Characterization of the human granulocyte-macrophage colony-stimulating factor receptor

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine derived from activated T cells, endothelial cells, fibroblasts, and macrophages. It stimulates myeloid and erythroid progenitors to form colonies in semisolid medium in vitro, as well as enhancing multiple differentiated functions of mature neutrophils, macrophages, and eosinophils. We have examined the binding of human GM-CSF to a variety of responsive human cells and cell lines. The most mature myelomonocytic cells, specifically human neutrophils, macrophages, and eosinophils, express the highest numbers of a single class of high affinity receptors (Kd approximately 37 pM, 293-1000 sites/cell). HL-60 and KG-1 cells exhibit an increase in specific binding at high concentrations of GM-CSF; computer analysis of the data is nonetheless consistent with a single class of high affinity binding sites with a Kd approximately 43 pM and 20-450 sites/cell. Dimethyl sulfoxide induces a 3-10-fold increase in high affinity receptors expressed in HL-60 cells, coincident with terminal neutrophilic differentiation. Finally, binding of 125I-GM-CSF to fresh peripheral blood cells from six patients with chronic myelogenous leukemia was analyzed. In three of six cases, binding was similar to the nonsaturable binding observed with HL-60 and KG-1 cells. GM-CSF binding was low, or in some cases, undetectable on myeloblasts obtained from eight patients with acute myelogenous leukemia. The observed affinities of the receptor for GM-CSF are consistent with all known biological activities. Affinity labeling of both normal neutrophils and dimethyl sulfoxide-induced HL-60 cells with unglycosylated 125I-GM-CSF yielded a band of 98 kDa, implying a molecular weight of approximately 84,000 for the human GM-CSF receptor.     

The cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), can deliver Bcl-XL as an extracellular fusion protein to protect cells from apoptosis and retain differentiation induction

Bcl-XL, a member of the Bcl-2 protein family, is able to suppress cell death induced by diverse stimuli in many cell types, including hematopoietic cells. Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that promotes the proliferation and maturation of neutrophils, eosinophils, and macrophages from bone marrow progenitors. We fused GM-CSF to Bcl-XL and examined the capacity of this chimera to bind human cells through the GM-CSF receptor and prevent apoptosis. We found that the chimeric protein increased the proliferation of human monocytes in culture from 24 h until at least 72 h. In the presence of different apoptotic agents, GM-CSF-Bcl-XL protected cells from induced cell death and promoted proliferation, whereas GM-CSF alone was completely inhibited. In the presence of cytarabine, GM-CSF-Bcl-XL was able also to promote the differentiation of the CD34+ myeloid precursor whereas Lfn-Bcl-XL, lacking the GM-CSF domain-stimulated cell proliferation and not differentiation. We conclude that recombinant GM-CSF-Bcl-XL binds the GM-CSF receptor on human monocyte/macrophage cells and bone marrow progenitors inducing differentiation and allowing Bcl-XL entry into cells where it blocks cell death and allows amplified cell proliferation. This fully human fusion protein has potential to prevent monocytopenia and represents a new strategy for engineering anti-apoptotic therapeutics.     

Biological activities of human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as an important regulation for hematopoietic cell development and function. Within the myeloid lineages, GM-CSF serves as a growth and developmental factor for intermediate-stage progenitors between early, interleukin 3-responsive and late granulocyte colony-stimulating factor-responsive precursors. GM-CSF also serves as an activator of circulating effector cells. The ability of GM-CSF to induce monocyte expression of tumor necrosis factor, interleukin 1 and other factors, further ties this hormone into a network of cytokines that interact to regulate many hematologic and immunologic responses. The availability of large quantities of recombinant GM-CSF now provides the opportunity and challenge not only for unraveling the mechanisms regulating hematopoiesis, but also for developing new therapies for enhancement of host defense against infection that were not previously possible.     

Characterization and partial purification of CD34+ progenitor cell ecto-phosphatidic acid phosphohydrolase

Phosphatidic acid and its hydrolysis product, diacylglycerol, play potentially vital roles as extracellular messengers in numerous cellular systems and may play a key role in regulating hematopoiesis. In this study, we describe an ecto-phosphatidic acid phosphohydrolase that potentially regulates cellular responses to phosphatidic acid on bone marrow derived human hematopoietic progenitors. We partially purified hematopoietic progenitor ecto-PAPase using a novel in-gel phosphatase assay and then characterized the enzyme on phenotypically defined subpopulations of hematopoietic CD34+ progenitors isolated by flow cytometry. The most pronounced PAPase activity was confined to uncommitted CD34+/CD38+ hematopoietic progenitors, which lacked the expression of other lineage-associated antigens. We conclude that hematopoietic progenitor cells at various stages of maturation possess a potent ecto-PAPase, an enzyme well positioned to regulate progenitor cell growth and differentiation induced by phosphatidic acid and related lipids.     

Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8

Differentiation mechanisms and inflammatory functions of neutrophils and macrophages are usually studied by genetic and biochemical approaches that require costly breeding and time-consuming purification to obtain phagocytes for functional analysis. Because Hox oncoproteins enforce self-renewal of factor-dependent myeloid progenitors, we queried whether estrogen-regulated Hoxb8 (ER-Hoxb8) could immortalize macrophage or neutrophil progenitors that would execute normal differentiation and normal innate immune function upon ER-Hoxb8 inactivation. Here we describe methods to derive unlimited quantities of mouse macrophages or neutrophils by immortalizing their respective progenitors with ER-Hoxb8 using different cytokines to target expansion of different committed progenitors. ER-Hoxb8 neutrophils and macrophages are functionally superior to those produced by many other ex vivo differentiation models, have strong inflammatory responses and can be derived easily from embryonic day 13 (e13) fetal liver of mice exhibiting embryonic-lethal phenotypes. Using knockout or small interfering RNA (siRNA) technologies, this ER-Hoxb8 phagocyte maturation system represents a rapid analytical tool for studying macrophage and neutrophil biology.     

The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic growth factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and is thought to play an important role in the mesoderm induction. Although hematopoietic cells derive from the mesoderm, relatively few studies have, until recently, addressed the role of FGF-2 in hematopoiesis. FGF-2 is expressed in cells of the bone marrow including stromal cells, and possibly cells from several hematopoietic cell lineages. It is stored in the bone marrow extra-cellular matrix and released by enzymes such as heparanase, plasmin, or phospholipase C and D. FGF-receptors (FGF-Rs) are expressed in leukemic cell lines and in hematopoietic cells. FGF-2 positively regulates hematopoiesis, by acting on stromal cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. The action of FGF-2 is most likely indirect since its action, on megakaryocytopoiesis for example, is abrogated by anti-IL6 antibodies. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of TGF-β Taken together, these results demonstrate that FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.     

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.     

Granulocyte-macrophage colony-stimulating factor （GM-CSF） and T-cell responses： what we do and don＇t know

Granulocyte-macrophage colony-stimulating factor （GM-CSF） is an important hematopoietic growth factor and immune modulator. GM-CSF also has profound effects on the functional activities of various circulating leukocytes. It is produced by a variety of cell types including T cells, macrophages, endothelial cells and fibroblasts upon receiving immune stimuli. Although GM-CSF is produced locally, it can act in a paracrine fashion to recruit circulating neutrophils, monocytes and lymphocytes to enhance their functions in host defense. Recent intensive investigations are centered on the application of GM-CSF as an immune adjuvant for its ability to increase dendritic cell （DC） maturation and function as well as macrophage activity. It is used clinically to treat neutropenia in cancer patients undergoing chemotherapy, in AIDS patients during therapy, and in patients after bone marrow transplantation. Interestingly, the hematopoietic system of GM-CSF-deficient mice appears to be normal; the most significant changes are in some specific T cell responses. Although molecular cloning of GM-CSF was carried out using cDNA library oft cells and it is well known that the T cells produce GM-CSF after activation, there is a lack of systematic investigation of this cytokine in production by T cells and its effect on T cell function. In this article, we will focus mainly on the immunobiology of GM-CSF in T cells.     

New relationships of human hematopoietic lineages facilitate detection of multipotent hematopoietic stem and progenitor cells

Three important goals of hematopoietic stem cell research are to understand of how hematopoietic stem cells (HSCs) self-renew, how lineage commitment takes place, and how HSCs can be expanded ex vivo. Research in this area requires a reliable model of hematopoiesis. Performing detailed functional analyses of human hematopoietic progenitor subsets, we recently gained evidence for new hematopoietic lineage relationships.¹ According to our data, neutrophils belong to the same branch of the hematopoietic tree as lymphocytes. In contrast, eosinophils and basophils derive from another branch, the erythro-myeloid branch. Here, after introducing the newly proposed hematopoietic model, we discuss its consequences for the identification and expansion of human multipotent progenitors and suggest a fast and reliable method to screen for multipotent hematopoietic cells in vitro.     

The construction of high efficiency human bone marrow tissue ex vivo

The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM-CSF and IL-3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.     

Expression of the GM-CSF receptor in ovine spermatozoa: GM-CSF effect on sperm viability and motility of sperm subpopulations after the freezing-thawing process

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of haematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed in myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other non-haematopoietic cells. We have previously demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and vitamin-C uptake and enhance several parameters of sperm motility in the presence of glucose or fructose substrates. In this study, we have analyzed the expression of GM-CSF receptors in ovine spermatozoa and studied the effect of GM-CSF on sperm viability and motility after the freezing-thawing process. Immunolocalization and immunoblotting analyses demonstrated that ovine spermatozoa (Xisqueta race) expressed GM-CSF receptors. In addition, GM-CSF partially counteracted the impairing action of freezing/thawing on the percentage of total motility, as well as on the specific motility patterns of each of the separate, motile sperm subpopulations of ram ejaculates subjected to this protocol. These results suggest that GM-CSF can play a role in the resistance of ram spermatozoa to environmental thermal stress.     

Macrophage-associated erythropoiesis and lymphocytopoiesis in mouse fetal liver: ultrastructural and ISH analysis

To elucidate the process of fetal liver hematopoiesis, the relationships between stroma and hematopoietic cells involved in maturation were investigated. Cultured mouse fetal liver explants were established for morphological analysis of the interactions between fetal liver stroma and hematopoietic cells ex vivo. Fetal liver stroma comprised epithelial cells and macrophages, which occupied most of the culture surface. Macrophages proliferated extensively in primary culture, but almost disappeared after 3 passages. Close morphological and functional relationships were established between macrophages and hemopoietic cells, whereas epithelial cells did not interact with blood cells. Scanning electron microscopy revealed that macrophages were in close contact with erythroblasts and formed a three-dimensional network. In each erythroblastic island, 2-3 lymphocytes were also in contact with the macrophages; erythroblasts, lymphocytes and macrophages formed close, firm associations through their cytoplasmic membranes. This cell orientation was confirmed by transmission electron microscopy of fetal liver in vivo. In situ hybridization revealed that the macrophages expressed jagged-1, an important ligand of the Notch signaling system in hematopoiesis.     

Nonhematopoietic cells are key players in innate control of bacterial airway infection

Airborne pathogens encounter several hurdles during host invasion, including alveolar macrophages (AMs) and airway epithelial cells (AECs) and their products. Although growing evidence indicates pathogen-sensing capacities of epithelial cells, the relative contribution of hematopoietic versus nonhematopoietic cells in the induction of an inflammatory response and their possible interplay is still poorly defined in vivo in the context of infections with pathogenic microorganisms. In this study, we show that nonhematopoietic cells, including AECs, are critical players in the inflammatory process induced upon airway infection with Legionella pneumophila, and that they are essential for control of bacterial infections. Lung parenchymal cells, including AECs, are not infected themselves by L. pneumophila in vivo but rather act as sensors and amplifiers of inflammatory cues delivered by L. pneumophila-infected AM. We identified AM-derived IL-1β as the critical mediator to induce chemokine production in nonhematopoietic cells in the lung, resulting in swift and robust recruitment of infection-controlling neutrophils into the airways. These data add a new level of complexity to the coordination of the innate immune response to L. pneumophila and illustrate how the cross talk between leukocytes and nonhematopoietic cells contributes to efficient host protection.     

The role of stroma in hematopoiesis and dendritic cell development

Development of the immune system is depicted as a hierarchical process of differentiation from hematopoietic stem cells (HSC) to lineage-committed precursors, which further develop into mature immune cells. In the case of dendritic cell (DC) development, this linear precursor-progeny approach has led to a confused picture of relationships between various subsets of DC identifiable in vivo. A possible reconciliation of the diversity of DC precursors and DC subsets in vivo encompasses the role of the microenvironment in DC hematopoiesis. We propose here that various niches for DC hematopoiesis within lymphoid organs could account for the diversity of DC in vivo. A tridimensional space consisting of stromal cells which produce a range of membrane-bound and secreted molecules providing signals to DC progenitors would define these niches.     

Characterization, chromosomal localization, and expression during hematopoietic differentiation of the gene encoding Arl6ip, ADP-ribosylation-like factor-6 interacting protein (ARL6)

Proliferation and differentiation of hematopoietic stem cells and progenitors are regulated by signals from the microenvironment, involving both secreted cytokines and adhesion molecules. The exact mechanisms by which cytokines act on hematopoietic development are still not well understood. To extend the molecular characterization of gene regulation during cytokine-induced hematopoiesis, we applied mRNA differential display to identify genes regulated when multipotent progenitor cells are allowed to differentiate into monocytes and neutrophils. Here we report the isolation and characterization of a gene that is downregulated during myeloid differentiation and encodes a 23-kDa protein with four putative transmembrane segments. The gene, which we named Arl6ip, is identical to a mouse gene recently identified by its physical interaction with ADP-ribosylation-like factor-6 (ARL6), belonging to the Ras superfamily. We add information on its full-length characterization as well as its regulation during hematopoiesis. It is expressed in all hematopoietic cell lineages, but the highest level of expression is found in early myeloid progenitor cells. Preliminary studies by immunofluorescence microscopy revealed that the ARL6IP protein is predominantly localized to intracytoplasmic membranes. This suggests an involvement of the Arl6ip gene in protein transport, membrane trafficking, or cell signaling during hematopoietic maturation.