The Experts below are selected from a list of 11118 Experts worldwide ranked by ideXlab platform
Hal E. Broxmeyer - One of the best experts on this subject based on the ideXlab platform.
-
regulation of Myeloid Progenitor Cell proliferation survival by il 31 receptor and il 31
Experimental Hematology, 2007Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Nico Ghilardi, Wen Tao, Charlie Mantel, Barbara Grahamevans, Frederic J De SauvageAbstract:Objective Interleukin (IL)-31 is a recently discovered helical cytokine. Its receptor consists of a ligand-specific IL-31 receptor (IL-31R) subunit and a receptor chain that is shared with Oncostatin M (OSM), called OSM-Rβ. Because OSM-Rβ–deficient animals have reduced hematopoietic Progenitor Cells (HPC) and OSM has effects on and is involved in homeostasis of HPC, we studied whether IL-31 and IL-31R play a role in hematopoiesis. Materials and Methods IL-31R −/− mice and their littermate wild-type (WT) controls were assessed for absolute numbers and cycling status of bone marrow and spleen HPC (colony-forming unit granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], colony-forming unit granulocyte, erythrocyte, macrophage, megakaryocyte). Recombinant IL-31 was evaluated for stimulation, enhancement, or inhibition of colony formation by HPC, and for survival-enhancing effects on HPC subjected to growth-factor withdrawal and delayed addition of grown factors. Hematopoietic stem Cells (HSC) from WT and IL-31R −/− mice were compared for competitive repopulating capacity in lethally irradiated congenic mice. Results IL-31R −/− mice demonstrated significantly decreased absolute numbers and cycling status of immature subsets of HPC in bone marrow bone and spleen compared to WT mice. There were no significant differences in absolute numbers of more mature subsets of WT and IL-31R −/− bone marrow CFU-GM. WT but not IL-31R −/− bone marrow CFU-GM responded to synergistic stimulation by combinations of cytokines. While IL-31 had neither colony-stimulating, -enhancing, or -inhibiting activity for bone marrow HPC, it did enhance survival of these HPC in the context of delayed addition of growth factors. No significant differences were detected in competitive repopulating HSC activity between WT and IL-31R −/− bone marrow Cells. Conclusion IL-31R is involved in positive regulation of absolute numbers and cycling status of immature subsets of HPC in vivo. While IL-31 in vitro does not modulate proliferation of HPC, it does enhance their survival, which may contribute to effects on cycling and numbers of HPC in vivo. Under steady-state conditions, loss of IL-31R on HPC does not appear to influence the activity of competitive repopulating HSC. These results with HPC may be of future utility for manipulation of hematopoiesis in a preclinical setting.
-
IL-31 Receptor and IL-31 in the Positive Regulation of Myeloid Progenitor Cell Proliferation/Survival.
Blood, 2005Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Nico Ghilardi, Wen Tao, Charlie Mantel, Barbara Graham-evans, Frederic DesauvageAbstract:Interleukin (IL)-31 receptor (R), also called gp130-like monocyte-receptor (GLM-R; Ghilardi et al. J. Biol. Chem.277:16831, 2002) is related to gp130 (~25% homology), and G-CSF-R (~24%). Its signaling activates STAT3 and STAT5. IL-31 is a four helix bundle cytokine preferentially produced by T helper 2 Cells. Nothing is known of the possible hematopoietic effects of IL-31R and IL-31. However, since: IL-31 signals through a receptor composed of IL-31RA and Oncostatin M R (Dillon et al. Nature Immunol. 5:752,2004), Oncostatin M is implicated in homeostasis of Myeloid Progenitor Cells (Broxmeyer et al. Immunity.16:815, 2002), and STAT3 and 5 are implicated by a number of groups in cytokine regulation of hematopoiesis, we hypothesized that the IL-31/IL-31R axis would be involved in regulation of hematopoiesis. We first compared Myeloid Progenitor Cell (MPC: CFU-GM, BFU-E, and CFU-GEMM) numbers and cycling status in marrow and spleen of IL-31R −/− vs. littermate control mice (+/+) using a combination of Epo, SCF and PWMSCM to stimulate in vitro the Cells taken from these mice. IL-31R −/− mice had significantly decreased numbers of MPC per femur (~51%) and spleen (~36–45%) with significantly decreased MPC cycling status in marrow (% MPC in S-phase: 0–3% in IL-31R −/− vs. 41–53% in +/+ mice). MPC in spleen of IL-31 −/− and +/+ were both in a slow or non cycling state (0–3% in S-phase). In contrast to CFU-GM from +/+ mice, CFU-GM from IL-31R −/− mice demonstrated little or no synergistic response to combined stimulation in vitro with GM-CSF or IL-3 with either SCF or Flt3-ligand (FL). This translated to decreased absolute numbers per femur of GM-CSF+SCF-, GM-CSF+FL-, IL-3+SCF-, and IL-3+FL- responsive CFU-GM in IL-31R −/− mice. However, there were no significant differences between GM-CSF- or IL-3- responsive CFU-GM per femur between IL-31R −/− vs. +/+ mice suggesting effects on immature subsets of MPC. Recombinant IL-31 was assessed for effects in vitro. IL-31, at concentrations up to 100ng/ml, did not stimulate colony formation by marrow MPC, nor did it enhance or suppress colony formation stimulated by GM-CSF, Epo, Epo+SCF, or Epo+SCF+GM-CSF. However, IL-31 did enhance survival of MPC subjected to delayed growth factor addition in a manner similar to, but not as potent as, that of SDF-1/CXCL12. IL-31 manifested no chemotaxis activity for +/+ MPC, and IL-31R −/− and +/+ MPC were equally responsive to the chemotactic effects of SDF-1/CXCL12. These results suggest that the IL-31R in vivo acts to positively regulate numbers and cycling of immature subsets of MPC in the marrow, and that IL-31 has survival enhancing effects on MPC in vitro.
-
il 31 receptor and il 31 in the positive regulation of Myeloid Progenitor Cell proliferation survival
Blood, 2005Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Nico Ghilardi, Wen Tao, Charlie Mantel, Barbara Grahamevans, Frederic DesauvageAbstract:Interleukin (IL)-31 receptor (R), also called gp130-like monocyte-receptor (GLM-R; Ghilardi et al. J. Biol. Chem.277:16831, 2002) is related to gp130 (~25% homology), and G-CSF-R (~24%). Its signaling activates STAT3 and STAT5. IL-31 is a four helix bundle cytokine preferentially produced by T helper 2 Cells. Nothing is known of the possible hematopoietic effects of IL-31R and IL-31. However, since: IL-31 signals through a receptor composed of IL-31RA and Oncostatin M R (Dillon et al. Nature Immunol. 5:752,2004), Oncostatin M is implicated in homeostasis of Myeloid Progenitor Cells (Broxmeyer et al. Immunity.16:815, 2002), and STAT3 and 5 are implicated by a number of groups in cytokine regulation of hematopoiesis, we hypothesized that the IL-31/IL-31R axis would be involved in regulation of hematopoiesis. We first compared Myeloid Progenitor Cell (MPC: CFU-GM, BFU-E, and CFU-GEMM) numbers and cycling status in marrow and spleen of IL-31R −/− vs. littermate control mice (+/+) using a combination of Epo, SCF and PWMSCM to stimulate in vitro the Cells taken from these mice. IL-31R −/− mice had significantly decreased numbers of MPC per femur (~51%) and spleen (~36–45%) with significantly decreased MPC cycling status in marrow (% MPC in S-phase: 0–3% in IL-31R −/− vs. 41–53% in +/+ mice). MPC in spleen of IL-31 −/− and +/+ were both in a slow or non cycling state (0–3% in S-phase). In contrast to CFU-GM from +/+ mice, CFU-GM from IL-31R −/− mice demonstrated little or no synergistic response to combined stimulation in vitro with GM-CSF or IL-3 with either SCF or Flt3-ligand (FL). This translated to decreased absolute numbers per femur of GM-CSF+SCF-, GM-CSF+FL-, IL-3+SCF-, and IL-3+FL- responsive CFU-GM in IL-31R −/− mice. However, there were no significant differences between GM-CSF- or IL-3- responsive CFU-GM per femur between IL-31R −/− vs. +/+ mice suggesting effects on immature subsets of MPC. Recombinant IL-31 was assessed for effects in vitro. IL-31, at concentrations up to 100ng/ml, did not stimulate colony formation by marrow MPC, nor did it enhance or suppress colony formation stimulated by GM-CSF, Epo, Epo+SCF, or Epo+SCF+GM-CSF. However, IL-31 did enhance survival of MPC subjected to delayed growth factor addition in a manner similar to, but not as potent as, that of SDF-1/CXCL12. IL-31 manifested no chemotaxis activity for +/+ MPC, and IL-31R −/− and +/+ MPC were equally responsive to the chemotactic effects of SDF-1/CXCL12. These results suggest that the IL-31R in vivo acts to positively regulate numbers and cycling of immature subsets of MPC in the marrow, and that IL-31 has survival enhancing effects on MPC in vitro.
-
transgenic expression of stromal Cell derived factor 1 cxc chemokine ligand 12 enhances Myeloid Progenitor Cell survival antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo
Journal of Immunology, 2003Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Charlie Mantel, Lisa L Kohli, Young Hee Lee, Wade D Clapp, Chang H KimAbstract:Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/Progenitor Cells. Exogenously added stromal Cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of Myeloid Progenitor Cells in vitro. To further evaluate SDF-1/CXCL12 effects on Progenitor Cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid Progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic Myeloid Progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and Gαi proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse Myeloid Progenitors. Consistent with in vitro results, Progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased Progenitor Cell cycling and significant increases in Progenitor Cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to Progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to Cell trafficking.
-
Regulation of Myeloid Progenitor Cell proliferation by STAT4- and STAT6-dependent CD4+ t helper Cell populations
Experimental Hematology, 2000Co-Authors: Mark H. Kaplan, S. Cooper, Giao Hangoc, Hal E. BroxmeyerAbstract:Abstract Signal Transducer and Activator of Transcription (STAT) proteins mediate growth, differentiation and gene activation following stimulation with cytokines. Gene deficient mice demonstrated that Stat4 is required for IL-12 stimulated functions including proliferation, interferon-γ induction and the development of T helper type 1 (Th1) Cells. Stat6-deficient mice lack IL-4 induced gene expression, proliferation and the differentiation of Th2 Cells. The absence of these genes also affects Myeloid Progenitor Cell function. In the absence of Stat4, there were decreases in the absolute numbers of CFU-GM, CFU-GEMM and BFU-E compared to control mice especially in the spleen. This was associated with decreased cycling (as determined by high specific activity 3 H-thymidine kill assay) of the Progenitor Cells. In contrast, mice deficient in Stat6 showed increased numbers of bone marrow and splenic CFU-GM, CFU-GEMM and BFU-E. These increases were associated with increases in cycling of the Progenitor Cells. Since Stat4 and Stat6 are required for reciprocal T helper Cell subsets, we speculated that alterations in Myeloid Progenitor numbers and cycling may be due to imbalances in Th Cell activity in Stat4-(Th2 only) and Stat6-(Th1 only) deficient mice. To test this, we depleted CD4+ or CD8+ Cells in vivo in Stat6-deficient mice using monoclonal antibodies. Treatment of Stat6-deficient mice with anti-CD4 resulted in Progenitor Cell numbers and cycling that were reduced below that observed in wildtype Cells. By contrast, anti-CD8 treated animals showed no decrease, and in some assays increases in Progenitor numbers and rats of cycling. Thus, CD4+ Cells positively regulate Myeloid Progenitor function in vivo. Stat6-deficient Progenitor Cells were as much as 10-fold more sensitive to co-stimulation with Steel factor in combination with GM-CSF. This heightened sensitivity was also diminished with in vivo anti-CD4 treatment, suggesting that one mechanism by which T Cells regulate Myeloid Progenitors is by altering responses to stimulatory cytokines. This suggest that Th1 Cells positively regulate Myeloid Progenitor Cells, which is consistent with the role of Th1 Cells in vivo in the regulation of inflammatory responses.
M Y Gordon - One of the best experts on this subject based on the ideXlab platform.
-
influence of pi 3k akt pathway on wnt signalling in regulating Myeloid Progenitor Cell proliferation evidence for a role of autocrine paracrine wnt regulation
British Journal of Haematology, 2009Co-Authors: Georgios Nteliopoulos, Stephen B Marley, M Y GordonAbstract:Summary The regulation of Myeloid Progenitor Cell (granulocyte-macrophage colonyforming units, CFU-GM) proliferation/differentiation by the Wnt and phosphatidylinositol-3 kinase (PI-3K) pathways was investigated using a colony-replating assay. The PI-3K pathway promoted differentiation of interleukin-3 (IL-3)-stimulated myelopoiesis via Akt, because inhibition of the PI-3K/Akt pathway with LY294002 or SH-5 increased proliferation. The involvement of canonical and non-canonical Wnt pathways was investigated using Wnt3a and Wnt5a respectively. Addition of the recombinant Wnts to IL-3 increased CFU-GM proliferation. Dkk-1, when combined with the Wnt proteins, abrogated the effects of Wnt3a but not Wnt5a. Surprisingly, the addition of Dkk-1 to LY294002 or SH-5 blocked their proliferative effects. We hypothesized that increased proliferation induced by PI-3K/Akt inhibitors was not mediated by downstream activation of the Wnt pathway but by induced endogenous production/release of Wnt proteins. The addition of SH-5 to IL-3 created an autocrine Wnt loop in CD34 + Cells, resulting in the phosphorylation of lipoprotein-receptor-related-protein 6. Furthermore, the addition of medium conditioned by CD34 + Cells cultured in IL-3 + SH-5 to IL-3 increased CFU-GM proliferation. This effect was abrogated by Dkk-1, suggesting that a Wnt in the conditioned medium increased proliferation. In summary, IL-3 via the PI-3K pathway promoted differentiation of Myeloid Progenitor Cells through a decrease of endogenous Wnt production/release.
-
influence of pi 3k akt pathway on wnt signalling in Myeloid Progenitor Cells evidence for a role of autocrine paracrine wnt regulation in cfu gm proliferation
Blood, 2008Co-Authors: Georgios Nteliopoulos, Stephen B Marley, M Y GordonAbstract:We have used a colony replating assay in conjunction with manipulation of the Wnt and PI-3K pathways to investigate regulation of Myeloid Progenitor Cell proliferation/differentiation. We found that PI-3 kinase pathway via Akt acts as a proliferative brake and promotes differentiation in IL3-driven myelopoiesis, since inhibition of PI-3K/Akt pathway with LY294002 (PI-3 kinase inhibitor) and SH-5 (Akt inhibitor) increased proliferation (reduced differentiation). We investigated the involvement of Wnt signalling in CFU-GM proliferation by using exogenous recombinant canonical Wnt3a and non-canonical Wnt5a. We showed that both of the Wnt members cannot support colony growth alone, but when added to IL3 they increased proliferation potential compared with the IL3 control, indicating an involvement of canonical Wnt/β-catenin and non-canonical Wnt pathways in the Myeloid Progenitor Cell proliferation. Immunoblotting analysis indicated that Wnt5a acts independently of β-catenin. Dkk-1 (Wnt-pathway inhibitor) alone did not affect IL-3 dependent proliferation but when combined with recombinant Wnts as expected it abrogated the effects of Wnt3a but not Wnt5a (acts as canonical-Wnt inhibitor). This was confirmed by β-catenin protein levels. Surprisingly, when Dkk-1 was added to LY294002 or SH-5, it blocked their proliferative effects. Dkk-1 acts as functional Wnt-receptor disabler and the finding that it blocks proliferation induced by PI-3K/Akt inhibitors’ indicates a link between the PI-3K and the Wnt signalling pathways. We hypothesised that increased proliferation induced by LY294002 or SH-5 was not mediated by downstream activation of the Wnt pathway but by induced endogenous expression of Wnt proteins and activation of the surface receptor. We conclude that there is a production of endogenous Wnt proteins that increases proliferation. Endogenous Wnt production has been reported in primitive haematopoietic Cells so there is potential for a paracrine or autocrine role for these Cell regulators. We tested this hypothesis in CD34 + Cells and found the addition of SH-5 to IL3 creates an autocrine loop of endogenous Wnt production, which results in the phosphorylation and activation of the LRP6 receptor and the initiation of the canonical signalling pathway. Furthermore, the conditioned medium of cultured CD34 + Cells was concentrated by using filtration devices (30kD cut-off) and added to IL3 to support the growth of Progenitors of another sample in a CFU-GM assay. We indicated that Wnt production and secretion can act in a paracrine way as well, since IL3+SH-5 conditioned medium increased the proliferative index of CFU-GM Cells whereas IL3 conditioned medium did not have significant effect. Dkk-1 abrogated the IL3+SH-5 conditioned medium’s induced proliferation, suggesting that the growth factors that had the proliferative effects were Wnt members. In conclusion, our data suggest that IL3 via PI-3K pathway promotes differentiation of Progenitor Myeloid Cells through inhibition of endogenous Wnt production.
-
chronic Myeloid leukaemia stem Cell derived but Progenitor Cell driven
Clinical Science, 2005Co-Authors: Stephen B Marley, M Y GordonAbstract:The biology of CML (chronic Myeloid leukaemia) has been extensively investigated as the disease is a paradigm of neoplasms induced when a translocation results in expression of a novel fusion protein, in this instance p210 BCR-ABL . Although CML manifests itself principally as unregulated expansion of the Myeloid lineage, the lesion is present in the stem Cell population and it has long been assumed that disregulated stem Cell kinetics must underlie the basic pathology of the disease. In this review, we present evidence that, in normal haemopoiesis, less primitive precursor Cells retain considerable flexibility in their capacity to undergo self-renewal, allowing them to maintain lineage-specific homoeostasis without inflicting proliferative stress upon the stem Cell population. This mechanism is dysregulated in CML and we have developed a self-renewal assay for CFU-GM (colony-forming unit-granulocyte/macrophage) which demonstrates that, in CML, the PI (proliferative index) of the Myeloid Progenitor Cell population is increased. The ability to measure the PI as an endpoint of p210 BCR-ABL expression gives considerable versatility to the in vitro investigation of putative therapeutic regimes in CML.
-
opposing effects of pi3 kinase pathway activation on human Myeloid and erythroid Progenitor Cell proliferation and differentiation in vitro
Experimental Hematology, 2004Co-Authors: J. L. Lewis, Stephen B Marley, Morenike Ojo, M Y GordonAbstract:Abstract Objective To investigate 1) the effects of lineage-specific cytokines (G-CSF and EPO) combined with ligands for different classes of cytokine receptors (common β chain, gp130, and tyrosine kinase) on proliferation by human Myeloid and erythroid Progenitor Cells; and 2) the signal transduction pathways associated with combinatorial cytokine actions. Materials and methods CFU-GM and BFU-E were cloned in vitro. Secondary colony formation by replated CFU-GM and subcolony formation by BFU-E provided measures of Progenitor Cell proliferation. Studies were performed in the presence of cytokine combinations with and without signal transduction inhibitors. Results Proliferation by CFU-GM and BFU-E was enhanced synergistically when common β chain receptor cytokines (IL-3 or GM-CSF) were combined with G-CSF or EPO, but not with gp130 receptor cytokines (LIF or IL-6) or tyrosine kinase receptor cytokines (SCF, HGF, Flt-3 ligand, or PDGF). Delayed addition studies with G-CSF+IL-3 and EPO+IL-3 demonstrated that synergy required the presence of both cytokines from the initiation of the culture. The Jak2-specific inhibitor, AG490, abrogated the effect of combining IL-3 with EPO but had no effect on the enhanced CFU-GM proliferation stimulated by IL-3+G-CSF. The PI3 kinase inhibitors LY294002 and wortmannin substituted for G-CSF in combination with IL-3 since proliferation in the presence of LY294002/wortmannin+IL-3 was enhanced to the same extent as in the presence of G-CSF+IL-3. In contrast, LY294002 and wortmannin inhibited proliferation in the presence of EPO and in the presence of EPO+IL-3. Conclusion 1) IL-3 may activate different signal transduction pathways when combined with G-CSF and when combined with EPO; 2) different signal transducing intermediates regulate erythroid and Myeloid Progenitor Cell proliferation; and 3) inhibition of the PI3 kinase pathway suppresses Myeloid Progenitor Cell differentiation and thereby increases proliferation.
-
a role for the fas fas ligand apoptotic pathway in regulating Myeloid Progenitor Cell kinetics
Experimental Hematology, 2002Co-Authors: Faris Q. Alenzi, Stephen B Marley, J. L. Lewis, Anil Chandrashekran, Anthony N. Warrens, John M. Goldman, M Y GordonAbstract:Abstract Objective Bone marrow from wild-type mice and mice with mutated Fas ( lpr ) or mutated Fas ligand ( gld ) was used to investigate the role of the Fas/FasL system in the regulation of Myeloid Progenitor Cell kinetics. Methods Granulocyte-macrophage colony-forming Cells (CFU-GM) were measured by a standard colony assay and the proliferative activity of CFU-GM was measured by replating primary colonies and observing secondary colony formation. Fas expression was restored to lpr mouse bone marrow Cells by retrovirus-mediated gene transfer and gld mouse marrow Cells were treated with soluble FasL. Wild-type marrow Cells were treated with YVAD (a caspase inhibitor) or anti-Fas monoclonal antibodies. Results There were greater frequencies of Myeloid Progenitor Cells (CFU-GM) in lpr and gld mouse marrow compared to wild-type (WT) marrow (p = 0.0008). The proliferative capacity of CFU-GM was also significantly greater for lpr and gld CFU-GM compared to WT CFU-GM (p = 0.0003 and 0.0001, respectively). Retrovirus-mediated restoration of Fas into lpr marrow, and provision of soluble FasL (sFasL) to gld CFU-GM reduced CFU-GM proliferation to WT levels. Treatment of WT CFU-GM with YVAD or anti-FasL monoclonal antibody increased CFU-GM proliferation to the levels found in lpr and gld CFU-GM. YVAD significantly increased and anti-Fas significantly reduced the proliferative capacity of human CFU-GM (p = 0.015 and 0.04, respectively). Conclusion Fas, FasL, and caspase activation may play an important role in regulating Myeloid Progenitor Cell kinetics.
Scott Cooper - One of the best experts on this subject based on the ideXlab platform.
-
regulation of Myeloid Progenitor Cell proliferation survival by il 31 receptor and il 31
Experimental Hematology, 2007Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Nico Ghilardi, Wen Tao, Charlie Mantel, Barbara Grahamevans, Frederic J De SauvageAbstract:Objective Interleukin (IL)-31 is a recently discovered helical cytokine. Its receptor consists of a ligand-specific IL-31 receptor (IL-31R) subunit and a receptor chain that is shared with Oncostatin M (OSM), called OSM-Rβ. Because OSM-Rβ–deficient animals have reduced hematopoietic Progenitor Cells (HPC) and OSM has effects on and is involved in homeostasis of HPC, we studied whether IL-31 and IL-31R play a role in hematopoiesis. Materials and Methods IL-31R −/− mice and their littermate wild-type (WT) controls were assessed for absolute numbers and cycling status of bone marrow and spleen HPC (colony-forming unit granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], colony-forming unit granulocyte, erythrocyte, macrophage, megakaryocyte). Recombinant IL-31 was evaluated for stimulation, enhancement, or inhibition of colony formation by HPC, and for survival-enhancing effects on HPC subjected to growth-factor withdrawal and delayed addition of grown factors. Hematopoietic stem Cells (HSC) from WT and IL-31R −/− mice were compared for competitive repopulating capacity in lethally irradiated congenic mice. Results IL-31R −/− mice demonstrated significantly decreased absolute numbers and cycling status of immature subsets of HPC in bone marrow bone and spleen compared to WT mice. There were no significant differences in absolute numbers of more mature subsets of WT and IL-31R −/− bone marrow CFU-GM. WT but not IL-31R −/− bone marrow CFU-GM responded to synergistic stimulation by combinations of cytokines. While IL-31 had neither colony-stimulating, -enhancing, or -inhibiting activity for bone marrow HPC, it did enhance survival of these HPC in the context of delayed addition of growth factors. No significant differences were detected in competitive repopulating HSC activity between WT and IL-31R −/− bone marrow Cells. Conclusion IL-31R is involved in positive regulation of absolute numbers and cycling status of immature subsets of HPC in vivo. While IL-31 in vitro does not modulate proliferation of HPC, it does enhance their survival, which may contribute to effects on cycling and numbers of HPC in vivo. Under steady-state conditions, loss of IL-31R on HPC does not appear to influence the activity of competitive repopulating HSC. These results with HPC may be of future utility for manipulation of hematopoiesis in a preclinical setting.
-
IL-31 Receptor and IL-31 in the Positive Regulation of Myeloid Progenitor Cell Proliferation/Survival.
Blood, 2005Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Nico Ghilardi, Wen Tao, Charlie Mantel, Barbara Graham-evans, Frederic DesauvageAbstract:Interleukin (IL)-31 receptor (R), also called gp130-like monocyte-receptor (GLM-R; Ghilardi et al. J. Biol. Chem.277:16831, 2002) is related to gp130 (~25% homology), and G-CSF-R (~24%). Its signaling activates STAT3 and STAT5. IL-31 is a four helix bundle cytokine preferentially produced by T helper 2 Cells. Nothing is known of the possible hematopoietic effects of IL-31R and IL-31. However, since: IL-31 signals through a receptor composed of IL-31RA and Oncostatin M R (Dillon et al. Nature Immunol. 5:752,2004), Oncostatin M is implicated in homeostasis of Myeloid Progenitor Cells (Broxmeyer et al. Immunity.16:815, 2002), and STAT3 and 5 are implicated by a number of groups in cytokine regulation of hematopoiesis, we hypothesized that the IL-31/IL-31R axis would be involved in regulation of hematopoiesis. We first compared Myeloid Progenitor Cell (MPC: CFU-GM, BFU-E, and CFU-GEMM) numbers and cycling status in marrow and spleen of IL-31R −/− vs. littermate control mice (+/+) using a combination of Epo, SCF and PWMSCM to stimulate in vitro the Cells taken from these mice. IL-31R −/− mice had significantly decreased numbers of MPC per femur (~51%) and spleen (~36–45%) with significantly decreased MPC cycling status in marrow (% MPC in S-phase: 0–3% in IL-31R −/− vs. 41–53% in +/+ mice). MPC in spleen of IL-31 −/− and +/+ were both in a slow or non cycling state (0–3% in S-phase). In contrast to CFU-GM from +/+ mice, CFU-GM from IL-31R −/− mice demonstrated little or no synergistic response to combined stimulation in vitro with GM-CSF or IL-3 with either SCF or Flt3-ligand (FL). This translated to decreased absolute numbers per femur of GM-CSF+SCF-, GM-CSF+FL-, IL-3+SCF-, and IL-3+FL- responsive CFU-GM in IL-31R −/− mice. However, there were no significant differences between GM-CSF- or IL-3- responsive CFU-GM per femur between IL-31R −/− vs. +/+ mice suggesting effects on immature subsets of MPC. Recombinant IL-31 was assessed for effects in vitro. IL-31, at concentrations up to 100ng/ml, did not stimulate colony formation by marrow MPC, nor did it enhance or suppress colony formation stimulated by GM-CSF, Epo, Epo+SCF, or Epo+SCF+GM-CSF. However, IL-31 did enhance survival of MPC subjected to delayed growth factor addition in a manner similar to, but not as potent as, that of SDF-1/CXCL12. IL-31 manifested no chemotaxis activity for +/+ MPC, and IL-31R −/− and +/+ MPC were equally responsive to the chemotactic effects of SDF-1/CXCL12. These results suggest that the IL-31R in vivo acts to positively regulate numbers and cycling of immature subsets of MPC in the marrow, and that IL-31 has survival enhancing effects on MPC in vitro.
-
il 31 receptor and il 31 in the positive regulation of Myeloid Progenitor Cell proliferation survival
Blood, 2005Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Nico Ghilardi, Wen Tao, Charlie Mantel, Barbara Grahamevans, Frederic DesauvageAbstract:Interleukin (IL)-31 receptor (R), also called gp130-like monocyte-receptor (GLM-R; Ghilardi et al. J. Biol. Chem.277:16831, 2002) is related to gp130 (~25% homology), and G-CSF-R (~24%). Its signaling activates STAT3 and STAT5. IL-31 is a four helix bundle cytokine preferentially produced by T helper 2 Cells. Nothing is known of the possible hematopoietic effects of IL-31R and IL-31. However, since: IL-31 signals through a receptor composed of IL-31RA and Oncostatin M R (Dillon et al. Nature Immunol. 5:752,2004), Oncostatin M is implicated in homeostasis of Myeloid Progenitor Cells (Broxmeyer et al. Immunity.16:815, 2002), and STAT3 and 5 are implicated by a number of groups in cytokine regulation of hematopoiesis, we hypothesized that the IL-31/IL-31R axis would be involved in regulation of hematopoiesis. We first compared Myeloid Progenitor Cell (MPC: CFU-GM, BFU-E, and CFU-GEMM) numbers and cycling status in marrow and spleen of IL-31R −/− vs. littermate control mice (+/+) using a combination of Epo, SCF and PWMSCM to stimulate in vitro the Cells taken from these mice. IL-31R −/− mice had significantly decreased numbers of MPC per femur (~51%) and spleen (~36–45%) with significantly decreased MPC cycling status in marrow (% MPC in S-phase: 0–3% in IL-31R −/− vs. 41–53% in +/+ mice). MPC in spleen of IL-31 −/− and +/+ were both in a slow or non cycling state (0–3% in S-phase). In contrast to CFU-GM from +/+ mice, CFU-GM from IL-31R −/− mice demonstrated little or no synergistic response to combined stimulation in vitro with GM-CSF or IL-3 with either SCF or Flt3-ligand (FL). This translated to decreased absolute numbers per femur of GM-CSF+SCF-, GM-CSF+FL-, IL-3+SCF-, and IL-3+FL- responsive CFU-GM in IL-31R −/− mice. However, there were no significant differences between GM-CSF- or IL-3- responsive CFU-GM per femur between IL-31R −/− vs. +/+ mice suggesting effects on immature subsets of MPC. Recombinant IL-31 was assessed for effects in vitro. IL-31, at concentrations up to 100ng/ml, did not stimulate colony formation by marrow MPC, nor did it enhance or suppress colony formation stimulated by GM-CSF, Epo, Epo+SCF, or Epo+SCF+GM-CSF. However, IL-31 did enhance survival of MPC subjected to delayed growth factor addition in a manner similar to, but not as potent as, that of SDF-1/CXCL12. IL-31 manifested no chemotaxis activity for +/+ MPC, and IL-31R −/− and +/+ MPC were equally responsive to the chemotactic effects of SDF-1/CXCL12. These results suggest that the IL-31R in vivo acts to positively regulate numbers and cycling of immature subsets of MPC in the marrow, and that IL-31 has survival enhancing effects on MPC in vitro.
-
transgenic expression of stromal Cell derived factor 1 cxc chemokine ligand 12 enhances Myeloid Progenitor Cell survival antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo
Journal of Immunology, 2003Co-Authors: Hal E. Broxmeyer, Giao Hangoc, Scott Cooper, Charlie Mantel, Lisa L Kohli, Young Hee Lee, Wade D Clapp, Chang H KimAbstract:Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/Progenitor Cells. Exogenously added stromal Cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of Myeloid Progenitor Cells in vitro. To further evaluate SDF-1/CXCL12 effects on Progenitor Cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid Progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic Myeloid Progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and Gαi proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse Myeloid Progenitors. Consistent with in vitro results, Progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased Progenitor Cell cycling and significant increases in Progenitor Cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to Progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to Cell trafficking.
-
Enhanced Myeloid Progenitor Cell Cycling and Apoptosis in Mice Lacking the Chemokine Receptor, CCR2
Blood, 1999Co-Authors: Suzanna Reid, Giao Hangoc, Scott Cooper, Alec Ritchie, Landin Boring, Jennifa Gosling, Israel F. Charo, Hal E. BroxmeyerAbstract:Chemokines regulate hematopoiesis in part by influencing the proliferative status of Myeloid Progenitor Cells (MPC). Human MCP-1/murine JE, a myelosuppressive chemokine, specifically binds C-C chemokine receptor 2 (CCR2). Transgenic mice containing a targeted disruption in CCR2 that prevents expression of CCR2 mRNA and protein and have MPC that are insensitive to inhibition by MCP-1 and JE in vitro were assessed for potential abnormalities in growth of bone marrow (BM) and spleen MPC. MPC in both unseparated and c-kit+lin- populations of BM from CCR2-deficient (-/-) mice were in a greatly increased proliferation state compared with CCR2 littermate control (+/+) mice, an effect not apparent with Progenitors from spleens of CCR2 (-/-) mice. Increased cycling status of CCR2 (-/-) BM MPC did not result in increased numbers of nucleated Cells or MPC in BM or spleens of CCR2 (-/-) mice. Possible reasons for this apparent discrepancy were highlighted by flow cytometric analysis of c-kit+lin- BM Cells and colony formation by MPC subjected to delayed addition of growth factors. The c-kit+lin- population of BM Cells from CCR2 (-/-) mice had a significantly higher percentage of apoptotic Cells than those from CCR2 (+/+) BM. However, elevated apoptosis was not associated with decreased numbers of c-kit+lin- Cells. The increased percentage of apoptotic c-kit+lin- Cells was due to elevated apoptosis within the c-kitdimlin-, but not the c-kitbrightlin-, subpopulations of Cells. Consistent with enhanced apoptosis of phenotypically defined Cells, MPC from CCR2 (-/-) BM and purified c-kit+lin- Cells demonstrated decreased Cell survival in vitro upon delayed addition of growth factors. The data suggest that signals received by CCR2 limit proliferation of Progenitor Cells in the BM, but also enhance survival of these Cells.
David B. Donner - One of the best experts on this subject based on the ideXlab platform.
-
macrophage stimulating protein a ligand for the ron receptor protein tyrosine kinase suppresses Myeloid Progenitor Cell proliferation and synergizes with vascular endothelial Cell growth factor and members of the chemokine family
Annals of Hematology, 1996Co-Authors: Hal E. Broxmeyer, Scott Cooper, David B. Donner, Andreas H Sarris, M H Wang, M S Chang, E J LeonardAbstract:Macrophage-stimulating protein (MSP), originally identified as an inducer of murine resident macrophage responsiveness to chemoattractants, is a ligand for human RON/murine STK receptor protein tyrosine kinases. Since STK was cloned from populations enriched for hematopoietic stem Cells, we initiated studies on the effects of MSP on colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) Myeloid Progenitor Cells. MSP alone had no colony stimulating activity. However, MSP caused about a 50% suppression of CFU-GM colony formation induced by synergistic combinations of SLF or Flt-L plus GM-CSF, G-CSF, or IL-3 and of BFU-E and CFU-GEMM colonies induced by SLF or Flt3-L plus Epo or Epo and IL-3. In contrast, MSP had no effect on Progenitors stimulated by one growth factor. MSP also suppressed colony formation by stimulated cord blood Progenitors, but only after preinduction to a rapidly cycling state. It was previously reported that several members of the chemokine family synergistically suppress Myeloid Progenitor proliferation. Likewise, synergistic suppression was observed when MSP was paired with VEGF, MIP-1α, IL-8, PF4, MCP-1, IP-10, or ENA-78, or when VEGF was paired with the chemokines; and the required MSP concentration was more than 100-fold less than for MSP alone. Additionally, MSP or VEGF inhibited proliferation of the human Myeloid growth factor-dependent Cell line, M07e, but a sustained effect required multiple additions over time. At the least, some of the MSP suppressive effects on Myeloid Progenitors, as assessed on single isolated CD34+++ marrow Cells, appeared to be directly on the Progenitors; sustained additions of MSP were required to see this effect. The suppressive action of MSP and its synergism with proteins of the chemokine family may be of relevance to regulation of blood Cell production.
-
Myeloid Progenitor Cell regulatory effects of vascular endothelial Cell growth factor.
International journal of hematology, 1995Co-Authors: Hal E. Broxmeyer, Scott Cooper, Ho Yeong Song, Byoung S. Kwon, Robert E. Warren, David B. DonnerAbstract:Vascular endothelial Cell growth factor (VEGF) is a ligand for the tyrosine kinase receptor Flk-1/KDR and Flt1 and is considered to be an endothelial Cell specific mitogen that plays an important role in angiogenesis. Since Flk-1 mRNA has been detected in primitive and more mature hematopoietic Cells, recombinant human VEGF was evaluated for its influence on hematopoiesis, which was assayed as in vitro colony formation by Myeloid Progenitor Cells from human bone marrow. VEGF enhanced colony formation by mature subsets of granulocyte-macrophage and erythroid Progenitor Cells that had been stimulated with a colony stimulating factor. In contrast, VEGF inhibited colony formation by more immature subsets of granulocyte-macrophage, erythroid and multipotential Progenitor Cells synergistically stimulated to proliferate with a colony stimulating factor and either steel factor or the ligand for the Flt-3 receptor tyrosine kinase. VEGF produced effects similar to those given above on purified CD34 Progenitor Cells from bone marrow and VEGF effects were neutralized by VEGF antibodies. However, when assessed for effects on single sorted CD34 Cells, VEGF only enhanced or suppressed colony formation by granulocyte-macrophage Progenitor Cells and the amplitude of the response was less than that observed when populations of these Cells were tested. In the single Cell assays, VEGF had no effect on colony formation by erythroid or multipotential Progenitors. These results suggest that the effects of VEGF, which were not species specific, are mediated by both direct and indirect actions on the Progenitors and thereby identify new activities for this important factor.
Stephen B Marley - One of the best experts on this subject based on the ideXlab platform.
-
influence of pi 3k akt pathway on wnt signalling in regulating Myeloid Progenitor Cell proliferation evidence for a role of autocrine paracrine wnt regulation
British Journal of Haematology, 2009Co-Authors: Georgios Nteliopoulos, Stephen B Marley, M Y GordonAbstract:Summary The regulation of Myeloid Progenitor Cell (granulocyte-macrophage colonyforming units, CFU-GM) proliferation/differentiation by the Wnt and phosphatidylinositol-3 kinase (PI-3K) pathways was investigated using a colony-replating assay. The PI-3K pathway promoted differentiation of interleukin-3 (IL-3)-stimulated myelopoiesis via Akt, because inhibition of the PI-3K/Akt pathway with LY294002 or SH-5 increased proliferation. The involvement of canonical and non-canonical Wnt pathways was investigated using Wnt3a and Wnt5a respectively. Addition of the recombinant Wnts to IL-3 increased CFU-GM proliferation. Dkk-1, when combined with the Wnt proteins, abrogated the effects of Wnt3a but not Wnt5a. Surprisingly, the addition of Dkk-1 to LY294002 or SH-5 blocked their proliferative effects. We hypothesized that increased proliferation induced by PI-3K/Akt inhibitors was not mediated by downstream activation of the Wnt pathway but by induced endogenous production/release of Wnt proteins. The addition of SH-5 to IL-3 created an autocrine Wnt loop in CD34 + Cells, resulting in the phosphorylation of lipoprotein-receptor-related-protein 6. Furthermore, the addition of medium conditioned by CD34 + Cells cultured in IL-3 + SH-5 to IL-3 increased CFU-GM proliferation. This effect was abrogated by Dkk-1, suggesting that a Wnt in the conditioned medium increased proliferation. In summary, IL-3 via the PI-3K pathway promoted differentiation of Myeloid Progenitor Cells through a decrease of endogenous Wnt production/release.
-
Influence of PI‐3K/Akt pathway on Wnt signalling in regulating Myeloid Progenitor Cell proliferation. Evidence for a role of autocrine/paracrine Wnt regulation
British journal of haematology, 2009Co-Authors: Georgios Nteliopoulos, Stephen B Marley, Myrtle Y. GordonAbstract:Summary The regulation of Myeloid Progenitor Cell (granulocyte-macrophage colonyforming units, CFU-GM) proliferation/differentiation by the Wnt and phosphatidylinositol-3 kinase (PI-3K) pathways was investigated using a colony-replating assay. The PI-3K pathway promoted differentiation of interleukin-3 (IL-3)-stimulated myelopoiesis via Akt, because inhibition of the PI-3K/Akt pathway with LY294002 or SH-5 increased proliferation. The involvement of canonical and non-canonical Wnt pathways was investigated using Wnt3a and Wnt5a respectively. Addition of the recombinant Wnts to IL-3 increased CFU-GM proliferation. Dkk-1, when combined with the Wnt proteins, abrogated the effects of Wnt3a but not Wnt5a. Surprisingly, the addition of Dkk-1 to LY294002 or SH-5 blocked their proliferative effects. We hypothesized that increased proliferation induced by PI-3K/Akt inhibitors was not mediated by downstream activation of the Wnt pathway but by induced endogenous production/release of Wnt proteins. The addition of SH-5 to IL-3 created an autocrine Wnt loop in CD34 + Cells, resulting in the phosphorylation of lipoprotein-receptor-related-protein 6. Furthermore, the addition of medium conditioned by CD34 + Cells cultured in IL-3 + SH-5 to IL-3 increased CFU-GM proliferation. This effect was abrogated by Dkk-1, suggesting that a Wnt in the conditioned medium increased proliferation. In summary, IL-3 via the PI-3K pathway promoted differentiation of Myeloid Progenitor Cells through a decrease of endogenous Wnt production/release.
-
influence of pi 3k akt pathway on wnt signalling in Myeloid Progenitor Cells evidence for a role of autocrine paracrine wnt regulation in cfu gm proliferation
Blood, 2008Co-Authors: Georgios Nteliopoulos, Stephen B Marley, M Y GordonAbstract:We have used a colony replating assay in conjunction with manipulation of the Wnt and PI-3K pathways to investigate regulation of Myeloid Progenitor Cell proliferation/differentiation. We found that PI-3 kinase pathway via Akt acts as a proliferative brake and promotes differentiation in IL3-driven myelopoiesis, since inhibition of PI-3K/Akt pathway with LY294002 (PI-3 kinase inhibitor) and SH-5 (Akt inhibitor) increased proliferation (reduced differentiation). We investigated the involvement of Wnt signalling in CFU-GM proliferation by using exogenous recombinant canonical Wnt3a and non-canonical Wnt5a. We showed that both of the Wnt members cannot support colony growth alone, but when added to IL3 they increased proliferation potential compared with the IL3 control, indicating an involvement of canonical Wnt/β-catenin and non-canonical Wnt pathways in the Myeloid Progenitor Cell proliferation. Immunoblotting analysis indicated that Wnt5a acts independently of β-catenin. Dkk-1 (Wnt-pathway inhibitor) alone did not affect IL-3 dependent proliferation but when combined with recombinant Wnts as expected it abrogated the effects of Wnt3a but not Wnt5a (acts as canonical-Wnt inhibitor). This was confirmed by β-catenin protein levels. Surprisingly, when Dkk-1 was added to LY294002 or SH-5, it blocked their proliferative effects. Dkk-1 acts as functional Wnt-receptor disabler and the finding that it blocks proliferation induced by PI-3K/Akt inhibitors’ indicates a link between the PI-3K and the Wnt signalling pathways. We hypothesised that increased proliferation induced by LY294002 or SH-5 was not mediated by downstream activation of the Wnt pathway but by induced endogenous expression of Wnt proteins and activation of the surface receptor. We conclude that there is a production of endogenous Wnt proteins that increases proliferation. Endogenous Wnt production has been reported in primitive haematopoietic Cells so there is potential for a paracrine or autocrine role for these Cell regulators. We tested this hypothesis in CD34 + Cells and found the addition of SH-5 to IL3 creates an autocrine loop of endogenous Wnt production, which results in the phosphorylation and activation of the LRP6 receptor and the initiation of the canonical signalling pathway. Furthermore, the conditioned medium of cultured CD34 + Cells was concentrated by using filtration devices (30kD cut-off) and added to IL3 to support the growth of Progenitors of another sample in a CFU-GM assay. We indicated that Wnt production and secretion can act in a paracrine way as well, since IL3+SH-5 conditioned medium increased the proliferative index of CFU-GM Cells whereas IL3 conditioned medium did not have significant effect. Dkk-1 abrogated the IL3+SH-5 conditioned medium’s induced proliferation, suggesting that the growth factors that had the proliferative effects were Wnt members. In conclusion, our data suggest that IL3 via PI-3K pathway promotes differentiation of Progenitor Myeloid Cells through inhibition of endogenous Wnt production.
-
chronic Myeloid leukaemia stem Cell derived but Progenitor Cell driven
Clinical Science, 2005Co-Authors: Stephen B Marley, M Y GordonAbstract:The biology of CML (chronic Myeloid leukaemia) has been extensively investigated as the disease is a paradigm of neoplasms induced when a translocation results in expression of a novel fusion protein, in this instance p210 BCR-ABL . Although CML manifests itself principally as unregulated expansion of the Myeloid lineage, the lesion is present in the stem Cell population and it has long been assumed that disregulated stem Cell kinetics must underlie the basic pathology of the disease. In this review, we present evidence that, in normal haemopoiesis, less primitive precursor Cells retain considerable flexibility in their capacity to undergo self-renewal, allowing them to maintain lineage-specific homoeostasis without inflicting proliferative stress upon the stem Cell population. This mechanism is dysregulated in CML and we have developed a self-renewal assay for CFU-GM (colony-forming unit-granulocyte/macrophage) which demonstrates that, in CML, the PI (proliferative index) of the Myeloid Progenitor Cell population is increased. The ability to measure the PI as an endpoint of p210 BCR-ABL expression gives considerable versatility to the in vitro investigation of putative therapeutic regimes in CML.
-
opposing effects of pi3 kinase pathway activation on human Myeloid and erythroid Progenitor Cell proliferation and differentiation in vitro
Experimental Hematology, 2004Co-Authors: J. L. Lewis, Stephen B Marley, Morenike Ojo, M Y GordonAbstract:Abstract Objective To investigate 1) the effects of lineage-specific cytokines (G-CSF and EPO) combined with ligands for different classes of cytokine receptors (common β chain, gp130, and tyrosine kinase) on proliferation by human Myeloid and erythroid Progenitor Cells; and 2) the signal transduction pathways associated with combinatorial cytokine actions. Materials and methods CFU-GM and BFU-E were cloned in vitro. Secondary colony formation by replated CFU-GM and subcolony formation by BFU-E provided measures of Progenitor Cell proliferation. Studies were performed in the presence of cytokine combinations with and without signal transduction inhibitors. Results Proliferation by CFU-GM and BFU-E was enhanced synergistically when common β chain receptor cytokines (IL-3 or GM-CSF) were combined with G-CSF or EPO, but not with gp130 receptor cytokines (LIF or IL-6) or tyrosine kinase receptor cytokines (SCF, HGF, Flt-3 ligand, or PDGF). Delayed addition studies with G-CSF+IL-3 and EPO+IL-3 demonstrated that synergy required the presence of both cytokines from the initiation of the culture. The Jak2-specific inhibitor, AG490, abrogated the effect of combining IL-3 with EPO but had no effect on the enhanced CFU-GM proliferation stimulated by IL-3+G-CSF. The PI3 kinase inhibitors LY294002 and wortmannin substituted for G-CSF in combination with IL-3 since proliferation in the presence of LY294002/wortmannin+IL-3 was enhanced to the same extent as in the presence of G-CSF+IL-3. In contrast, LY294002 and wortmannin inhibited proliferation in the presence of EPO and in the presence of EPO+IL-3. Conclusion 1) IL-3 may activate different signal transduction pathways when combined with G-CSF and when combined with EPO; 2) different signal transducing intermediates regulate erythroid and Myeloid Progenitor Cell proliferation; and 3) inhibition of the PI3 kinase pathway suppresses Myeloid Progenitor Cell differentiation and thereby increases proliferation.
