Thrombopoietin signaling is required for in vivo expansion of IL-11–responsive hematopoietic progenitor cells in the steady state

Experimental Hematology 29 (2001) 138–145

Thrombopoietin signaling is required for in vivo expansion of IL-11–responsive hematopoietic progenitor cells in the steady state Clare L. Scotta,b, Lorraine Robba,b, Harshal H. Nandurkara,b, Rachel Mansfieldc, Warren S. Alexandera,b, and C. Glenn Begleya,b,c,d a The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria, Australia; The Cooperative Research Centre for Cellular Growth Factors, P.O. Royal Melbourne Hospital, Victoria, Australia; c Rotary Bone Marrow Research Laboratories, P.O. Royal Melbourne Hospital, Victoria, Australia; d The Western Australian Institute for Medical Research, Perth, Australia

b

(Received 17 December 1999; revised 14 September 2000; accepted 27 September 2000)

Objective. mpl⫺/⫺ mice have a profound defect in platelets and megakaryocytes and a defect in hematopoietic progenitor cells and stem cells. However, no specific subset of the progenitor/ stem cell compartment has been shown to be particularly affected by this deficiency in mpl⫺/⫺ mice. In this article, we identified a specific subset of bone marrow progenitor/stem cells that was altered in mpl⫺/⫺ mice. Materials and Methods. In vitro and in vivo hematopoietic assays were utilized to examine the response to interleukin-11 in mice lacking the receptor for thrombopoietin (TPO) (mpl⫺/⫺ mice). Results. The interleukin (IL)-11–responsive subset of progenitor cells was not detected in clonal cultures of bone marrow cells from mpl⫺/⫺ mice. However, mpl⫺/⫺ mice responded to IL-11 in vivo as evidenced by a rise in platelet count and an increase in spleen weight. Experiments were performed to address this paradox: administration of 5-fluorouracil with consequent “expansion” of early hematopoietic cells resulted in the appearance of IL-11–responsive cells in mpl⫺/⫺ mice when assayed in in vitro cultures. Conclusion. Thus, although mpl⫺/⫺ mice have the capacity to produce IL-11–responsive progenitor cells, under steady state conditions their expansion is dependent on TPO. This is the first evidence that a specific subset of bone marrow progenitor/stem cells is altered in mpl⫺/⫺ mice. © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: mpl—Thrombopoietin—Interleukin-11—Platelet—Progenitor cell

Introduction The cloning of thrombopoietin (TPO), followed by characterization of the recombinant protein, confirmed its role as the primary regulator of megakaryocyte and platelet development [1,2]. TPO has been shown to stimulate both megakaryocyte colony growth from bone marrow progenitor cells and the maturation of immature megakaryocytes, and to support the formation of functional platelets [3–9]. The analysis of mice lacking TPO or its receptor, c-mpl (mpl⫺/⫺ mice), confirmed the thrombopoietic action of this growth factor [10–12]. These mice were not only profoundly thrombocytopenic, with platelet counts in the order of 10–20% of

Offprint requests to: C.L. Scott, MB, BS, The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria 3050 Australia; E-mail: [email protected]

those seen in wild-type mice, but also had a marked reduction in bone marrow megakaryocytes. In addition to these defects, TPO was found to have a broader role in hematopoiesis than initially anticipated, with profound effects on primitive hematopoietic cells [2]. Thus, in c-mpl– and TPO-deficient mice, there was a 50% reduction in the number of multipotential hematopoietic progenitor cells in the bone marrow [11,13]. Preprogenitor cells and spleen colony-forming cells (CFU-S) were reduced by 90% and hematopoietic repopulating cells were deficient by an even greater magnitude [14,15]. Several additional lines of evidence have provided support for TPO signaling in early hematopoiesis: 1) c-mpl is expressed on a subset of immature hematopoietic cells (CD34⫹ cells), 2) the viral homologue, v-mpl, induces an acute myeloproliferative leukemia with massive expansion of maturing cells of multiple hematopoietic lineages [16], 3)

0301-472X/01 $–see front matter. Copyright © 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(00)0 0 6 2 2 - 6

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TPO, alone or in combination with early-acting cytokines (stem cell factor (SCF), IL-3, or Flt-3 ligand), stimulates the in vitro proliferation of early hematopoietic multilineage progenitor cells [3,17,18], and 4) in vivo administration of TPO expanded the bone marrow and spleen progenitor pools of all hematopoietic lineages [19]. However, despite the clear role for TPO in the genesis of hematopoietic progenitor cells, it remains unclear whether the action of TPO on this compartment is a global one, or whether particular subsets of cells are specifically influenced by TPO signaling. Another cytokine, IL-11, has recently become available for use in the clinic because of its ability to accelerate platelet recovery after chemotherapy [20,21]. However, despite these data, mice lacking the IL-11–specific receptor ␣ chain (IL11Ra⫺/⫺ mice) showed no defect in hematopoiesis, indicating that IL-11 was dispensable in the steady state [22,23]. Mice deficient for both c-mpl and either the growth factor or receptor for IL-3, IL-6, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL11 (mpl⫺/⫺/IL11Ra⫺/⫺ mice) have also been examined and were indistinguishable from mpl⫺/⫺ mice [24–26]. This indicated that even in the absence of TPO signaling, IL-11 did not play an essential role in thrombopoiesis. However, in contrast to these results, the actions of IL-11 on hematopoietic progenitor cells are well documented. IL-11 acts synergistically with other cytokines to stimulate the proliferation of primitive stem cells, multipotential and committed progenitor cells from bone marrow, peripheral blood, and cord blood [27–29]. Similarly, ex vivo expansion of bone marrow cells by IL-11 and SCF enhanced short-term engraftment potential of these cells and hematopoietic recovery was better sustained during serial transplants in lethally irradiated mice [30]. In this study, we elected to specifically examine the interaction between TPO and IL-11 by analyzing the response to IL-11 both in vitro and in vivo in mpl⫺/⫺ mice. We identified a specific progenitor “stem” cell defect in the IL11–responsive subset of progenitor cells in mpl⫺/⫺ mice.

Materials and methods Mice The generation of mpl⫺/⫺ mice and IL11Ra⫺/⫺ mice have been described previously [11,22]. As controls, C57BL/6 or 129/Sv (wild-type) mice were used. All experiments were conducted on mice 8–16 weeks of age. In vitro cultures Bone marrow and spleen progenitor cells were assayed using semisolid agar and methyl cellulose cultures as previously described [22,31,32]. Semisolid 1-mL agar cultures containing 5 ⫻ 104 bone marrow cells or 105 spleen cells in 0.3% agar in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% newborn calf serum were plated in triplicate and stimulated by multiple combinations of purified recombinant growth factors at the following final concentrations: murine (m) SCF (produced by expression in Pichia pastoris) at 100 or 10 ng/mL; mIL-3 (Peprotech, Rocky Hill,

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NJ) at 1000 or 100 U/mL; murine GM-CSF (a gift from DNAX Corporation, San Diego, CA) at 10 ng/mL; human (h) granulocyte colony-stimulating factor (G-CSF) (a gift from AMRAD Pharmaceuticals, Melbourne, Australia) at 10 ng/mL; hIL-11 (a gift from Genetics Institute, Boston, MA) at 10 ng/mL; hIL-6 (a gift from Amgen, Thousand Oaks, CA) at 100 ng/mL; human erythropoietin (EPO) (a gift from Amgen, Thousand Oaks, CA) at 4 U/mL; and pegylated recombinant human megakaryocyte growth and differentiation factor (MGDF) (PEG-rhuMGDF, Amgen, Thousand Oaks, CA) at 200 ng/mL. After 7 days of incubation at 37⬚C in a fully humidified atmosphere of 10% CO2 in air, colonies were enumerated by examination of cultures at 35⫻ magnification. The investigator was blinded to the genotype of the animals from which the cells were derived. Cultures were fixed with 2.5% glutaraldehyde in normal saline and stained using acetylcholinesterase, luxol fast blue, and hematoxylin, and the colonies classified at 100⫻ or 400⫻ magnification. Megakaryocyte colonies were defined as those containing three or more megakaryocyte cells. In vivo administration of IL-11 Mice were injected intraperitoneally with rhIL-11, 100 ␮g/kg/day, or carrier (0.1% bovine serum albumin) in 0.2 mL for 7 days following a previously described protocol [33]. Mice were bled from the retro-orbital sinus prior to injection and again at the end of the administration period. The peripheral blood hematocrit, white blood cell count, and platelet count were determined using either manual techniques or automated hematological analysis (Technicon H.1 System, Technicon Instrument Corporation, Tarrytown, NY). Splenic weight was recorded and cell suspensions from femoral bone marrow and spleen were prepared and the cell number enumerated in a hemocytometer after eosin staining. Colony-forming unit-spleen assay CFU-S assays were performed using 75,000 (wild-type) or 150,000 (mpl⫺/⫺) bone marrow cells from donor mice previously injected with either IL-11 or carrier. Cells were washed in serum-free DMEM and injected intravenously via the retro-orbital sinus into 4–5 wild-type recipients. Several hours before transplantation, the recipient mice were irradiated with 11 Gy of ␥-irradiation in two equal doses given three hours apart from a 137Cs source (Atomic Energy, Ottawa, Canada) at a dose rate of 31 cGy/min. Transplanted mice were maintained on oral antibiotic (1.1 g/liter of neomycin sulfate, Sigma, St. Louis, MO, USA). Spleens were removed after 12 days and fixed in Carnoy’s solution (60% ethanol, 30% chloroform, 10% acetic acid) and the numbers of macroscopic colonies were enumerated [14]. 5-flurouracil administration A single dose of 5-flurouracil (5-FU), 150 mg/kg, was administered intravenously to wild-type or mpl⫺/⫺ mice. Bone marrow and spleen were harvested at various time points from day 0 to day 17 post–5-FU injection. Progenitor cells were assayed in clonal culture as previously described.

Results Lack of in vitro response to IL-11 with bone marrow cells from mpl⫺/⫺ mice During our analysis of hematopoietic progenitor cells in the IL11Ra⫺/⫺ mice, we noticed a failure of mpl⫺/⫺ bone mar-

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row cells to respond to IL-11 in in vitro cultures. This result was unexpected. The mpl⫺/⫺ cells had been included as a control population for those experiments and were consistently, and incorrectly, designated as of IL11Ra⫺/⫺ origin by the blinded investigator. We therefore sought to characterize the failure of mpl⫺/⫺ cells to respond to IL-11. The response of bone marrow progenitor cells from mpl⫺/⫺ mice or wild-type mice to an array of cytokines was assayed in clonal culture. As previously reported [11], the total number of granulocyte-macrophage (GM) colonies seen when mpl⫺/⫺ bone marrow cells were stimulated with a combination of growth factors was reduced in the order of 50%. To specifically address the effect of IL-11 and IL-6 on mpl⫺/⫺ hematopoietic cells, assays were established to exploit the synergy between these factors and SCF or IL-3. At least two concentrations of SCF or IL-3 were assayed, with or without IL-11 or IL-6. Consistent with previous studies [22], in cultures of wild-type cells, IL-11 alone did not stimulate colony formation while IL-6 alone stimulated small numbers of colonies (Table 1). Using wild-type bone marrow cells, the addition of IL-6 to either SCF or IL-3 resulted in a “superadditive” or synergistic effect on colony number when compared with the effect of either SCF or IL-3 alone (Table 1). This synergistic response was even more apparent if a submaximal concentration of either SCF or IL-3 was used (data not shown). A similar superadditive response was seen when IL-11 was combined with SCF or IL-3. In addition to the effect on colony number, the synergistic effect of IL-6 and IL-11 was also manifest as an increase in colony size. Colony size was measured as cell number per 50 colonies with a minimum of 50 colonies examined per stimulus per mouse. When cultures were stimulated with SCF alone, average colony size was 1.45 ⫾ 0.29 ⫻ 104 cells per 50 colonies, compared with 5.17 ⫾ 1.30 ⫻ 104 cells when SCF and IL-6 were added, or 4.33 ⫾ 2.80 ⫻ 104 cells when SCF and IL-11 were added. For IL-3, colony size was 3.67 ⫾ 1.10 ⫻ 104 cells vs 10.22 ⫾

2.52 ⫻ 104 cells with IL-3 ⫹ IL-6, or 7.11 ⫾ 0.95 ⫻ 104 with IL-3 ⫹ IL-11. This increase in colony number and in colony size resulted in a striking difference between the appearance of control cultures stimulated with SCF or IL-3 alone, and cultures to which IL-6 or IL-11 was added. In keeping with the results for wild-type cells, mpl⫺/⫺ bone marrow cells also showed a consistent enhancement of colony number in response to stimulation with IL-6 combined with either SCF or IL-3 (Table 1). Given the well-described deficit in the hematopoietic progenitor cell compartment in mpl⫺/⫺ mice, it was perhaps not surprising that this synergistic response to IL-6 was somewhat attenuated. However, a synergistic response to IL-6 in mpl⫺/⫺ cultures was clearly present. Again, this synergistic response to IL-6 was associated with an increase in colony size (for SCF alone, ⬍103 cells vs SCF ⫹ IL-6, 1.22 ⫾ 1.73 ⫻ 104 cells per 50 colonies). In contrast, however, no significant synergy was seen in response to IL-11. This was true for both colony number and colony size. This is illustrated in Figure 1, which shows results for eight individual experiments.

Table 1. Increased colony formation is not observed with addition of IL-11 to cultures of mpl⫺/⫺ bone marrow cells Stimulus 1

Wild-type

mpl⫺/⫺

Stimulus 2

Saline

SCF 10 ng/mL

IL-3 100U/mL

Saline ⫹IL-6 ⫹IL-11 Saline ⫹IL-6 ⫹IL-11

0⫾0 4⫾4 0⫾0 0⫾0 1⫾1 ND

3⫾4 26 ⫾ 7* 17 ⫾ 9* 1⫾1 8 ⫾ 2* 1⫾1

34 ⫾ 23 54 ⫾ 26* 55 ⫾ 25* 23 ⫾ 9 28 ⫾ 12† 21 ⫾ 10

Results are number of colonies (mean ⫾ standard deviation) per 50,000 bone marrow cells (n ⫽ 6–8 mice of each genotype per stimulus). Note the lack of response to IL-11 in cultures of mpl⫺/⫺ cells. ND: not done. *p ⬍ 0.0004, †p ⬍ 0.02 (paired t-test comparing cultures containing either SCF or IL-3 in the presence or absence of IL-6 and IL-11).

Figure 1. Lack of synergistic response to IL-11 in cultures of mpl⫺/⫺ bone marrow cells. Mean colony number for cultures of 50,000 bone marrow cells from mice with the genotypes shown. Cultures were stimulated with SCF (10 ng/mL) alone (the left point of each pair) or SCF plus either IL-6 or IL-11(the right point of each pair) (Panel A) or IL-3 (100 U/mL) alone plus either IL-6 or IL-11 (Panel B). Each line represents data from one mouse. * p ⬍ 0.0005, ** p ⬍ 0.02, n ⫽ 8 mice (paired t-test comparing cultures containing either SCF or IL-3 in the presence or absence of IL-6 and IL-11).

C.L. Scott et al./Experimental Hematology 29 (2001) 138–145

Thus, while a synergistic response in terms of colony number and colony size was evident with IL-6 for wild-type and mpl⫺/⫺ cells, there was no such response for mpl⫺/⫺ cells with IL-11. The absence of synergy with the addition of IL11 in mpl⫺/⫺ bone marrow cultures mimicked that observed in IL11Ra⫺/⫺ bone marrow cultures and in mpl⫺/⫺/ IL11Ra⫺/⫺ bone marrow cultures (Fig. 2). The failure to detect a response to IL-11 was not simply the consequence of the reduced numbers of colonies per dish in the cultures of mpl⫺/⫺ cells, as an IL-11 response was still clearly evident for wild-type cells when cultured under conditions designed to replicate this situation (data not shown). The synergistic activity of IL-6 and IL-11 affects predominantly megakaryocyte progenitors Experiments were performed to determine the types of colonies stimulated as a result of the synergistic activity of IL11 on wild-type cells, and therefore, by extrapolation, the types of colonies absent in mpl⫺/⫺ bone marrow. Stimulation of wild-type bone marrow cells with IL-11 or IL-6 increased the proportion of megakaryocyte colonies approximately threefold (Table 2). Given that stimulation with IL-11 or IL6 increased the total colony number by twofold, the absolute increase in megakaryocyte colonies was approximately sixfold and GM colonies also increased approximately fourfold in wild-type bone marrow cells. In contrast, there was

Figure 2. Lack of synergistic response to IL-11 in cultures of IL11Ra⫺/⫺ and IL11Ra⫺/⫺/mpl⫺/⫺ bone marrow cells. Mean colony number for cultures of 50,000 bone marrow cells from mice with the genotypes shown. Cultures were stimulated with IL-3 alone (the left point of each pair) or IL3 plus IL-11 (the right point of each pair). Each line represents data from one mouse. 첣: No significant increase in colony number observed with addition of IL-11.

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Table 2. Analysis of types of colonies stimulated by IL-6 or IL-11 Colony Type (%)

Wild-type IL-3 100/U/mL ⫹IL-6 ⫹IL-11 mpl⫺/⫺ IL-3 100 U/mL ⫹IL-6 ⫹IL-11

Colony Number

G

GM

Mac

Meg

38 ⫾ 14 70 ⫾ 24 74 ⫾ 19

27 ⫾ 11 17 ⫾ 5 21 ⫾ 4

9⫾6 26 ⫾ 10 15 ⫾ 5

43 ⫾ 10 24 ⫾ 7 28 ⫾ 5

11 ⫾ 2 27 ⫾ 3* 32 ⫾ 3*

40 ⫾ 18 52 ⫾ 23 42 ⫾ 22

6⫾2 13 ⫾ 5 14 ⫾ 5

14 ⫾ 5 18 ⫾ 11 16 ⫾ 8

53 ⫾ 5 36 ⫾ 14 47 ⫾ 5

17 ⫾ 4 24 ⫾ 9 18 ⫾ 4

Results are number of colonies (mean ⫾ standard deviation) per 50,000 bone marrow cells or percent of each colony type (n ⫽ 3–4 mice per genotype). Colony type was determined by examination of stained cultures at 100⫻ or 400⫻ magnification (⬎90 colonies scored per genotype). G; granulocyte, GM; granulocyte/macrophage, Mac; macrophage, Meg; megakaryocyte. *p ⬍ 0.01 for wild-type cultures.

no increase in the total number of colonies and no increase in the proportion of megakaryocyte- or GM-containing colonies when mpl⫺/⫺ cells were stimulated by IL-11 (Table 2). Taken together, these experiments demonstrated that there was no direct action of IL-11 on mpl⫺/⫺ hematopoietic cells. mpl⫺/⫺ mice respond to IL-11 administered in vivo Previous studies have demonstrated that mpl⫺/⫺ mice responded when IL-11 was administered in vivo [13]. Given the results presented, we sought to confirm these findings. IL-11 was administered intraperitoneally to wild-type and mpl⫺/⫺ mice twice daily for seven days. The following parameters were monitored: platelet count, spleen weight, bone marrow, and spleen GM-CFC and CFU-S. In wildtype mice an increase in all parameters was seen in response to IL-11 (Fig. 3A and C; Fig. 4). mpl⫺/⫺ mice responded to the administration of IL-11 with an increase in platelet count and in splenic weight (Fig. 3B and C). However, in mpl⫺/⫺ mice, increases in bone marrow CFC, splenic CFC, and CFU-S were not evident (Fig. 4). In addition, cells from mpl⫺/⫺ mice failed to show a “synergistic” response to IL11 in vitro, although such synergy was enhanced as a result of prior IL-11 treatment in wild-type animals (data not shown). Thus, there was inconsistency between the in vitro findings, where there was no evidence of a direct response to IL-11, and the in vivo findings, where a biological response to IL-11 with elevation of the platelet count was evident within seven days. In vitro responses of progenitor cells from mpl⫺/⫺ mice post–5-fluorouracil (5-FU) administration We sought to reconcile the in vitro and in vivo findings by determining whether a direct action of IL-11 could be demonstrated in vitro. We speculated that such an effect might be revealed as a result of “expansion” of the progenitor/stem

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Figure 3. In vivo response to IL-11 is observed in mpl⫺/⫺ mice. (A) Results for wild-type mice; (B) Results for mpl⫺/⫺ mice. Results shown for individual mice (9–11 mice per group). Pre: platelet count prior to treatment with either IL-11 or phosphate-buffered saline (PBS); Post: platelet count after treatment with either IL-11 or PBS. (C) Spleen weight postinjection with either PBS or IL-11. Results shown for individual mice (4–7 mice per group). * p ⬍ 0.002, ** p ⬍ 0.018, n ⫽ 5–11 mice (Paired t-test for pre- and postplatelet counts, Unpaired t-test for comparisons of spleen weight).

cell compartment associated with depletion of more mature hematopoietic cells. To this end, cohorts of wild-type and mpl⫺/⫺ mice were treated with 5-FU, 150 mg/kg, and individual mice were sacrificed daily and bone marrow was harvested for clonogenic assays. For wild-type bone marrow cells post–5-FU exposure, both IL-6 and IL-11–responsive populations were routinely detected (Table 3). The maximum number of GM-CFC and the maximum synergistic response was seen 10–17 days after 5-FU treatment. The kinetics of recovery of GM-CFC was similar for mpl⫺/⫺ mice. As before, in cultures of mpl⫺/⫺ cells, IL-6–responsive cells were clearly seen. In addition, there was an in vitro synergistic response to IL-11 post–5-FU. As with wild-type bone marrow cells, this was most evident 10–17 days after treatment with 5-FU: there was an increase in colony number with addition of IL-6 to SCF, and also following addition of IL-11 to SCF (time-course experiment, Table 3, and Day 13 and Day 15 from all experiments, Fig. 5). For wildtype cells, the synergistic action of IL-6 in this context resulted in a maximum 15-fold increase in colony formation compared with the effect of SCF alone. A sevenfold increase was seen with IL-11. For mpl⫺/⫺ cells, an increase

in colony number was also observed; addition of IL-6 to SCF resulted in a maximum fourfold increase in colony number compared to a maximum sixfold increase with IL-11. Thus, IL-11–responsive cells were clearly evident in mpl⫺/⫺ bone marrow.

Discussion A number of investigators have shown that while growth factors such as IL-3, SCF, and TPO can support the growth of hematopoietic progenitor cells in vitro, IL-6 and IL-11, while supporting little or no colony growth alone, can augment the response to these cytokines [34]. We used these observations to design a series of experiments to establish the lack of IL-11 response in IL11Ra⫺/⫺ bone marrow cells [22]. During these experiments we made a surprising observation. Bone marrow cells from mpl⫺/⫺ mice failed to respond to IL-11. This observation was therefore examined in more detail. Mice lacking TPO or its receptor c-mpl have deficits in both the megakaryocytic lineage [10–12] and in the progenitor cell compartments. [11,13–15]. These multilineage deficits are most evident in the more immature subpopulations.

C.L. Scott et al./Experimental Hematology 29 (2001) 138–145

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Table 3. Action of IL-11 on hematopoietic progenitor cells of mpl⫺/⫺ mice following treatment with 5-FU mpl⫺/⫺

Wild-type SCF Day 10 ng/mL 0 0⫾1 3 0⫾0 6 2⫾0 9 0⫾0 10 0⫾0 13 0⫾0 15 17 ⫾ 7 17 28 ⫾ 11 20 9⫾8 22 3⫾1

⫹IL-6

⫹IL-11

34 ⫾ 3 14 ⫾ 4 1⫾1 0⫾1 51 ⫾ 1 28 ⫾ 8 96 ⫾ 6 20 ⫾ 6 16 ⫾ 7 131 ⫾ 27 15 ⫾ 1 ND 67 ⫾ 1 52 ⫾ 17 77 ⫾ 10 65 ⫾ 24 63 ⫾ 9 43 ⫾ 14 31 ⫾ 4 17 ⫾ 1

SCF

⫹IL-6

⫹IL-11

10 ng/mL 0⫾0 0⫾1 0⫾0 0⫾0 1⫾1 0⫾0 12 ⫾ 2 4⫾1 4⫾1 6⫾1

4⫾2 3⫾1 4⫾3 20 ⫾ 1 3⫾0 3⫾3 22 ⫾ 3 14 ⫾ 0 10 ⫾ 2 17 ⫾ 7

0⫾0 2⫾1 0⫾1 4⫾4 3⫾1 ND 23 ⫾ 6 7⫾1 11 ⫾ 4 8⫾1

mpl⫺/⫺

Wild-type IL-3 Figure 4. In vivo response to IL-11 was not seen for all parameters in mpl⫺/⫺ mice. (A) GM-CFC cultured in SCF/IL-3. (B) Splenic GM-CFC cultured in GM-CSF/SCF/IL-3; Mean ⫾ SD of 3–4 mice per group. Cultures were incubated in 0.03% agar in 10% CO2 in air for 7 days and colonies were enumerated by examination of cultures at 35⫻ magnification. (C) CFU-S experiment: recipient spleen weight. (D) Day 12 CFU-S colonies; Mean ⫾ SD of 2–4 donors per group, each donor injected into 4–5 recipients. Error bars represent SD.

However, irradiated recipient mpl⫺/⫺ mice were able to support normal hematopoietic cells from wild-type bone marrow, suggesting that these deficits are intrinsic to mpl⫺/⫺ hematopoietic cells [14]. Data from several studies suggested that the progenitor cell potential of mpl⫺/⫺ mice was globally reduced, compared with wild-type mice. The in vitro response of mpl⫺/⫺ myeloid and erythroid bone marrow progenitor cells to a wide range of cytokine combinations was impaired [11,13]. In each case, however, responsive cell populations were evident in clonal culture. In this study we demonstrated a complete lack of synergistic response to IL-11 for mpl⫺/⫺ cells when cultured with IL-11 in combination with either SCF or IL-3. This contrasted with the synergistic response clearly demonstrable in the steady state for both wild-type and mpl⫺/⫺ cells in response to IL-6, a molecule closely related to IL-11 both in terms of receptor signaling properties and shared biological and hematopoietic function [34,35]. The lack of response to IL-11 by mpl⫺/⫺ cells conflicted with reports, confirmed in this study, of in vivo responses to IL-11 as evidenced by a rise in platelet count following IL-11 administration [12,13]. Although an in vivo response to IL-11 was not observed for all indices tested in mpl⫺/⫺ mice, this probably reflects the low basal level for some of these parameters in mpl⫺/⫺ mice. In particular, the in vivo response to IL-11 was not associated with the generation of IL-11–responsive cells in vitro. In wild-type mice, treatment with 5-FU resulted in “expansion” of the progenitor/“stem” cell compartment overall, as has been described previously [36], and “expansion” of

⫹IL-6

⫹IL-11

Day 100 U/mL 0 3⫾1 21 ⫾ 2 25 ⫾ 6 3 0⫾0 1⫾1 0⫾1 6 23 ⫾ 10 34 ⫾ 6 29 ⫾ 7 9 15 ⫾ 4 54 ⫾ 0 49 ⫾ 1 10 9 ⫾ 3 133 ⫾ 36 57 ⫾ 16 13 21 ⫾ 5 43 ⫾ 10 54 ⫾ 6 15 78 ⫾ 3 98 ⫾ 9 109 ⫾ 17 17 125 ⫾ 53 154 ⫾ 8 185 ⫾ 10 20 122 ⫾ 5 159 ⫾ 2 152 ⫾ 16 22 49 ⫾ 0 55 ⫾ 5 68 ⫾ 12

IL-3

⫹IL-6

100 U/mL 5⫾1 10 ⫾ 4 6⫾3 10 ⫾ 1 2⫾2 8⫾1 22 ⫾ 2 23 ⫾ 10 61 ⫾ 18 116 ⫾ 17 48 ⫾ 8 88 ⫾ 3 21 ⫾ 30 25 ⫾ 2 32 ⫾ 3 30 ⫾ 0 24 ⫾ 6 23 ⫾ 0 34 ⫾ 1 36 ⫾ 8

⫹IL-11 4⫾1 8⫾3 11 ⫾ 2 39 ⫾ 5 95 ⫾ 16 91 ⫾ 7 31 ⫾ 3 38 ⫾ 7 30 ⫾ 4 39 ⫾ 4

Time course experiment: results are number of GM-CSF colonies (mean ⫾ SD) per 50,000 bone marrow cells for one wild-type and one mpl⫺/⫺ mouse harvested on each day post injection of 5-FU. ND, not done.

the IL-11- and IL-6–responsive subsets in particular. Strikingly, when mpl⫺/⫺ mice were treated with 5-FU, IL-11– responsive progenitor cells were also observed. There are two potential explanations for this result. Firstly, IL-11–responsive hematopoietic progenitor cells were present, but at an undetectably low level in bone marrow from mpl⫺/⫺ mice. In this situation, the IL-11–responsive subset of progenitor/“stem” cells would require TPO signaling for in vivo expansion in steady state, but not for “expansion” following hematopoietic stress. Alternatively, it is possible that in the absence of c-mpl signaling, this subset of cells was not present at all in the steady state. However in contrast to the situation in IL11R⫺/⫺ animals, where IL-11– responsive cells are never detected, treatment with 5-FU provided a stimulus for hematopoietic cell “expansion” in mpl⫺/⫺ animals. Both models are consistent with data suggesting an important interaction between c-mpl and other thrombopoietic cytokines including IL-11 [9]. Although unable to distinguish between these possibilities, the synergistic response to IL-11 implies that a functional IL-11 receptor complex was present on the mpl⫺/⫺ cells [37,38]. Kaushansky et al. neutralized TPO signaling in in vitro progenitor cell assays by addition of an antibody, and re-

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Figure 5. Synergistic response to IL-11 seen in cultures of mpl⫺/⫺ bone marrow cells following 5-FU exposure. Mean colony number for cultures of 50,000 bone marrow cells from wild-type (open circles) and four mpl⫺/⫺ mice (closed circles) on day 13 and on day 15 following 5-FU exposure. Cultures were stimulated with SCF (10 ng/mL) alone or SCF plus either IL-6 or IL-11 (Panels A and B) or IL-3 (100 U/mL) alone plus either IL-6 or IL-11 (Panels C and D). Each line represents data from one mouse. * p ⬍ 0.004, ** p ⬍ 0.043, n ⫽ 4 mice (paired t-test comparing cultures of mpl⫺/⫺ bone marrow cells containing either SCF or IL-3 in the presence or absence of IL-6 and IL-11).

vealed the existence of a population of megakaryocytic progenitor cells that was absolutely dependent on the presence of TPO [9]. This TPO-dependent progenitor cell population, which comprised about 70% of megakaryocytic progenitors found in cultures of adult murine bone marrow, was absent in mpl⫺/⫺ mice. The deficit in in vitro response of mpl⫺/⫺ hematopoietic progenitor cells to IL-11 observed in this study suggests that it is this TPO-dependent component of the megakaryocytic progenitor cell population that is responsive to IL-11 stimulation. It is now well recognized that TPO, in addition to being the key growth factor for cells of the megakaryocytic lineage, also acts on hematopoietic stem cells and multipotential hematopoietic progenitor cells [2]. This study, using in vitro assays of mpl⫺/⫺ hematopoietic progenitor cells, is the first demonstration that, in the absence of TPO signaling, there is, in addition to the global deficit, a selective deficit in IL-11–responsive hematopoietic progenitor cells. Acknowledgments The authors thank Bette Papaevangeliou for technical assistance. This work was supported in part by the Anti-Cancer Council of

Victoria, the Bone Marrow Donor Institute, the Sylvia and Charles Viertel Charitable Foundation, National Institute of Health Grants nos. CA22556 and HL62275, the National Health and Medical Research Council of Australia, and the Cooperative Research Centre for Cellular Growth Factors.

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