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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Jegalian, A. G. Articles by Wu, H. Search for Related Content PubMed PubMed Citation Articles by Jegalian, A. G. Articles by Wu, H. Related Collections Hematopoiesis and Stem Cells Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 April 2002, Vol. 99, No. 7, pp. 2603-2605 BRIEF REPORT Erythropoietin receptor haploinsufficiency and in vivo interplay with granulocyte-macrophage colony-stimulating factor and interleukin 3 Armin G. Jegalian, Adriana Acurio, Glenn Dranoff, and Hong Wu From the Molecular Biology Institute, Department of Molecular and Medical Pharmacology, and Howard Hughes Medical Institute, University of California Los Angeles School of Medicine; and the Department of Adult Oncology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA.     Abstract Top Abstract Introduction Study design Results and discussion References Erythropoietin (EPO) and its receptor (EPOR) are critical for definitive erythropoiesis, as mice lacking either gene product die during embryogenesis with severe anemia. Here we demonstrate that mice expressing just one functional allele of the EpoR have lower hematocrits and die more frequently than do wild-type littermates on anemia induction. Furthermore, EpoR+/ erythroid colony-forming unit (CFU-E) progenitors are reduced both in frequency and in responsiveness to EPO stimulation. To evaluate the interaction between EPO and granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3), GM-CSF/ or IL-3/ mice were interbred with EpoR+/ mice. Deletion of either GM-CSF or IL-3 also leads to reduction in CFU-E numbers and hematocrits but does not significantly alter steady-state erythroid burst-forming unit numbers. These results suggest EpoR haploinsufficiency and promotion of in vivo erythropoiesis by GM-CSF and IL-3. (Blood. 2002;99:2603-2605) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Erythropoietin (EPO) and the EPO receptor (EPOR) are indispensable for the proliferation and survival of erythroid colony-forming unit (CFU-E) progenitors and their terminal differentiation into mature erythrocytes.1-3 Mice lacking either Epo or EpoR die at about embryonic day 13 with defects in definitive erythropoiesis and cardiac development.1-4 Furthermore, both types of knockout mice exhibit identical phenotypes, suggesting that no other ligands or receptors can replace EPO or the EPOR.1,5 In the steady state, both heterozygotes exhibit normal hematologic parameters, viability, and fertility. However, the compensatory capacity of one copy of Epo or EpoR in stress or emergency situations remained to be studied. In concert with EPO, other cytokines have been implicated in regulating erythropoiesis. Early experiments suggest that interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) primarily act on early multipotent progenitors to enhance the formation of the erythroid burst-forming unit (BFU-E) in the presence of EPO.6,7 In addition, ectopic expression of c (the subunit common to receptors for GM-CSF and IL-3) enhances EPO responsiveness in Ba/F3 cells transfected with EpoR, and a direct interaction between EPOR and c has been described by coimmunoprecipitation.8 In UT-7 cells, however, GM-CSF induces a rapid down-regulation of EpoR messenger RNA and, thus, opposes EPO activity.9 Furthermore, in vitro studies reveal that EPO counteracts bone marrow CFU-GM colony growth, whereas colony-stimulating factors may inhibit BFU-E colony growth.10-12 Although GM-CSF and IL-3 stimulate proliferation and differentiation of hematopoietic cells in vitro,13 gene inactivation studies suggest that neither factor is essential for steady-state hematopoiesis. Instead, GM-CSF is important in pulmonary homeostasis,14,15 whereas IL-3 plays a physiologically relevant role in delayed-type hypersensitivity16 and the generation of mast cells or basophils in response to parasites.17 Baseline hematopoiesis in mice lacking c, c and IL-3, or GM-CSF and IL-3, is substantially intact, although modest changes in eosinophil homeostasis have been observed, suggesting that the normalcy of hematopoiesis in the GM-CSF or IL-3 single knockout is not due to compensation by one cytokine for the lack of the other.18-20 To assess whether both EpoR alleles are required for animals to compensate for erythroid perturbation and to analyze the nature and extent of physiologically relevant interplay between the lineage-dominant EPO and the early-acting GM-CSF and IL-3 in erythropoiesis, GM-CSF- or IL-3-deficient mice were interbred with EpoR heterozygous mutant mice and subjected to hemolysis. Our results suggest that (1) EpoR is haploinsufficient in response to phenylhydrazine (PHZ) treatment and (2) GM-CSF and IL-3 promote, rather than oppose, EPO activity in vivo.     Study design Top Abstract Introduction Study design Results and discussion References Mice EpoR+/, IL-3/, and GM-CSF/ mice and genotyping have been described.1,14,16 Because the original mice were derived from C57BL/6/129 or BALB/c/129 F1 intercrossing, they were backcrossed for 7 generations to pure C57BL/6 mice to minimize the confounding effects of outbreeding. PHZ administration and hematocrit measurement PHZ (ICN Biomedicals, Aurora, OH), which causes erythrocyte lysis in vivo,21 was injected intraperitoneally (60 mg/kg). For hematocrit measurements, mice anesthetized with Avertin (Sigma Chemical, St Louis, MO; 0.015 mL 2.5% stock/g) were bled from the retro-orbital plexus (20-40 µL) with the use of heparin-coated capillary tubes, which were then spun in a microcentrifuge. Progenitor cell assays Cells were isolated from femurs or spleens, washed thrice in Iscoves modified Dulbecco media supplemented with 2% fetal calf serum, and counted according to Wu et al.1 Nucleated cells were plated at 100 000/mL in methylcellulose (0.9% wt/vol) (M3236; StemCell Technologies, Vancouver, BC) supplemented with 15% fetal calf serum (Omega, Tarzana, CA) and recombinant hEPO (Amgen, Thousand Oaks, CA) at the indicated concentrations. For BFU-E analysis, 1 U/mL rhEPO and 100 ng/mL stem cell factor were used. Colonies were scored according to Wu et al.1 Statistical analysis Statistical analysis was performed by using single-factor analysis of variance.     Results and discussion Top Abstract Introduction Study design Results and discussion References Anemia was induced in 6 groups of mice matched for genetic background, sex, age, and weight, with more than 30 animals per group: (1) wild type (WT), (2) EpoR+/, (3) GM-CSF/, (4) GM-CSF/, EpoR+/, (5) IL-3/, and (6) IL-3/, EpoR+/. Two PHZ injections were administered 48 hours apart, and hematocrit was recorded from blood collected on alternate days (Figure 1A). The differences in hematocrit among genotypes were highly reproducible and enhanced by the degree of anemia (Figure 1B). WT mice had the highest hematocrits, followed by GM-CSF/ and IL-3/ mice, followed by EpoR+/ mice. Deleting one allele of EpoR has a significantly greater effect on hematocrit than deleting 2 alleles of GM-CSF, suggesting that EPO/EPOR plays a more dominant role in erythropoiesis. IL-3/, EpoR+/ and GM-CSF/, EpoR+/ mice had the lowest hematocrits, and the day 5 hematocrits are likely overestimates, because mice in these 2 groups had the highest mortality (Figure 1C). Indeed, hematocrits of below 15% are often lethal, even in WT mice. View larger version (31K): [in this window] [in a new window]   Figure 1. Anemia induction and lethality in gene-targeted mice. (A) Schematic of anemia induction regime. (B) Hematocrits of 10- to 12-week-old, weight-matched female mice having undergone the aforementioned treatment. Results (average ± SEM) are based on 5 separate experiments using at least 5 mice per genotype per experiment. Compared with same-day WT hematocrit, * and ** denote P < .05 and < .005, respectively. # represents P < .05 for IL-3/;EpoR+/ or GM-CSF/;EpoR+/, when compared with EpoR+/ hematocrit; % and @ represent P < .05 when comparing WT mice with mice lacking GM-CSF or IL-3, respectively. (C) Survival rates after PHZ injection. Results are based on the cumulative studies of at least 30 mice per genotype. We next examined bone marrow erythroid progenitor cells from the aforementioned mice without PHZ treatment. In addressing the haploinsufficiency of EpoR, various concentrations of EPO were used (Figure 2A). CFU-E-derived colonies occurred for WT cells at a 60% greater frequency than for EpoR+/ cells at the highest EPO concentration (1 U/mL) but at a 150% greater frequency at the lowest EPO concentration tested (0.03 U/mL). Similar trends were noted with spleen-derived erythroid progenitors (Figure 2B). These results suggest that CFU-E progenitors are not only fewer but also less responsive to EPO in EpoR+/ mice compared with WT mice. BFU-E numbers did not vary between WT and EpoR+/ (Figure 2C), in agreement with previous findings that EPO promotes the transition from the BFU-E to CFU-E stage.1,2 View larger version (38K): [in this window] [in a new window]   Figure 2. In vitro colony formation of erythroid progenitors from mice of the indicated genotypes. Numbers refer to scored colonies/105 nucleated bone marrow cells from the femurs (A,C,D) or spleens (B) of 10- to 12-week-old female mice. Results are based on 3 sets of independent experiments per study. (A) CFU-E frequencies, for either WT (black) or EpoR+/ (white) bone marrow cells, grown in the indicated concentration of rhEPO. * and ** denote P < .05 and < .005, respectively, when each value is compared with the same genotype value with 1.0 U/mL EPO. In comparing WT with EpoR+/, P < .002 at each EPO concentration. (B) CFU-E frequencies, spleen. Statistical analysis was performed and indicated as in part A. (C) BFU-E frequencies, bone marrow. (D) CFU-E frequencies, bone marrow. Compared with WT values at the same EPO concentration, * and ** denote P < .05 and < .005, respectively. % and @ represent P < .05 when comparing WT mice with mice lacking GM-CSF or IL-3, respectively. We then tested the effects of disrupting either GM-CSF or IL-3 on BFU-E and CFU-E rates. BFU-E frequencies do not vary significantly (Figure 2C), although with BFU-E values so small, significant changes might not be discernible. In contrast, CFU-E frequencies were reduced, either on WT or EpoR+/ background, especially at the minimal EPO concentration (0.03 U/mL) (Figure 2D), at which the ratio is 0.4 in EpoR+/ versus WT, 0.44 in EpoR+, GM-CSF/ versus GM-CSF/, and 0.44 in EpoR+/;IL-3/ versus IL-3/. Thus, we conclude that GM-CSF and IL-3 support, rather than oppose, steady-state CFU-E progenitor formation in vivo and may potentially do so in an additive manner with EPO. Together, our results indicate that both copies of EpoR are required for emergent erythropoiesis and that responsiveness of CFU-E progenitors to EPO stimulation depends on EPOR dosage. Whereas the reduction in CFU-E frequency in EpoR+/ mice is dramatic, the effect on hematocrit is relatively mild, suggestive of compensation for reduced CFU-E levels in the animal. These results also highlight the importance of physiologically challenging genetically engineered mice to discern functional deficiencies. Furthermore, we have shown that GM-CSF and IL-3 appear to promote erythropoiesis in vivo. The role of GM-CSF or IL-3 in erythropoiesis and the haploinsufficiency of EpoR have clinical implications for EPO therapy in combination with other growth factors or for individuals with defective EPOR function.22     Acknowledgments We thank Dr Harvey Lodish for supporting the initial phase of this study.     Footnotes Submitted September 14, 2001; accepted November 13, 2001. Supported in part by the Medical Scientist Training Program (A.G.J.), the Leukemia and Lymphoma Society (G.D.), the Howard Hughes Medical Institute (H.W.), and the V Foundation and CA74886. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Hong Wu, Howard Hughes Medical Institute and Department of Molecular and Medical Pharmacology, UCLA School of Medicine, CHS 23-214, Los Angeles, CA 90095-1735; e-mail: hwu{at}mednet.ucla.edu.     References Top Abstract Introduction Study design Results and discussion References 1. Wu H, Liu X, Jaenisch R, Lodish HF. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell. 1995;83:59-67[CrossRef][Medline] [Order article via Infotrieve]. 2. Lin CS, Lim SK, D'Agati V, Costantini F. Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis. Genes Dev. 1996;10:154-164[Abstract/Free Full Text]. 3. Kieran MW, Perkins AC, Orkin SH, Zon LI. Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor. Proc Natl Acad Sci U S A. 1996;93:9126-9131[Abstract/Free Full Text]. 4. 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Nishinakamura R, Miyajima A, Mee PJ, Tybulewicz VL, Murray R. Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions. Blood. 1996;88:2458-2464[Abstract/Free Full Text]. 19. Scott CL, Robb L, Papaevangeliou B, Mansfield R, Nicola NA, Begley CG. Reassessment of interactions between hematopoietic receptors using common beta-chain and interleukin-3-specific receptor beta-chain-null cells: no evidence of functional interactions with receptors for erythropoietin, granulocyte colony-stimulating factor, or stem cell factor. Blood. 2000;96:1588-1590[Abstract/Free Full Text]. 20. Gillessen S, Mach N, Small C, Mihm M, Dranoff G. Overlapping roles for granulocyte-macrophage colony-stimulating factor and interleukin-3 in eosinophil homeostasis and contact hypersensitivity. Blood. 2001;97:922-928[Abstract/Free Full Text]. 21. Rencricca NJ, Rizzoli V, Howard D, Duffy P, Stohlman F Jr. Stem cell migration and proliferation during severe anemia. Blood. 1970;36:764-771[Abstract/Free Full Text]. 22. Goyal RK, Longmore GD. Abnormalities of cytokine receptor signalling contributing to diseases of red blood cell production. Ann Med. 1999;31:208-216[Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: Y. Liu, R. Pop, C. Sadegh, C. Brugnara, V. H. Haase, and M. Socolovsky Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo Blood, July 1, 2006; 108(1): 123 - 133. [Abstract] [Full Text] [PDF] G. Terszowski, C. Waskow, P. Conradt, D. Lenze, J. Koenigsmann, D. Carstanjen, I. Horak, and H.-R. 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