Cytoplasmic Domains of the Human Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Receptor β Chain (hβc) Responsible for Human GM-CSF-induced Myeloid Cell Differentiation

Defects in macrophage colony-stimulating factor (M-CSF) signaling disrupt myeloid cell differentiation in nonobese diabetic (NOD) mice, blocking myeloid maturation into tolerogenic antigen-presenting cells (APCs). In the absence of M-CSF signaling, NOD myeloid cells have abnormally high granulocyte macrophage colony-stimulating factor (GM-CSF) expression, and as a result, persistent activation of signal transducer/activator of transcription 5 (STAT5). Persistent STAT5 phosphorylation found in NOD macrophages is not affected by inhibiting GM-CSF. However, STAT5 phosphorylation in NOD bone marrow cells is diminished if GM-CSF signaling is blocked. Moreover, if M-CSF signaling is inhibited, GM-CSF stimulationin vitrocan promote STAT5 phosphorylation in nonautoimmune C57BL/6 mouse bone marrow cultures to levels seen in the NOD. These findings suggest that excessive GM-CSF production in the NOD bone marrow may interfere with the temporal sequence of GM-CSF and M-CSF signaling needed to mediate normal STAT5 function in myeloid cell differentiation gene regulation.

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A Truncated Isoform of the Human β Chain Common to the Receptors for Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-3 (IL-3), and IL-5 With Increased mRNA Expression in Some Patients With Acute Leukemia

Blood ◽

10.1182/blood.v91.1.54.54_54_63 ◽

1998 ◽

Vol 91 (1) ◽

pp. 54-63 ◽

Cited By ~ 4

Author(s):

Rosemary E. Gale ◽

Robin W. Freeburn ◽

Asim Khwaja ◽

Rajesh Chopra ◽

David C. Linch

Keyword(s):

Amino Acids ◽

Acute Leukemia ◽

Myeloid Cells ◽

Colony Stimulating Factor ◽

Interleukin 3 ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Β Chain ◽

Stimulating Factor

We report here a naturally occurring isoform of the human β chain common to the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (GMRβC) with a truncated intracytoplasmic tail caused by deletion of a 104-bp exon in the membrane-proximal region of the chain. This β intracytoplasmic truncated chain (βIT) has a predicted tail of 46 amino acids, instead of 432 for βC, with 23 amino acids in common with βC and then a new sequence of 23 amino acids. In primary myeloid cells, βIT comprised approximately 20% of the total β chain message, but was increased up to 90% of total in blast cells from a significant proportion of patients with acute leukemia. Specific anti-βITantibodies demonstrated its presence in primary myeloid cells and cell lines. Coexpression of βIT converted low-affinity GMRα chains (KD 2.5 nmol/L) to higher-affinity αβ complexes (KD 200 pmol/L). These could bind JAK2 that was tyrosine-phosphorylated by stimulation with GM-CSF. βITdid not support GM-CSF–induced proliferation when cotransfected with GMRα into CTLL-2 cells. Therefore, it may interfere with the signal-transducing properties of the βC chain and play a role in the pathogenesis of leukemia.

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The Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Exists as a Preformed Receptor Complex That Can Be Activated by GM-CSF, Interleukin-3, or Interleukin-5

Blood ◽

10.1182/blood.v90.8.3005 ◽

1997 ◽

Vol 90 (8) ◽

pp. 3005-3017 ◽

Cited By ~ 52

Author(s):

Joanna M. Woodcock ◽

Barbara J. McClure ◽

Frank C. Stomski ◽

Michael J. Elliott ◽

Christopher J. Bagley ◽

...

Keyword(s):

Myeloid Cell ◽

Cytokine Receptor ◽

Activation Mechanism ◽

Receptor Complex ◽

Colony Stimulating Factor ◽

Interleukin 3 ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract The granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor α chain (GMRα) and to the common β chain of the GM-CSF, IL-3, and IL-5 receptors (βc ) immunoprecipitated both GMRα and βc from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMRα and βc , as shown by the ability of soluble βc to associate with membrane-anchored GMRα or soluble GMRα. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMRα-associated βc . These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.

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A Truncated Isoform of the Human β Chain Common to the Receptors for Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-3 (IL-3), and IL-5 With Increased mRNA Expression in Some Patients With Acute Leukemia

Blood ◽

10.1182/blood.v91.1.54 ◽

1998 ◽

Vol 91 (1) ◽

pp. 54-63 ◽

Cited By ~ 24

Author(s):

Rosemary E. Gale ◽

Robin W. Freeburn ◽

Asim Khwaja ◽

Rajesh Chopra ◽

David C. Linch

Keyword(s):

Amino Acids ◽

Acute Leukemia ◽

Myeloid Cells ◽

Colony Stimulating Factor ◽

Interleukin 3 ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Β Chain ◽

Stimulating Factor

Abstract We report here a naturally occurring isoform of the human β chain common to the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (GMRβC) with a truncated intracytoplasmic tail caused by deletion of a 104-bp exon in the membrane-proximal region of the chain. This β intracytoplasmic truncated chain (βIT) has a predicted tail of 46 amino acids, instead of 432 for βC, with 23 amino acids in common with βC and then a new sequence of 23 amino acids. In primary myeloid cells, βIT comprised approximately 20% of the total β chain message, but was increased up to 90% of total in blast cells from a significant proportion of patients with acute leukemia. Specific anti-βITantibodies demonstrated its presence in primary myeloid cells and cell lines. Coexpression of βIT converted low-affinity GMRα chains (KD 2.5 nmol/L) to higher-affinity αβ complexes (KD 200 pmol/L). These could bind JAK2 that was tyrosine-phosphorylated by stimulation with GM-CSF. βITdid not support GM-CSF–induced proliferation when cotransfected with GMRα into CTLL-2 cells. Therefore, it may interfere with the signal-transducing properties of the βC chain and play a role in the pathogenesis of leukemia.

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Activation of JAK2 in Human Vascular Endothelial Cells by Granulocyte-Macrophage Colony-Stimulating Factor

Blood ◽

10.1182/blood.v89.3.863 ◽

1997 ◽

Vol 89 (3) ◽

pp. 863-872 ◽

Cited By ~ 32

Author(s):

Raffaella Soldi ◽

Luca Primo ◽

Maria Felice Brizzi ◽

Fiorella Sanavio ◽

Massimo Aglietta ◽

...

Keyword(s):

Endothelial Cells ◽

Tyrosine Kinase ◽

Janus Kinase ◽

Colony Stimulating Factor ◽

Functional Program ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Β Chain ◽

Stimulating Factor

Abstract Besides the regulation of hematopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the expression of a functional program in endothelial cells (ECs) related to angiogenesis and to their survival in the bone marrow microenvironment. ECs express specific GM-CSF high-affinity binding sites, which mediate the proliferative and migratory response. We now report that ECs express the α and β subunits of GM-CSF receptor (GM-CSFR), and that GM-CSF is able to activate the Janus kinase (JAK)2, a member of the cytosolic tyrosine kinase family, which is known to mediate signals of several non–tyrosine kinase receptors. JAK2 tyrosine phoshorylation, as well as activation of its catalytic activity, is induced by subnanomolar concentrations of GM-CSF and occurs within 3 minutes of stimulation and persists at least for 10 minutes. The effect is specific as inferred by the lack of effect of heat-inactivated GM-CSF or neutralized by specific antibodies and by the finding that interleukin-5, which utilizes a specific α chain and the same β chain of GM-CSFR, does not phosphorylate JAK2. Furthermore, we show that the amount of JAK2 physically associated with GM-CSFR β chain is increased after GM-CSF stimulation and that GM-CSF triggers both β chain and JAK2 tyrosine phosphorylation. Taken together, these results suggest that biologic activities of GM-CSF in vascular endothelium may, in part, be elicited by GM-CSFR–mediated JAK2 activation.

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Synergistic effects of nerve growth factor and granulocyte-macrophage colony-stimulating factor on human basophilic cell differentiation

Blood ◽

10.1182/blood.v77.5.971.971 ◽

1991 ◽

Vol 77 (5) ◽

pp. 971-979 ◽

Cited By ~ 55

Author(s):

T Tsuda ◽

D Wong ◽

J Dolovich ◽

J Bienenstock ◽

J Marshall ◽

...

Keyword(s):

Cell Differentiation ◽

Nerve Growth Factor ◽

Growth Factor ◽

Colony Stimulating Factor ◽

Nerve Growth ◽

Basophilic Cell ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract We have recently shown that nerve growth factor (NGF) promotes human granulopoiesis, specifically augmenting basophilic cell differentiation observed in methylcellulose hematopoietic colony assays of human peripheral blood. Because the NGF effect was seen in the presence of conditioned medium derived from a human T-cell line (Mo-CM) containing granulocyte-macrophage colony-stimulating factor (GM-CSF), we examined interactions of purified NGF and recombinant human GM-CSF (rhGM-CSF) on granulocyte growth and differentiation. rhGM-CSF stimulated a dose- dependent increase in methylcellulose colony growth at concentrations between 0.1 U/mL and 10 U/mL, and in the presence of NGF at 500 ng/mL this effect was enhanced. The number of basophilic cell colony-forming units (CFU-Baso) and histamine-positive colonies increased synergistically when NGF was added to rhGM-CSF. Furthermore, because Mo- CM acts with sodium butyrate to promote basophilic differentiation of alkaline-passaged myeloid leukemia cells, HL-60, we also examined the interaction of NGF and Mo-CM or rhGM-CSF using this assay. In the presence of NGF, Mo-CM at concentrations of 0.5% to 20% vol/vol, and rhGM-CSF at concentrations of 0.1 U/mL to 100 U/mL synergistically increased histamine production by butyrate-induced, alkaline-passaged HL-60 cells; this was associated with the appearance of metachromatic, tryptase-negative, IgE receptor-positive cells. The effects of rhGM-CSF or Mo-CM were completely abrogated by a specific anti-rhGM-CSF neutralizing antibody in methylcellulose, with or without NGF; the NGF synergy with rhGM-CSF in the HL-60 assay was also inhibited by either anti-rhGM-CSF or anti-NGF antibody. These studies support the notion that differentiation in the basophilic lineage may be enhanced by NGF acting to increase the number of GM-CSF-responsive basophilic cell progenitors.

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Human Interleukin-3/Granulocyte Macrophage-Colony Stimulating Factor Knock-In Mice Support Human Myeloid Cell Reconstitution and Human Immune Responses In the Lung.

Blood ◽

10.1182/blood.v116.21.3789.3789 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3789-3789

Author(s):

Tim Willinger ◽

Anthony Rongvaux ◽

Hitoshi Takizawa ◽

Elizabeth E. Eynon ◽

Sean Stevens ◽

...

Keyword(s):

Immune Responses ◽

Myeloid Cell ◽

Humanized Mice ◽

Colony Stimulating Factor ◽

Interleukin 3 ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Human Immune ◽

Macrophage Colony ◽

Stimulating Factor

Abstract Abstract 3789 Humanized mice, i.e. mice with a functional human immune system, have great potential to study human immunology in vivo and to allow vaccine testing. To this end, mice need to fully support engraftment with human immune cells, allow infection with human pathogens, and mount effective human immune responses to pathogens. A major limitation of current humanized mice is the poor development and function of human myeloid cells. Here we report a novel strategy to overcome this limitation by generating human interleukin-3/granulocyte macrophage colony stimulating factor knock-in (hIL-3/GM-CSF KI) mice to create a better environment for human myeloid cells. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis (PAP) due to elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the PAP syndrome. In addition, these mice showed an enhanced systemic inflammatory response to LPS. Finally, humanization of IL-3 and GM-CSF lead to a stronger innate immune response against influenza virus infection. In summary, hIL-3/GM-CSF KI mice represent a new mouse model to study human immune responses in the lung and against pathogens such as influenza. Disclosures: Stevens: Regeneron Pharmaceuticals: Employment; AnaptysBio Inc: Employment.

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Down-regulation of interleukin-3/granulocyte-macrophage colony-stimulating factor receptor β-chain in BCR-ABL+human leukemic cells: association with loss of cytokine-mediated Stat-5 activation and protection from apoptosis after BCR-ABL inhibition

Blood ◽

10.1182/blood.v97.9.2846 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2846-2853 ◽

Cited By ~ 33

Author(s):

Nicholas J. Donato ◽

Ji Y. Wu ◽

Ling Zhang ◽

Hagop Kantarjian ◽

Moshe Talpaz

Keyword(s):

Map Kinase ◽

Leukemic Cells ◽

Signaling Cascades ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Sti 571 ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Β Chain ◽

Stimulating Factor

Abstract Several signaling cascades are engaged by expression of the p210 bcr-abl tyrosine kinase, and evidence suggests that these signals drive leukemogenesis. In this report, signaling pathways were examined and compared between cells derived from leukemic patients and cells expressing a bcr-abl construct (MBA). The effects of acute inhibition of bcr-abl with STI-571 on these signals and the survival of bcr-abl–expressing cells were also evaluated. Expression of bcr-abl in interleukin-3 (IL-3)/granulocyte-macrophage colony-stimulating factor (GM-CSF)–dependent Mo7e cells (MBA) resulted in growth factor independence, constitutive activation of Stat-5 phosphorylation, engagement of mitogen-activated protein (MAP) kinase signals, and increased expression of PTP1B and bcl-xL. STI-571 inhibited cell growth and induced apoptosis in bcr-abl–expressing cells (MBA, K562, BV-173, KBM5) but not in bcr-abl− tumor cells (Mo7e, KG-1, ME-180, Daudi). STI-571–mediated apoptosis correlated with the inhibition of Stat-5 and MAP kinase activation and a reduction in overexpressed bcl-xL but not in PTP1B. Inhibitor had no effect on IL-3/GM-CSF–dependent Mo7e cell signaling and did not prevent activation of the other Jak/Stat pathways (interferon α, IL-3/GM-CSF). However, neither IL-3 nor GM-CSF could reactivate Stat-5 after the STI-571–mediated inhibition of bcr-abl. Expression of the common β-chain of the IL-3/GM-CSF receptor was down-regulated in Stat-5–activated myeloid leukemic cells, suppressing IL-3/GM-CSF signal transduction and the ability of these cytokines to provide apoptotic protection. These studies suggest that bcr-abl activates cytokine-independent mechanisms of survival while inactivating intrinsic cytokine signaling cascades, making bcr-abl+myeloid cells vulnerable to apoptosis after bcr-abl inactivation.

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Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line

Blood ◽

10.1182/blood.v77.2.243.243 ◽

1991 ◽

Vol 77 (2) ◽

pp. 243-248 ◽

Cited By ~ 57

Author(s):

Y Kanakura ◽

B Druker ◽

KW Wood ◽

HJ Mamon ◽

K Okuda ◽

...

Keyword(s):

Signal Transduction ◽

Cell Line ◽

Human Factor ◽

Myeloid Cell ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Myeloid Cell Line ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

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Identification of a 14-3-3 Binding Sequence in the Common β Chain of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-3 (IL-3), and IL-5 Receptors That Is Serine-Phosphorylated by GM-CSF

Blood ◽

10.1182/blood.v94.6.1933 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1933-1942 ◽

Cited By ~ 35

Author(s):

F.C. Stomski ◽

M. Dottore ◽

W. Winnall ◽

M.A. Guthridge ◽

J. Woodcock ◽

...

Keyword(s):

Binding Site ◽

Signaling Molecules ◽

Colony Stimulating Factor ◽

Interleukin 3 ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

The Common ◽

Macrophage Colony ◽

Β Chain ◽

Stimulating Factor

Abstract The common β chain (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of βc. However, the contribution of serine phosphorylation in βc to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of βc that interacts with the adaptor protein 14-3-3ζ. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3ζ fusion protein showed that 14-3-3 directly associates with βc but not the GM-CSF receptor  chain. C-terminal truncation mutants of βcfurther showed that a region between amino acids 544 and 626 in βc was required for its association with 14-3-3ζ. This region contains the sequence 582HSRSLP587, which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of582HSRSLP587 for EFAAAA completely abolished interaction of βc with GST–14-3-3ζ. Furthermore, the interaction of βc with GST–14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when 585Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated 585Ser bound 14-3-3ζ with an affinity of 150 nmol/L. To study the regulation of 585S phosphorylation in vivo, we raised antibodies that specifically recognized 585Ser-phosphorylated βc. Using these antibodies, we showed that GM-CSF stimulation strongly upregulated 585Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site (577Tyr) to the 14-3-3–binding site (582HSRSLP587) and their conservation between mouse, rat, and human βc but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

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