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1.
Proc Natl Acad Sci U S A ; 105(47): 18501-6, 2008 Nov 25.
Article in English | MEDLINE | ID: mdl-19011094

ABSTRACT

Two types of blast colonies can be stimulated to develop in semisolid agar cultures of murine bone marrow cells. Typically, these are either multicentric colonies stimulated by stem cell factor (SCF) plus interleukin-6 (IL-6) or dispersed colonies stimulated by Flt3 ligand (FL) plus IL-6. Both types of blast colony-forming cells (BL-CFCs) can generate large numbers of lineage-committed granulocyte-macrophage progenitor cells and exhibit some capacity for self-generation and the formation of eosinophil and megakaryocyte progenitor cells. However, the two populations of BL-CFCs are largely distinct and partially separable by fluorescence-activated cell sorting and are distinguished by differing capacity to form granulocyte-committed progeny. Both types of BL-CFCs can generate dendritic cells and small numbers of lymphocytes but the FL-responsive BL-CFCs have a greater capacity to form both B and T lymphocytes. Both types of blast colonies offer remarkable opportunities to analyze multilineage commitment at a clonal level in vitro.


Subject(s)
Hematopoietic Stem Cells/cytology , Animals , B-Lymphocytes/cytology , Cell Differentiation , Cells, Cultured , Interleukin-6/physiology , Membrane Proteins/physiology , Mice , Mice, Inbred Strains , Stem Cell Factor/physiology , T-Lymphocytes/cytology
2.
J Immunol ; 167(8): 4661-7, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11591796

ABSTRACT

Organs from neonatal mice dying from IFN-gamma-dependent inflammatory disease initiated by loss of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) had a normal capacity to produce G-CSF in vitro but a reduced capacity to produce GM-CSF, most evident with the lung, and some reduction in the production of M-CSF by muscle tissue. In contrast, organs from mice lacking the genes for both SOCS-1 and IFN-gamma had a normal capacity to produce CSFs. Organs from young adult mice dying with polymyositis and myocarditis that lacked SOCS-1 but were heterozygous for IFN-gamma had a normal capacity to produce GM-CSF and M-CSF, but muscle tissue produced significantly increased amounts of G-CSF and IL-5 with IL-5 production also being elevated for the salivary gland, thymus, and heart. Loss of the IFN-gamma gene alone had no impact on organ production of these cytokines in vitro. In none of the inflammatory disease models was IL-3 production detected. The SOCS-1 protein appears to have no direct influence on the cellular production of these cytokines and the abnormalities observed either depend on the coaction of IFN-gamma, or more likely, are linked with the invasion and destruction of tissue by T lymphocytes, macrophages, eosinophils, and neutrophils. The ability of local organs to produce these proinflammatory cytokines could contribute to the development and progression of these inflammatory lesions.


Subject(s)
Carrier Proteins/genetics , Colony-Stimulating Factors/biosynthesis , Inflammation/immunology , Interleukin-5/biosynthesis , Repressor Proteins , Animals , Bone and Bones/immunology , Granulocyte Colony-Stimulating Factor/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Interferon-gamma/immunology , Lung/immunology , Macrophage Colony-Stimulating Factor/biosynthesis , Mice , Mice, Mutant Strains , Muscle, Skeletal/immunology , Myocarditis/immunology , Myocardium/immunology , Myositis/immunology , Organ Culture Techniques , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins , Thymus Gland/immunology
3.
Leukemia ; 15(8): 1248-55, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11480567

ABSTRACT

Murine Ba/F3 cells were transfected with cDNA for the alpha-chain of the murine interleukin-5 (IL-5) receptor and cloned lines of these cells were able to proliferate in response to as little as 2.5 pg/ml of IL-5. The bioassay was demonstrated to be specific for IL-5 and was able to measure IL-5 produced in culture by organs from adult C57BL/6 and BALB/c mice. The highest levels of IL-5 were produced by lung tissue but thymus and bladder consistently produced IL-5 and more variable production was observed by the heart, spleen, muscle, bone shaft, uterus and testes. Bone marrow cells produced no detectable IL-5. Observed levels of production of IL-5 were similar when using organs from mice lacking high-affinity receptors for IL-5 and from nu/nu, RAG-1-/- and NOD/SCID mice lacking T lymphocytes. In inflammatory peritoneal exudates induced by the injection of casein plus bacteria, levels of induced IL-5 were higher if the mice lacked high-affinity receptors for IL-5. The data indicate that T lymphocytes are not the dominant cellular source of IL-5 in organ-conditioned media and that local IL-5 production can occur with a wide range of normal murine organs.


Subject(s)
Interleukin-5/analysis , Animals , Biological Assay , DNA, Complementary/analysis , DNA, Complementary/genetics , Gene Expression Regulation , Interleukin-5/biosynthesis , Interleukin-5/genetics , Mice , Organ Specificity
4.
Leukemia ; 14(10): 1785-95, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11021754

ABSTRACT

The cloned pro-B-lymphocyte murine leukemic cell line GB2, was established from a leukemic Max41 x Emu-myc double transgenic mouse. Its Igh alleles are rearranged and its surface markers are primarily B-lymphoid, but a small proportion of the cells also express surface Gr-1 and some cells develop the morphology of maturing granulocytes. The cell line grows continuously in suspension culture without the addition of growth factors, but expresses mRNA for M-CSF, TPO and Flt-3-ligand. When stimulated in agar cultures by GM-CSF, G-CSF, M-CSF, IL-3, SCF, IL-6, leukemia inhibitory factor (LIF), IL-5 or IFNgamma, GB2 cells generated blast colonies or colonies of maturing granulocytes and macrophages. There was a striking similarity in colony types, relative colony numbers and maturation of colony cells to those formed by normal bone marrow cells in response to the same stimuli. GB2 blast colony-forming cells exhibited self-renewal as well as an ability to form granulocyte-macrophage colony-forming progeny, with evidence that a hierarchical sequence of clonogenic cells is generated in the cell line even after subcloning. Factor-specific maturation was clearly initiated by the action of the added growth factors. In contrast, FACS-sorting experiments showed that commitment to various types of colony-forming cell occurs in maintenance suspension cultures in the apparent absence of potentially relevant growth factors.


Subject(s)
Cell Differentiation , Granulocytes/cytology , Leukemia, B-Cell/pathology , Macrophages/cytology , Animals , Cell Division/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Mice , Phenotype , Tumor Cells, Cultured
5.
Proc Natl Acad Sci U S A ; 97(16): 9174-9, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10908669

ABSTRACT

Mice lacking the gene encoding the suppressor of cytokine signaling-1 (SOCS-1 -/-) and heterozygous for the IFN-gamma gene (IFN-gamma +/-) avoided the IFN-gamma-dependent preweaning death of SOCS-1 -/- IFN-gamma +/+ mice but did not exhibit the good health of young adult SOCS-1 -/- IFN-gamma -/- mice. SOCS-1 -/- IFN-gamma +/- mice died within 160 days of birth with massive T lymphocyte, macrophage, and eosinophil infiltration of all skeletal muscles and a similar severe myocarditis. The cornea also developed inflammatory infiltration and often a corneal ulcer. The mice exhibited evidence of selective CD8 T lymphocyte activation in populations in the thymus, spleen, and lymph nodes and focal T- and B-lymphoid infiltrates developed in the lung and salivary gland without apparent tissue damage. Comparison of SOCS-1 -/- IFN-gamma +/- mice with various control mice indicated that the development of tissue-damaging T lymphocyte, macrophage, and eosinophil infiltrates required loss of SOCS-1 and the presence of some IFN-gamma, but that the lung lymphoid infiltrates required only loss of SOCS-1 to develop.


Subject(s)
Carrier Proteins/physiology , Heterozygote , Interferon-gamma/genetics , Myocarditis/genetics , Polymyositis/genetics , Repressor Proteins , Animals , Carrier Proteins/genetics , Mice , Mice, Inbred C57BL , Myocarditis/pathology , Polymyositis/pathology , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins
6.
Leukemia ; 13(6): 926-34, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10360382

ABSTRACT

Mice with homozygous inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) protein die within 21 days of birth with low body weight, fatty degeneration and necrosis of the liver, infiltration of the lung, pancreas, heart and skin by macrophages and granulocytes and a profound depletion of T- and B-lymphocytes. In the present study, SOCS-1 -/- mice were found to have a moderate neutrophilia, and reduced platelet and hematocrit levels. Replacement of the SOCS-1 gene by a lac-Z reporter gene allowed documentation by FACS sorting that at least a proportion of granulocyte-macrophage progenitor cells transcribe SOCS-1. Most hematopoietic progenitor cell frequencies were normal in -/- marrow as were the size and cellular content of colonies formed by -/- progenitor cells in response to various stimulating factors. However, there was an increased frequency of macrophage progenitor cells in -/- mice and, abnormally, one quarter of all progenitor cells were located in the liver. Progenitor cells from -/- mice were hyper-responsive to stimulation by GM-CSF but not by M-CSF or Multi-CSF (IL-3). Progenitor cells from -/- mice were also hypersensitive to inhibition by interferon-gamma (IFN-gamma), the degree of inhibition varying markedly with the stimulating factor used. The suppressive effects of IFN-gamma therefore appear to involve interactions with particular growth factor-initiated signals in -/- cells--interactions that are strongly modulated by the action of the SOCS-1 protein.


Subject(s)
Carrier Proteins/genetics , Hematopoiesis/genetics , Repressor Proteins , Animals , Bone Marrow/metabolism , Bone Marrow/pathology , Female , Hematopoietic Stem Cells/pathology , Interferon-gamma/physiology , Leukocytes, Mononuclear/pathology , Male , Mice , Mice, Knockout , Spleen/pathology , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins
7.
Blood ; 93(5): 1579-85, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10029586

ABSTRACT

Marrow cells from mice lacking high-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF; betac-/- mice) were shown to bind and internalize much less GM-CSF than cells from normal (betac+/+) mice. betac-/- mice were used to determine the effect of negligible receptor-mediated clearance on detectible GM-CSF responses to the intravenous injection of endotoxin or the intraperitoneal injection of casein plus microorganisms. Unlike the minor serum GM-CSF responses to endotoxin seen in betac+/+ mice, serum GM-CSF levels rose 30-fold to 9 ng/mL in betac-/- mice even though loss of GM-CSF in the urine was greater than in betac+/+ mice. Organs from betac-/- and betac+/+ mice had a similar capacity to produce GM-CSF in vitro, as did peritoneal cells from both types of mice when challenged in vitro by casein. However, when casein was injected intraperitoneally, betac-/- mice developed higher and more sustained levels of GM-CSF than did betac+/+ mice. The data indicated that receptor-dependent removal of GM-CSF masks the magnitude of GM-CSF responses to endotoxin and local infections. Because of this phenomenon, serum GM-CSF concentrations can be a misleading index of the occurrence or nonoccurrence of GM-CSF responses to infections.


Subject(s)
Bacterial Infections/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Lipopolysaccharides/pharmacology , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Animals , Caseins/administration & dosage , Chelating Agents/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Granulocyte-Macrophage Colony-Stimulating Factor/urine , Injections, Intraperitoneal , Mice , Mice, Knockout
8.
Leukemia ; 12(3): 353-62, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9529130

ABSTRACT

GM-CSF transgenic mice were crossed with mice with homozygous inactivation of the gene encoding the common beta chain (beta c) of the GM-CSF receptor to produce mice with constitutively elevated GM-CSF levels but no high-affinity GM-CSF receptors. GM-CSF transgenic beta c -/- mice had exceptionally elevated serum GM-CSF levels but failed to develop the abnormal peritoneal cell population, eye destruction or tissue lesions characteristic of GM-CSF transgenic beta c +/+ mice. The alveolar proteinosis of beta c -/- mice was not altered in GM-CSF transgenic beta c -/- mice. Levels of GM-CSF mRNA in transgenic GM-CSF beta c -/- were elevated but lower than in transgenic beta +/+ mice and the higher serum GM-CSF levels were traced in part to the longer serum half-life of GM-CSF in beta c -/- than in beta c +/+ mice although urinary loss of GM-CSF was higher in beta c -/- than in +/+ mice. The data indicate that the transgenic phenotype was due to stimulation by GM-CSF and not an insertional effect, that low-affinity receptors are not capable of initiating tissue pathology even in the presence of excess GM-CSF levels and that autocrine production of GM-CSF by GM-CSF-responsive cells also fails to induce changes in these cells. The results support current dogma that the action of polypeptide regulators is mediated exclusively by activation of high-affinity membrane receptors.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/deficiency , Animals , Crosses, Genetic , Eye/pathology , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Homozygote , Leukocyte Count , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Mice, Transgenic , Organ Specificity , Polymerase Chain Reaction , Pulmonary Alveolar Proteinosis/genetics , Pulmonary Alveolar Proteinosis/pathology , RNA, Messenger/biosynthesis , Transcription, Genetic
9.
Blood ; 88(10): 3755-64, 1996 Nov 15.
Article in English | MEDLINE | ID: mdl-8916939

ABSTRACT

The intraperitoneal injection into mice of casein preparations containing bacteria induced a rapid accumulation of neutrophils within 3 hours due to selective release of mature cells from the bone marrow. Significant increases in the concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) occurred in the peritoneal cavity during the process, but the intraperitoneal injection of neither CSF induced a significant accumulation of neutrophils and the coinjection of G-CSF and casein failed to enhance the neutrophil response. The lack of involvement of either CSF in the neutrophil migration was confirmed by the development of typical neutrophil exudates when casein was injected into mice with inactivation of the genes encoding GM-CSF, G-CSF, or the beta-common chain of the GM-CSF receptor. However, preinjection of G-CSF increased the number of marrow neutrophils available for migration and did result in increased numbers of neutrophils in the peritoneal cavity after casein injection. Typical eosinophil inflammatory responses to the injection of casein or thioglycollate occurred in GM-CSF -/ -mice but not in beta c -/- mice, suggesting that interleukin-5 was necessary for this response.


Subject(s)
Chemotaxis, Leukocyte/physiology , Granulocyte Colony-Stimulating Factor/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Neutrophils/physiology , Peritonitis/physiopathology , Animals , Ascitic Fluid/chemistry , Ascitic Fluid/cytology , Bacterial Infections/physiopathology , Bone Marrow/pathology , Caseins/toxicity , Eosinophils , Granulocyte Colony-Stimulating Factor/analysis , Granulocyte-Macrophage Colony-Stimulating Factor/analysis , Leukocyte Count , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Peritonitis/chemically induced , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Thioglycolates/toxicity
10.
Leukemia ; 10(4): 713-9, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8618452

ABSTRACT

Double transgenic mice were produced by mating granulocyte-macrophage colony-stimulation factor (GM-CSF) transgenic mice with max 41 transgenic mice that exhibit excess granulopoiesis and a predisposition to thymic lymphoma development. Although only two-thirds of the double transgenic mice had elevated circulating GM-CSF levels, double transgenic mice maintained significantly higher blood granulocytes and monocytes and more extreme granulopoietic hypercellularity in the marrow and spleen than max 41 transgenic mice. In double transgenic mice, early death occurred from the GM-CSF transgenic syndrome. Because of these early deaths, the incidence of thymic and generalized lymphomas was artificially lower than in max 41 mice but those lymphomas that did develop occurred earlier than in max 41 mice. While the excess GM-CSF levels in double transgenic mice stimulated increased granulocyte and monocyte formation and peritoneal dendritic cells were excessive, this failed to prevent the spontaneous development of T lymphomas, suggesting that dendritic cell-initiated suppression of tumor development may not be effective with this type of tumor.


Subject(s)
DNA-Binding Proteins/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Leukemia, Experimental/genetics , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Bone Marrow/immunology , Bone Marrow Cells , Cells, Cultured , Crosses, Genetic , DNA-Binding Proteins/genetics , Dendritic Cells/cytology , Dendritic Cells/immunology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Hematopoietic Stem Cells/cytology , Leukemia, Experimental/immunology , Leukemia, Experimental/physiopathology , Lymph Nodes/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Transgenic , Spleen/cytology , Spleen/immunology , Transcription Factors/biosynthesis
11.
Leukemia ; 9(9): 1556-64, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7544853

ABSTRACT

Factor-specific cell line bioassays were used to monitor the production in vitro by adult and fetal mouse organs of GM-CSF, G-CSF, M-CSF, Multi-CSF (IL-3), IL-6 and leukemia inhibitory factor (LIF). No tissue was observed to produce Multi-CSF. Highest producers of the other regulators were lung, muscle, thymus, heart and bone shaft and all tissues producing detectable growth factors produced all five with the same rank order of activity. Adult tissues produced more GM-CSF than G-CSF and less M-CSF than either, no differences being observed between male, female and pregnant female tissues. In contrast, the pregnant uterus produced high levels of M-CSF as did the fetal membranes and tissues with only low GM-CSF and no G-CSF production. Pre-irradiation did not alter the pattern of regulator production by adult tissues. The intravenous injection of endotoxin elevated serum levels of GM-CSF, G-CSF, M-CSF and IL-6 but the dominant rise was in G-CSF levels. The data indicating that multiple organs produce the regulators monitored in a common rank order suggest some overall linkage in their production with major differences between adult and fetal tissues.


Subject(s)
Colony-Stimulating Factors/biosynthesis , Growth Inhibitors/biosynthesis , Lymphokines/biosynthesis , Animals , Base Sequence , Biological Assay , Cell Line , Colony-Stimulating Factors/blood , Culture Media, Conditioned , Female , Fetus/metabolism , Granulocyte Colony-Stimulating Factor/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Growth Inhibitors/blood , Interleukin-3/biosynthesis , Interleukin-6/biosynthesis , Leukemia Inhibitory Factor , Lymphokines/blood , Macrophage Colony-Stimulating Factor/biosynthesis , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred CBA , Molecular Sequence Data
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