/+mpl−/−
Salivary gland  0  0  0  0  0  0  0  2 ± 4 12 ± 14  
Thymus  0  0  3 ± 0  8 ± 4 28 ± 15  83 ± 61  2 ± 1  2 ± 1 24 ± 11  24 ± 11  
Lung  0  0  50 ± 42 100 ± 85  660 ± 877  800 ± 679  8 ± 4 10 ± 0  1,312 ± 925  1,792 ± 512  
Heart  0  4 ± 2  6 ± 5  18 ± 17  55 ± 70 0  0  48 ± 23  64 ± 0 
Liver  0  0  0  0  0  0  0  0  0  
Spleen  0  0  5 ± 7 10 ± 0  163 ± 223  100 ± 85  0  6 ± 3  5 ± 4  
Kidney  0  2 ± 1  3 ± 4  10 ± 14  4 ± 2  0  40 ± 34  36 ± 40  
Muscle  0  47 ± 40  94 ± 80  305 ± 417  375 ± 225 0  0  105 ± 99  338 ± 424  
Bone marrow  0  0  4 ± 1  6 ± 3  8 ± 7 61 ± 84  0  0  0  
Bone shaft  0  0  19 ± 16  30 ± 0 248 ± 329  300 ± 225  13 ± 4  20 ± 14 0  0  
Bladder  0  0  8 ± 4 10 ± 0  85 ± 106  80 ± 0  10 ± 0 10 ± 0  30 ± 25  48 ± 18  
Brain  0  1 ± 1  1 ± 2  6 ± 5  8 ± 4  0  1 ± 1  1 ± 1  
Testes  0  4 ± 2  5 ± 0  11 ± 12  6 ± 5  0  12 ± 6  12 ± 6 
Serum  0  0  0  0  0  0  0  0  0  
Detection limit (pg) 20  200  100  100  100  
Organ IL-3 (ng)LIF (ng) IL-6 (ng)IL-11 (ng) GM-CSF (ng)
mpl+/+mpl−/−mpl+/+mpl−/−mpl+/+mpl−/−mpl+/+mpl−/−mpl+/+mpl−/−
Salivary gland  0  0  0  0  0  0  0  2 ± 4 12 ± 14  
Thymus  0  0  3 ± 0  8 ± 4 28 ± 15  83 ± 61  2 ± 1  2 ± 1 24 ± 11  24 ± 11  
Lung  0  0  50 ± 42 100 ± 85  660 ± 877  800 ± 679  8 ± 4 10 ± 0  1,312 ± 925  1,792 ± 512  
Heart  0  4 ± 2  6 ± 5  18 ± 17  55 ± 70 0  0  48 ± 23  64 ± 0 
Liver  0  0  0  0  0  0  0  0  0  
Spleen  0  0  5 ± 7 10 ± 0  163 ± 223  100 ± 85  0  6 ± 3  5 ± 4  
Kidney  0  2 ± 1  3 ± 4  10 ± 14  4 ± 2  0  40 ± 34  36 ± 40  
Muscle  0  47 ± 40  94 ± 80  305 ± 417  375 ± 225 0  0  105 ± 99  338 ± 424  
Bone marrow  0  0  4 ± 1  6 ± 3  8 ± 7 61 ± 84  0  0  0  
Bone shaft  0  0  19 ± 16  30 ± 0 248 ± 329  300 ± 225  13 ± 4  20 ± 14 0  0  
Bladder  0  0  8 ± 4 10 ± 0  85 ± 106  80 ± 0  10 ± 0 10 ± 0  30 ± 25  48 ± 18  
Brain  0  1 ± 1  1 ± 2  6 ± 5  8 ± 4  0  1 ± 1  1 ± 1  
Testes  0  4 ± 2  5 ± 0  11 ± 12  6 ± 5  0  12 ± 6  12 ± 6 
Serum  0  0  0  0  0  0  0  0  0  
Detection limit (pg) 20  200  100  100  100  

Mean ± standard deviation of the calculated total amount of each cytokine produced in medium conditioned by the entire organ over 4 days. Results from 2 to 4 mice for each assay are shown. Toxicity of media conditioned by liver, kidney, salivary gland, and brain reduced the detection limit for those samples by eightfold.

In vivo activity of megakaryocytopoietic cytokines in mpl−/− mice.

To investigate whether alternative cytokines are capable of stimulating megakaryocytopoiesis in the absence of TPO signalling, the response ofmpl−/− mice to daily administration of IL-6 or LIF was compared with that of normal animals. Presumably reflecting their thrombocytopenic state, at analysis, severalmpl−/− mice displayed evidence of subcutaneous hemorrhage at the injection site and two LIF-injected animals that exhibited hematocrit values below 40 were excluded from analysis.

Consistent with previous reports,16 IL-6 injections for 6 days in normal mice elevated spleen weight and induced a 1.6-fold increase in platelet numbers. Similar findings were observed inmpl−/− mice, with the platelet count increasing by approximately twofold (Table2). In mice of both genotypes, the increase in platelet counts was accompanied by significant increases in megakaryocytes and megakaryocyte progenitor cells, particularly in the spleen, but also in the bone marrow (Table 2). Little alteration in the hematocrit or white blood cell count were observed in IL-6–injected mice of either genotype (Table 2).

Table 2.

Effects of IL-6 Administration inmpl−/− Mice

mpl+/+mpl−/−
Carrier IL-6Carrier IL-6
Spleen weight (mg)  106 ± 14 188 ± 25* 128 ± 23  179 ± 38 
Platelets  (×10−6/mL)  758 ± 116 1,213 ± 170* 48 ± 11  107 ± 28* 
Hematocrit (%)  46 ± 2  44 ± 3  46 ± 2  42 ± 2 
WCC (×10−6/mL)  4.1 ± 0.3  4.7 ± 1.9 5.1 ± 1.1  4.5 ± 1.9  
Megakaryocytes  
 Bone marrow (per 30 hpf)  103 ± 8  169 ± 28* 7.5 ± 3.0  17 ± 6 
 Spleen (per 60 hpf) 36 ± 26  72 ± 25  1.0 ± 1.0  3.5 ± 2.0 
Megakaryocyte progenitor cells  
 Bone marrow (×10−3 per femur)  10.9 ± 4.7 18.9 ± 3.3 2.2 ± 0.9  4.8 ± 1.2 
 Spleen (×10−3)  8.5 ± 4.3 48.7 ± 6.4* 3.2 ± 1.4  8.6 ± 3.9* 
mpl+/+mpl−/−
Carrier IL-6Carrier IL-6
Spleen weight (mg)  106 ± 14 188 ± 25* 128 ± 23  179 ± 38 
Platelets  (×10−6/mL)  758 ± 116 1,213 ± 170* 48 ± 11  107 ± 28* 
Hematocrit (%)  46 ± 2  44 ± 3  46 ± 2  42 ± 2 
WCC (×10−6/mL)  4.1 ± 0.3  4.7 ± 1.9 5.1 ± 1.1  4.5 ± 1.9  
Megakaryocytes  
 Bone marrow (per 30 hpf)  103 ± 8  169 ± 28* 7.5 ± 3.0  17 ± 6 
 Spleen (per 60 hpf) 36 ± 26  72 ± 25  1.0 ± 1.0  3.5 ± 2.0 
Megakaryocyte progenitor cells  
 Bone marrow (×10−3 per femur)  10.9 ± 4.7 18.9 ± 3.3 2.2 ± 0.9  4.8 ± 1.2 
 Spleen (×10−3)  8.5 ± 4.3 48.7 ± 6.4* 3.2 ± 1.4  8.6 ± 3.9* 

Mean ± standard deviation of data from 4 mice in each group. Progenitor cell numbers were enumerated from stained semisolid agar cultures containing IL-3, SCF, and EPO and incubated in 5% CO2 in air at 37°C for 7 days.

Abbreviation: hpf, high power field.

*

P < .01 for comparison with carrier-injected controls of the same phenotype.

P = .06.

P < .05.

Similarly, LIF administration for 7 days also increased the number of circulating platelets in both normal andmpl−/− mice (Table 3). The magnitude of the increase, 1.6-fold, was consistent with that observed in previous studies of LIF activity in normal animals.17 In contrast to IL-6–treated mice, little if any significant elevation in the number of mature megakaryocytes was observed in LIF-injectedmpl−/− mice. No significant elevation in megakaryocyte progenitor cells was observed in either wild-type ormpl−/− mice receiving LIF (Table 3).

Table 3.

Effects of LIF Administration inmpl−/− Mice

mpl+/+mpl−/−
Carrier LIFCarrier LIF
Spleen weight (mg)  88 ± 37 96 ± 21  92 ± 34  128 ± 30* 
Platelets  (×10−6/mL)  857 ± 200 1,431 ± 386 61 ± 18  103 ± 26 
Hematocrit (%)  51 ± 3  50 ± 3  48 ± 3  45 ± 3 
WCC (×10−6/mL)  4.1 ± 1.1  3.4 ± 1.4 3.7 ± 0.6  3.4 ± 0.6  
Megakaryocytes  
 Bone marrow (per 30 hpf)  118 ± 25  145 ± 25* 7.9 ± 5.8  10.9 ± 4.8  
 Spleen (per 60 hpf) 26 ± 27  36 ± 24  0.4 ± 0.5  0.4 ± 0.5 
Megakaryocyte progenitor cells  
 Bone marrow (×10−3 per femur)  12.1 ± 3.3  16.4 ± 6.0 4.1 ± 1.5  3.7 ± 1.7  
 Spleen (×10−3)  8.7 ± 5.4  11.8 ± 5.9 2.6 ± 2.0  5.0 ± 4.7 
mpl+/+mpl−/−
Carrier LIFCarrier LIF
Spleen weight (mg)  88 ± 37 96 ± 21  92 ± 34  128 ± 30* 
Platelets  (×10−6/mL)  857 ± 200 1,431 ± 386 61 ± 18  103 ± 26 
Hematocrit (%)  51 ± 3  50 ± 3  48 ± 3  45 ± 3 
WCC (×10−6/mL)  4.1 ± 1.1  3.4 ± 1.4 3.7 ± 0.6  3.4 ± 0.6  
Megakaryocytes  
 Bone marrow (per 30 hpf)  118 ± 25  145 ± 25* 7.9 ± 5.8  10.9 ± 4.8  
 Spleen (per 60 hpf) 26 ± 27  36 ± 24  0.4 ± 0.5  0.4 ± 0.5 
Megakaryocyte progenitor cells  
 Bone marrow (×10−3 per femur)  12.1 ± 3.3  16.4 ± 6.0 4.1 ± 1.5  3.7 ± 1.7  
 Spleen (×10−3)  8.7 ± 5.4  11.8 ± 5.9 2.6 ± 2.0  5.0 ± 4.7 

Mean ± standard deviation of data from 7 to 11 mice in each group. Progenitor cell numbers were enumerated from stained semisolid agar cultures containing IL-3, SCF, and EPO and incubated in 5% CO2 in air at 37°C for 7 days.

*

P < .05.

P < .01 for comparison with carrier-injected controls of the same phenotype.

Thrombopoiesis in mice deficient in both c-Mpl and the IL-3Rα chain.

To determine whether IL-3 plays an essential role in the residual megakaryocytopoiesis observed in the absence of TPO signalling, mice deficient in both c-Mpl and the IL-3Rα chain were generated. NZB mice are homozygous for a mutation in the IL-3Rα chain gene (Il3ra), and their cells are markedly hyporesponsive to IL-3 in vitro23 (see Table 4). A total of 98 F2 mice from an intercross betweenmpl−/− and NZB mice were generated. Twenty-seven (27.5%) of these animals were thrombocytopenic and were genotyped as mpl−/−. As expected with normal Mendelian segregation of alleles, of the 27mpl-deficient animals, seven (26%) were also homozygous for the mutant IL-3Rα chain (Il3ran/n). Although one of the seven mpl−/−Il3ran/n mice died before analysis of an undetermined cause at 3 weeks of age, the mutant IL-3Rα gene appeared to have no significant impact on the survival ofmpl−/− mice. Moreover, thempl−/−Il3ran/nanimals were no more thrombocytopenic thanmpl−/−Il3ra+/+ ormpl−/−Il3ra+/nmice (Table 4) suggesting that IL-3 does not contribute significantly to the maintenance of residual platelet numbers inmpl−/− mice. As expected, cells from mice homozygous for the Il3ran allele displayed only a residual response to IL-3, while normal colony numbers were observed in response to GM-CSF (Table 4).

Table 4.

Platelet Counts and In Vitro Hematopoietic Colony Formation in Mice Defective for Both c-Mpl and IL-3Rα

Genotype No. Platelet Count3-150 (×109/L) Colony Formation per 50,000 BM Cells3-151
Culture Stimulus
IL-3 GM-CSFSaline
NZB (Il3ran/n)  870 ± 149  2.5 ± 1.53-152 48 ± 10 0.5 ± 0.5  
mpl−/− 56 ± 17  ND  ND  ND 
F1 (mpl+/−Il3ra+/n16  1,060 ± 163  ND  ND  ND 
F2mpl−/−Il3ran/n 6  71 ± 43  3.5 ± 2.53-152 30 ± 3 0.5 ± 0.5 
mpl−/−Il3ra+/(+ or n) 20  69 ± 33  26 ± 7  28 ± 5 0 ± 0 
Genotype No. Platelet Count3-150 (×109/L) Colony Formation per 50,000 BM Cells3-151
Culture Stimulus
IL-3 GM-CSFSaline
NZB (Il3ran/n)  870 ± 149  2.5 ± 1.53-152 48 ± 10 0.5 ± 0.5  
mpl−/− 56 ± 17  ND  ND  ND 
F1 (mpl+/−Il3ra+/n16  1,060 ± 163  ND  ND  ND 
F2mpl−/−Il3ran/n 6  71 ± 43  3.5 ± 2.53-152 30 ± 3 0.5 ± 0.5 
mpl−/−Il3ra+/(+ or n) 20  69 ± 33  26 ± 7  28 ± 5 0 ± 0 

Abbreviation: ND, not determined.

F3-150

Mean ± standard deviation of data for the number of mice indicated for each genotype. Mice were analyzed at 6 to 8 weeks of age for F1 and F2 mice and at 3 to 5 months of age for NZB andmpl−/− mice.

F3-151

Mean ± standard deviation of data from colony assays containing no stimulus (saline), IL-3, or GM-CSF and maintained in a fully humidified atmosphere of 5% CO2 in air for 7 days.

F3-152

P < .01 for comparison with GM-CSF–stimulated cultures from the same mice.

Hematopoiesis in mice deficient in both c-Mpl and IL-3.

Given that some residual IL-3 responsiveness may still exist in NZB mice (Table 4), mice created through gene targeting to definitively lack IL-324 were bred with mpl-deficient animals for analysis of hematopoiesis in mpl−/−IL-3−/− double mutant offspring. Genotyping of 276 weanlings from matings ofmpl+/-IL-3+/- parents showed normal ratios of offspring of each of the expected genotypes (Table 5). In addition, no adult lethality was observed, indicating that the loss of IL-3 had no significant effect on the survival of mpl−/− mice. Peripheral blood analysis showed that, like thempl−/−Il3ran/nmice, mpl−/−IL-3−/− animals displayed the thrombocytopenia typical of mpl−/− mice. However, the superimposed lack of IL-3 did not reduce the platelet count further, or was any thrombocytopenia observed in mice lacking IL-3 alone (Table 6). Similarly, the reduction in megakaryocytes evident in histologic sections ofmpl−/− mice was not exacerbated inmpl−/− IL-3−/−animals and megakaryocyte numbers were normal in IL-3−/− mice (Table 6). Both IL-3−/− andmpl−/− IL-3−/−mice also displayed normal hematocrits and total white blood cell counts, as well as levels of circulating lymphocytes, monocytes, neutrophils, and eosinophils that were within the normal range (Table6). The number of peritoneal cells, the cellularity of the femoral bone marrow and spleen, and the distribution of morphologically recognizable precursor cells in these populations was not significantly different in mutant mice of all genotypes from that in normal controls (Table 6). Moreover, flow cytometric analysis of cells from bone marrow, spleen and thymus, using antibodies directed against a range of T lymphoid, B lymphoid, myeloid, and erythroid markers,20 showed no perturbations in mutant animals of any genotype (data not shown).

Table 5.

Production of mpl−/−IL-3−/− Mice

Genotype No. of Mice
Observed*Expected4-151
mpl+/+IL-3+/+ 15  17  
mpl+/+IL-3+/− 44  34  
mpl+/+IL-3−/− 14  17 
mpl+/− IL-3+/+ 35  34 
mpl+/− IL-3+/− 74  69 
mpl+/− IL-3−/− 28 34  
mpl−/− IL-3+/+ 17 17  
mpl−/− IL-3+/− 40  34  
mpl−/−IL-3−/− 11  17 
Genotype No. of Mice
Observed*Expected4-151
mpl+/+IL-3+/+ 15  17  
mpl+/+IL-3+/− 44  34  
mpl+/+IL-3−/− 14  17 
mpl+/− IL-3+/+ 35  34 
mpl+/− IL-3+/− 74  69 
mpl+/− IL-3−/− 28 34  
mpl−/− IL-3+/+ 17 17  
mpl−/− IL-3+/− 40  34  
mpl−/−IL-3−/− 11  17 

*Genotypes of 276 offspring from matings betweenmpl+/− IL-3+/− parents.

F4-151

Expected Mendelian frequency from 276 offspring assuming 1:2:1 ratio for production of wild-type (+/+): heterozygous (+/−): homozygous mutant (−/−) mice. χ2 analysis showed no significant different from observed frequencies (P = .4).

Table 6.

Hematologic Profile of mpl−/−IL-3−/− Mutant Mice

Genotype
Wild-Typempl+/+IL-3−/−mpl−/−IL-3+/+mpl−/−IL-3−/−
Peripheral blood  
 Platelets (×10−6/mL)  792 ± 80  769 ± 74 76 ± 39  70 ± 25  
 Hematocrit (%)  49 ± 4 47 ± 1  52 ± 2  47 ± 3  
 White cell count (×10−6/mL)  5.8 ± 2.9  3.8 ± 2.0 4.6 ± 0.6  4.6 ± 2.4  
  Neutrophils (%) 9 ± 6  13 ± 3  9 ± 2  10 ± 5 
  Lymphocytes (%)  82 ± 7  83 ± 4  85 ± 3 85 ± 5  
  Monocytes (%)  6 ± 2  3 ± 1 5 ± 2  4 ± 1  
  Eosinophils (%)  3 ± 2 1 ± 1  1 ± 1  1 ± 1  
Bone marrow 
 Cellularity (×10−6/femur)  15.6 ± 5.4 15.0 ± 1.4  23.0 ± 3.0  13.4 ± 5.25-151 
  Blasts (%)  3 ± 2  3 ± 1  2 ± 1 2 ± 2  
  Promyelocytes/myelocytes (%)  7 ± 4 6 ± 1  6 ± 2  6 ± 2 
  Metamyelocytes/neutrophils (%)  28 ± 5  30 ± 4 21 ± 3  23 ± 10  
  Lymphocytes (%)  26 ± 8 33 ± 4  35 ± 4  30 ± 13  
  Monocytes (%) 10 ± 2  4 ± 25-151 3 ± 2  3 ± 2 
  Eosinophils (%)  5 ± 2  1 ± 1  4 ± 2 1 ± 1  
  Nucleated erythroid cells (%)  21 ± 9 23 ± 4  29 ± 3  35 ± 17  
  Megakaryocytes (per 10 fields)5-150 66 ± 17  83 ± 5  8 ± 3 7 ± 2  
Spleen  
 Weight (mg)  98 ± 17 83 ± 5  73 ± 13  67 ± 3  
 Cellularity (×10−6)  77 ± 20  79 ± 17 70 ± 20  65 ± 15  
  Blasts (%)  2 ± 1 2 ± 0  3 ± 1  1 ± 2 
  Promyelocytes/myelocytes (%)  0 ± 0  0 ± 0 0 ± 0  0 ± 1  
  Metamyelocytes/neutrophils (%) 2 ± 1  1 ± 2  2 ± 2  3 ± 2 
  Lymphocytes (%)  80 ± 19  87 ± 2  87 ± 8 80 ± 15  
  Monocytes (%)  1 ± 1  3 ± 15-151 1 ± 1  1 ± 1  
  Eosinophils (%)  0 ± 1 0 ± 0  0 ± 0  0 ± 0  
  Nucleated erythroid cells (%)  15 ± 19  7 ± 0  7 ± 0  15 ± 11 
  Megakaryocytes (per 10 fields)5-150 9 ± 5  11 ± 3 0.7 ± 0.6  0.7 ± 0.6  
Peritoneal cavity 
 Cellularity (×10−6)  5.4 ± 1.9 4.1 ± 0.5  7.4 ± 3.4  4.6 ± 1.1 
  Neutrophils (%)  0 ± 0  0 ± 0  0 ± 0 0 ± 1  
  Lymphocytes (%)  45 ± 13  39 ± 16 42 ± 4  43 ± 13  
  Monocytes (%)  52 ± 12 57 ± 14  55 ± 3  56 ± 13  
  Eosinophils (%) 1 ± 1  1 ± 1  1 ± 2  0 ± 1  
  Mast cells (%)  2 ± 3  3 ± 2  2 ± 1 1 ± 1 
Genotype
Wild-Typempl+/+IL-3−/−mpl−/−IL-3+/+mpl−/−IL-3−/−
Peripheral blood  
 Platelets (×10−6/mL)  792 ± 80  769 ± 74 76 ± 39  70 ± 25  
 Hematocrit (%)  49 ± 4 47 ± 1  52 ± 2  47 ± 3  
 White cell count (×10−6/mL)  5.8 ± 2.9  3.8 ± 2.0 4.6 ± 0.6  4.6 ± 2.4  
  Neutrophils (%) 9 ± 6  13 ± 3  9 ± 2  10 ± 5 
  Lymphocytes (%)  82 ± 7  83 ± 4  85 ± 3 85 ± 5  
  Monocytes (%)  6 ± 2  3 ± 1 5 ± 2  4 ± 1  
  Eosinophils (%)  3 ± 2 1 ± 1  1 ± 1  1 ± 1  
Bone marrow 
 Cellularity (×10−6/femur)  15.6 ± 5.4 15.0 ± 1.4  23.0 ± 3.0  13.4 ± 5.25-151 
  Blasts (%)  3 ± 2  3 ± 1  2 ± 1 2 ± 2  
  Promyelocytes/myelocytes (%)  7 ± 4 6 ± 1  6 ± 2  6 ± 2 
  Metamyelocytes/neutrophils (%)  28 ± 5  30 ± 4 21 ± 3  23 ± 10  
  Lymphocytes (%)  26 ± 8 33 ± 4  35 ± 4  30 ± 13  
  Monocytes (%) 10 ± 2  4 ± 25-151 3 ± 2  3 ± 2 
  Eosinophils (%)  5 ± 2  1 ± 1  4 ± 2 1 ± 1  
  Nucleated erythroid cells (%)  21 ± 9 23 ± 4  29 ± 3  35 ± 17  
  Megakaryocytes (per 10 fields)5-150 66 ± 17  83 ± 5  8 ± 3 7 ± 2  
Spleen  
 Weight (mg)  98 ± 17 83 ± 5  73 ± 13  67 ± 3  
 Cellularity (×10−6)  77 ± 20  79 ± 17 70 ± 20  65 ± 15  
  Blasts (%)  2 ± 1 2 ± 0  3 ± 1  1 ± 2 
  Promyelocytes/myelocytes (%)  0 ± 0  0 ± 0 0 ± 0  0 ± 1  
  Metamyelocytes/neutrophils (%) 2 ± 1  1 ± 2  2 ± 2  3 ± 2 
  Lymphocytes (%)  80 ± 19  87 ± 2  87 ± 8 80 ± 15  
  Monocytes (%)  1 ± 1  3 ± 15-151 1 ± 1  1 ± 1  
  Eosinophils (%)  0 ± 1 0 ± 0  0 ± 0  0 ± 0  
  Nucleated erythroid cells (%)  15 ± 19  7 ± 0  7 ± 0  15 ± 11 
  Megakaryocytes (per 10 fields)5-150 9 ± 5  11 ± 3 0.7 ± 0.6  0.7 ± 0.6  
Peritoneal cavity 
 Cellularity (×10−6)  5.4 ± 1.9 4.1 ± 0.5  7.4 ± 3.4  4.6 ± 1.1 
  Neutrophils (%)  0 ± 0  0 ± 0  0 ± 0 0 ± 1  
  Lymphocytes (%)  45 ± 13  39 ± 16 42 ± 4  43 ± 13  
  Monocytes (%)  52 ± 12 57 ± 14  55 ± 3  56 ± 13  
  Eosinophils (%) 1 ± 1  1 ± 1  1 ± 2  0 ± 1  
  Mast cells (%)  2 ± 3  3 ± 2  2 ± 1 1 ± 1 

Mean ± standard deviations of data from 3 to 6 mice of each genotype.

F5-150

Enumerated from histological sections at 200× (spleen) or 400× (bone marrow) magnification, with a minimum of 20 fields counted per mouse.

F5-151

P < .05 for pairwise comparison of data frommpl+/+ IL-3−/− with wild-type mice or for comparison of data from mpl−/−IL-3−/− mice with mpl−/−IL-3+/+ mice. No other significant differences existed (P > .05).

To determine whether IL-3 plays a physiologic role in earlier stages of hematopoiesis, progenitor cells from mutant animals were assayed in semisolid agar cultures containing a combination of SCF, IL-3, and EPO, which stimulates a broad range of erythroid and myeloid colonies.20 The total numbers of hematopoietic progenitor cells were normal in IL-3−/− mice and the low levels evident in mpl−/−animals20 were not further lowered inmpl−/− IL-3−/−mice (Table 7). The enumeration of individual colony types also indicated that loss of IL-3 did not alter the numbers of progenitor cells committed to specific hematopoietic lineages, including megakaryocyte progenitors, either in normal ormpl−/− mice (Table 7). As expected, in cultures stimulated solely by TPO, no colony formation was evident with the cells from mpl−/− ormpl−/− IL-3−/−mice, while equal numbers of small megakaryocyte colonies developed from wild-type and IL-3−/− bone marrow cells (wild-type: 9 ± 4; IL-3−/−: 8 ± 5 colonies per 2.5 × 105 cells plated, n = 3).

Table 7.

Bone Marrow Progenitor Cell Analysis ofmpl−/− IL-3−/− Mutant Mice

Genotype Progenitor Cells per 2.5 × 104 Bone Marrow Cells
Total GranulocyteGM Macrophage Eosinophil Megakaryocyte Meg/EErythroid Blast
Wild-type  90 ± 40  26 ± 2 16 ± 11  21 ± 14  1 ± 1  8 ± 5 3 ± 2  4 ± 6  11 ± 4 
mpl+/+ IL-3−/− 79 ± 20  23 ± 5  13 ± 1  13 ± 4 1 ± 2  6 ± 2  5 ± 2  7 ± 8  11 ± 5 
mpl−/− IL-3+/+ 35 ± 13  8 ± 3  8 ± 8  9 ± 1  2 ± 2 3 ± 2  3 ± 2  1 ± 1  1 ± 1 
mpl−/− IL-3−/− 27 ± 8  8 ± 5  7 ± 4  6 ± 4  1 ± 1 2 ± 2  2 ± 1  1 ± 1  
Genotype Progenitor Cells per 2.5 × 104 Bone Marrow Cells
Total GranulocyteGM Macrophage Eosinophil Megakaryocyte Meg/EErythroid Blast
Wild-type  90 ± 40  26 ± 2 16 ± 11  21 ± 14  1 ± 1  8 ± 5 3 ± 2  4 ± 6  11 ± 4 
mpl+/+ IL-3−/− 79 ± 20  23 ± 5  13 ± 1  13 ± 4 1 ± 2  6 ± 2  5 ± 2  7 ± 8  11 ± 5 
mpl−/− IL-3+/+ 35 ± 13  8 ± 3  8 ± 8  9 ± 1  2 ± 2 3 ± 2  3 ± 2  1 ± 1  1 ± 1 
mpl−/− IL-3−/− 27 ± 8  8 ± 5  7 ± 4  6 ± 4  1 ± 1 2 ± 2  2 ± 1  1 ± 1  

Mean ± standard deviations of colony numbers in replicate cultures stimulated with the combination of SCF, IL-3, and EPO from 3 mice per genotype. Cultures were incubated in 5% CO2 in air for 7 days. Colony numbers and composition were determined by examination of stained cultures at 200× magnification. There were no significant differences for comparison of data frommpl+/+ IL-3−/− mice with wild-type mice, or for comparison of data frommpl−/− IL-3−/− mice withmpl−/− IL-3+/+ mice (P > .1).

Abbreviations: GM, mixed granulocyte/macrophage; Meg/E, mixed megakaryocyte/erythroid.

Interestingly, despite development in the absence of IL-3, normal numbers of IL-3–responsive cells had developed in IL-3−/− bone marrow (wild-type: 88 ± 28; IL-3−/−: 71 ± 30 colonies per 2.5 × 105 cells plated, n = 3) and no further reduction from that already evident in mpl−/− mice was evident in the mpl−/−IL-3−/− double mutants (mpl−/−: 29 ± 9;mpl−/− IL-3−/−: 33 ± 12 colonies per 2.5 × 105 cells plated, n = 3). In addition, colony formation in response to stimulation by single cytokines (IL-5, IL-6, GM-CSF, G-CSF, M-CSF, or SCF) was similar between bone marrow cells from mpl−/−and mpl−/−IL-3−/− mice, as well as between IL-3−/− and wild-type animals (data not shown). Consistent with these observations of bone marrow progenitor cells, the lack of IL-3 also failed to alter the numbers of erythroid, mixed erythroid, or myeloid colony-forming cells in the spleens of these animals (data not shown). Together, these analyses suggest that even in the absence of signalling from the major regulator of megakaryocytopoiesis, TPO, IL-3 does not contribute to the residual megakaryocyte and platelet production inmpl−/− mice. Moreover, these data confirm and extend previous observations24 to suggest that IL-3 has no essential physiologic role in the maintenance of other mature hematopoietic cells or their committed progenitor cells, in otherwise normal animals or in mpl−/−mice.

The dominant role of TPO in the regulation of platelet production has been convincingly demonstrated by the severe thrombocytopenia evident in mice lacking this regulator or its receptor.19-21However, as TPO−/− andmpl−/− mice retain the capacity to produce sufficient platelets to prevent bleeding, an important contribution to thrombopoiesis in vivo is likely to be made by other cytokines with megakaryocytopoietic potential. Candidate cytokines include IL-6, IL-11, IL-3, and LIF, which significantly elevate platelet numbers when injected into animals.15-18 Although gene targeting studies have shown that in the presence of TPO, the loss of such cytokines does not significantly affect platelet levels,28-31 subtle actions of these factors may exist and be shown or amplified in mice lacking the dominant TPO signalling system. We initially investigated this possibility by examiningmpl−/− mice for evidence of elevated production of known megakaryocytopoietic factors. Although we found, as previously described,19 that serum TPO was elevated, we did not detect biologically active IL-6, IL-11, IL-3, LIF, or GM-CSF in the circulation of mice of either genotype. A measure of production of these cytokines from individual tissue sources was obtained by analyzing organ conditioned medium. As evident in a previous survey,25 factors were produced by multiple tissues of normal adult mice, in particular the lungs, muscle, thymus, and bone shaft. However, no significant differences in the organ distribution or level of cytokine production were observed in analyses of media conditioned by the organs of mpl−/−mice (Table 1). Thus, dramatic elevation in production of alternative megakaryocytopoietic cytokines does not appear to be a major mechanism by which mice lacking an intact TPO signalling system maintain residual platelet production. An interesting characteristic ofmpl−/− mice is that despite producing reduced numbers of progenitors cells for essentially all hematopoietic lineages, deficits in mature blood cells are restricted to platelets.19,20 This observation complements previous studies, including those of cytokine administration32 and other mutant mouse models of hematopoiesis,33 in which alterations in the number of committed progenitor cells are not necessarily reflected by the numbers of circulating blood cells. In the case of the granulocyte lineage, for example, we also found no evidence that elevated cytokine production provides a compensatory mechanism: the concentrations and spectra of organs producing G-CSF (data not shown) and GM-CSF (Table 1) were not significantly different between normal and mpl-deficient mice.

To determine the in vivo megakaryocytopoietic potential of alternative cytokines in the absence of TPO signalling, control andmpl−/− mice were injected with LIF or IL-6. Previous studies with these cytokines in normal mice showed a capacity to elevate platelet numbers up to twofold.16 17Our studies confirmed these observations and demonstrated that LIF and IL-6 retain this activity in mpl−/−mice. Although the platelet numbers in LIF- or IL-6–injectedmpl−/− mice did not recover to normal levels, reflecting the reduced numbers of megakaryocytes and their progenitors available for stimulation, the magnitude of the platelet increase was at least as significant in mpl-deficient mice as in the wild-type controls (IL-6: mpl+/+ 1.6-fold,mpl−/− 2.2-fold; LIF:mpl+/+ 1.7-fold,mpl−/− 1.6 fold, Table 2).

Our results confirm previous studies demonstrating the thrombopoietic potential of IL-6, and also of IL-11 and SCF, inmpl−/− and TPO−/− mice22 and further demonstrate the potency of LIF in the absence of TPO signalling. These observations suggest that, in vivo, such cytokines act substantially independently of TPO, in contrast to their reported action in certain in vitro assays.34 The elevation in platelet numbers inmpl−/− mice injected with IL-6 was also accompanied by significant increases in the numbers of mature megakaryocytes and their progenitors in hematopoietic organs, a response consistent with that in wild-type animals (Table 2). Thus, our data also extend previous observations to show that in vivo, alternative cytokines cannot only elevate platelet numbers, but can stimulate the full process of megakaryocytopoiesis in the absence of TPO signalling.

These analyses provide important proof that alternative cytokines have the capacity to stimulate megakaryocytopoiesis in the absence of TPO signalling, but do not provide direct evidence that any of these factors control the residual steady-state megakaryocyte and platelet development in mpl−/− mice. To further address this issue, we are conducting genetic crosses betweenmpl−/− mice and other genetically modified mice unable to produce alternative megakaryocytopoietic cytokines or their receptors. As IL-3 is the most potent single stimulus for megakaryocyte colony formation in vitro,1 we initiated studies with mice deficient in either IL-3 or the IL-3Rα chain. NZB mice have a naturally-occurring mutation in the IL-3Rα chain gene preventing normal cell-surface expression of the receptor and resulting in a markedly reduced response to IL-3.23Interbreeding of mpl−/− and NZB mice yielded the expected number of mpl−/−Il3ran/n double mutant offspring and platelet numbers in these mice were equivalent to those in theirmpl−/−Il3ra+/(+/n)littermates (Table 4), suggesting little or no role for IL-3 inmpl-deficient platelet development.

As our data and that of others23 suggested that some residual IL-3 responsiveness may exist in NZB mice (Table 4), we definitively addressed the role of IL-3 in normal and residualmpl−/− megakaryocytopoiesis using animals genetically modified to lack this cytokine.24 The numbers of circulating platelets, mature megakaryocytes, and megakaryocyte progenitor cells were normal in IL-3−/− mice (Tables 6 and 7). Moreover, even in the absence of TPO signalling, lack of IL-3 did not exacerbate the megakaryocyte deficiency or the thrombocytopenia of c-mpl−/− mice (Tables 6 and 7). In previous studies, initial indications that IL-3 may not play a prominent role in megakaryocytopoiesis came from the normal megakaryocyte and platelet production evident in athymic nu/numice, which lack T lymphocytes, considered a prominent source of IL-3.35 Our data from genetically modified mice specifically lacking the cytokine provide definitive evidence that, despite its potent in vitro activity on megakaryocyte proliferation1 and its ability to stimulate this lineage on administration in vivo,15 physiologically IL-3 has no significant essential role in megakaryocytopoiesis, or is it involved in the residual megakaryocyte and platelet production inmpl−/− mice. Of broader significance, IL-3−/− mice displayed normal numbers of hematopoietic progenitor cells in the bone marrow and spleen, as well as their mature progeny in the circulation, marrow, spleen, and peritoneal cavity24 (Tables 6 and 7). Similarly, the reduction in committed progenitor cells characteristic ofmpl−/− mice was not exacerbated in the absence of IL-3. Thus, in addition to providing no evidence of a role for IL-3 in megakaryocyte and platelet production, our studies confirm previous analyses24 to suggest that IL-3 plays little physiologic role in the development of blood cells of other lineages.

We thank Ladina DiRago and Sandra Mifsud for excellent technical assistance and Jodie deWinter and Anne Chow for animal husbandry.

Supported by the National Health and Medical Research Council, Canberra, the Anti-Cancer Council of Victoria, the Cooperative Research Centre for Cellular Growth Factors, the National Institutes of Health, Bethesda, Grant No. CA22556, and by support to S.K. by the Naito Foundation.

Address reprint requests to Warren S. Alexander, PhD, The Walter and Eliza Hall Institute for Medical Research, PO Royal Melbourne Hospital, Victoria 3050, Australia.

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.

1
Gordon
MS
Hoffman
R
Growth factors affecting human thrombopoiesis: Potential agents for treatment of thrombocytopenia.
Blood
80
1992
302
2
Kaushansky
K
Thrombopoietin: The primary regulator of platelet production.
Blood
86
1995
419
3
Broudy
VC
Lin
NL
Kaushansky
K
Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance megakaryocyte colony growth and increases megakaryocyte ploidy in vitro.
Blood
85
1995
1719
4
Hunt
P
Li
Y-S
Nichol
JL
Hokom
MM
Bogenberger
JM
Swift
SE
Skrine
JD
Hornkohl
AC
Lu
H
Clogston
C
Merewether
LA
Johnson
MJ
Parker
V
Knudten
A
Farese
A
Hsu
RY
Garcia
A
Stead
R
Bosselman
RA
Bartley
TD
Purification and biologic characterization of plasma-derived megakaryocyte growth and development factor.
Blood
86
1995
540
5
Banu
N
Wang
JF
Deng
B
Groopman
JE
Avraham
H
Modulation of megakaryocytopoiesis by thrombopoietin: The c-Mpl ligand.
Blood
86
1995
1331
6
Kaushansky
K
Lok
S
Holly
RD
Broudy
VC
Lin
N
Bailey
MC
Forstrom
JW
Buddle
MM
Oort
PJ
Hagen
FS
Roth
GJ
Papayannopoulou
T
Foster
DC
Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.
Nature
369
1994
568
7
Zeigler
FC
deSauvage
F
Widmer
HR
Keller
GA
Donahue
C
Schreiber
RD
Malloy
B
Hass
P
Eaton
D
Matthews
W
In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells.
Blood
84
1994
4045
8
Choi
ES
Hokom
M
Bartley
T
Li
Y-S
Ohashi
H
Kato
T
Nichol
JL
Skrine
J
Knudten
A
Chen
J
Hornkohl
A
Grampp
G
Sleeman
L
Cole
S
Trail
G
Hunt
P
Recombinant human megakaryocyte growth and development factor (MGDF), a ligand for c-Mpl, produces functional human platelets in vitro.
Stem Cells
13
1995
317
9
Debili
N
Wendling
F
Katz
A
Guichard
J
Breton-Gorius
J
Hunt
P
Vainchenker
W
The Mpl ligand or thrombopoietin or megakaryocyte growth and differentiation factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood
86
1995
2516
10
Lok
S
Kaushansky
K
Holly
RD
Kuijper
JL
Lofton-Day
CE
Oort
PJ
Grant
FJ
Heipel
MD
Burkhead
SK
Kramer
JM
Bell
LA
Sprecher
CA
Blumberg
H
Johnson
R
Prunkard
D
Ching
AFT
Mathewes
SL
Bailey
MC
Forstrom
JW
Buddle
MM
Osborn
SG
Evans
SJ
Sheppard
PO
Presnell
SR
O'Hara
PJ
Hagen
FS
Roth
GJ
Foster
DC
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369
1994
565
11
Ulich
TR
del Castillo
J
Yin
S
Swift
S
Padilla
D
Senaldi
G
Bennett
L
Shutter
J
Bogenberger
J
Sun
D
Samal
B
Shimamoto
G
Lee
R
Steinbrink
R
Boone
T
Sheridan
WT
Hunt
P
Megakaryocyte growth and development factor ameliorates carboplatin-induced thrombocytopenia in mice.
Blood
86
1995
971
12
Farese
AM
Hunt
P
Boone
T
MacVittie
TJ
Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates.
Blood
86
1995
54
13
Basser
RL
Rasko
JEJ
Clarke
K
Cebon
J
Green
MD
Hussein
S
Alt
C
Menchaca
D
Tomita
D
Marty
J
Fox
RM
Begley
CG
Thrombopoietic effects of pegylated recominant human megakaryocyte growth and development factor (PEG-rHuMGDF) in patients with advanced cancer.
Lancet
348
1996
1279
14
Rasko
JEJ
Basser
RL
Boyd
J
Mansfield
R
O'Malley
CJ
Hussein
S
Berndt
MC
Clarke
K
O'Byrne
J
Sheridan
WP
Grigg
AP
Begley
CG
Multilineage mobilisation of peripheral blood progenitor cells in humans following administration of PEG-rHuMGDF.
Br J Haematol
97
1997
871
15
Metcalf
D
Begley
CG
Johnson
GR
Nicola
NA
Lopez
AF
Williamson
DJ
Effects of bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice.
Blood
68
1986
46
16
Hill
RJ
Warren
MK
Levin
J
Stimulation of thrombopoiesis in mice by human recombinant interleukin 6.
J Clin Invest
85
1990
1242
17
Metcalf
D
Nicola
NA
Gearing
DP
Effects of injected leukemia inhibitory factor on hematopoietic and other tissues in mice.
Blood
76
1990
50
18
Neben
TY
Loebelenz
J
Hayes
L
McCarthy
K
Stoudemire
J
Schaub
R
Goldman
SJ
Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice.
Blood
81
1993
901
19
Gurney
AL
Carver-Moore
K
de Sauvage
FJ
Moore
MW
Thrombocytopenia in c-mpl-deficient mice.
Science
265
1994
1445
20
Alexander
WS
Roberts
AR
Nicola
NA
Li
R
Metcalf
D
Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietin receptor c-Mpl.
Blood
87
1996
2162
21
deSauvage
FJ
Carver-Moore
K
Luoh
S-M
Ryan
A
Dowd
M
Eaton
DL
Moore
MW
Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.
J Exp Med
183
1996
651
22
Carver-Moore
K
Broxmeyer
HE
Luoh
S-M
Cooper
S
Peng
J
Burstein
SA
Moore
MW
deSauvage
FJ
Low levels of erythroid and myeloid progenitors in thrombopoietin and c-mpl-deficient mice.
Blood
88
1996
803
23
Ichihara
M
Hara
T
Takagi
M
Cho
LC
Gorman
DM
Miyajima
A
Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor α subunit gene.
EMBO J
14
1995
939
24
Mach N, Lantz CS, Galli SJ, Reznikoff G, Mihm M, Small C, Granstein R, Beissert S, Sadelain M, Mulligan RC, Dranoff G: Involvement of interleukin-3 in delayed type hypersensitivity. Blood (in press)
25
Metcalf
D
Willson
TA
Hilton
DJ
DiRago
L
Mifsud
S
Production of hematopoietic regulatory factors in cultures of adult and fetal mouse organs: Measurement by specific bioassay.
Leukemia
9
1995
1556
26
Alexander
WS
Metcalf
D
Dunn
AR
Point mutations within a dimer interface homology domain of c-Mpl induce constitutive receptor activity and tumorigenicity.
EMBO J
14
1995
5569
27
Fielder
PJ
Gurney
AL
Stefanich
E
Marian
M
Moore
MW
Carver-Moore
K
de Sauvage
FJ
Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets.
Blood
87
1996
2154
28
Escary
J-L
Perreau
J
Dumenil
D
Ezine
S
Brulet
P
Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation.
Nature
363
1993
361
29
Bernad
A
Kopf
M
Kulbacki
R
Weich
N
Koehler
G
Gutierrez-Ramos
JC
Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system.
Immunity
1
1994
725
30
Nishinakamura
R
Miyajima
A
Mee
PJ
Tybulewicz
VLJ
Murray
R
Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions.
Blood
88
1996
2458
31
Nandurkar
HH
Robb
L
Tarlinton
D
Barnett
L
Kontgen
F
Begley
CG
Adult mice with targeted mutation of the IL-11 receptor (Il11Ra) display normal hematopoiesis.
Blood
90
1997
2148
32
Kaushansky
K
Lin
N
Grossmann
A
Humes
J
Sprugel
KH
Broudy
VC
Thrombopoietin expands erythroid, granulocyte-macrophage and megakaryocytic progenitor cells in normal and myelosuppressed mice.
Exp Hematol
23
1996
265
33
Cheng
J
Baumhueter
S
Cacalano
G
Thibodeaux
H
Thomas
R
Broxmeyer
HE
Cooper
S
Hague
N
Moore
M
Lasky
LA
Hematopoietic defects in mice lacking the sialomucin CD34.
Blood
87
1996
479
34
Kaushansky
K
Broudy
VC
Lin
N
Jorgensen
MJ
McCarty
J
Fox
N
Zucker-Franklin
D
Lofton-Day
C
Thrombopoietin, the Mpl ligand, is essential for full megakaryocyte development.
Proc Natl Acad Sci USA
92
1995
3234
35
Ebbe
S
Levin
J
Miller
K
Yee
T
Levin
F
Phalen
E
Thrombocytopoietic response to immunothrombocytopenia in nude mice.
Blood
69
1987
192
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