The hematopoietic system of the mouse arises from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac at day 7.0 of gestation. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Previous studies demonstrated that the development kinetics and growth factor responsiveness of hematopoietic precursors derived from embryonic stem cells (ES cells) is similar to that found in the yolk sac, indicating that the onset of hematopoiesis within the embryoid bodies (EBs) parallels that found in the embryo. Furthermore, in vitro differentiation of ES cells to hematopoietic cells is valuable for establishment of therapeutic clone against a variety of hematological disorders. Despite the identification of multipotential hematopoietic progenitors in EBs, a subset of more primitive progenitors, identical to the high proliferative potential colony-forming cells (HPP-CFC) derived from human and murine hematopoietic tissues, have not been clearly identified regarding particular their replating potential in vitro. HPP-CFC is among the most primitive hematopoietic multipotent precursors cultured in vitro. In this study, our aim was to investigate the in vitro and in vivo hematopoietic capacity of HPP-CFC within the day 12 EBs, rather than the expansion of more committed progenitors. In this study the HPP-CFC could be detected within EBs differentiated for 5 to 14 days of murine ES cells, but the development dynamics of the HPP-CFC differed greatly among distinct serum lots. Qualitatively HPP-CFC is capable of forming secondary colonies. As to our expectation the ES cells-derived HPP-CFC demonstrated similar regeneration capacity to those from yolk sac, giving rise to secondary granulocyte, erythrocyte, macrophage and mast cells, however largely differed from the counterparts of adult bone marrow. In addition, by RT-PCR ES cells-derived HPP-CFC were found to express transcription factors associated closely with stem cell proliferation including SCL, GATA-2 and AML1 as well as various receptors of hematopoietic growth factors such as c-kit, GM-CSF receptor and interleukin 3 receptor et al. Finally, in order to understand the in vivo hematopoietic capacity of the ES cells-derived HPP-CFC, spleen colony-forming unit (CFU-S) assay was performed. Nevertheless, typical CFU-S was not observed after transplantation of the day 12 EB cells or HPP-CFC colonies into lethally irradiated adult murine. In conclusion the HPP-CFC differentiated from murine ES cells displayed robust hematopoietic activity in vitro, however their in vivo reconstitution ability was not detected. The difference between in vitro and in vivo hematopoietic activities of ES cells-derived primitive hematopoietic precursors deserves further investigation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Cell Differentiation ; genetics ; physiology ; Colony-Forming Units Assay ; Core Binding Factor Alpha 2 Subunit ; genetics ; Embryonic Stem Cells ; cytology ; GATA2 Transcription Factor ; genetics ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Mice ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Interleukin-3 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; T-Cell Acute Lymphocytic Leukemia Protein 1

PURPOSE: Granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF) are principal cytokines in granulopoiesis and their physiologic effects are mediated through binding to specific cell surface receptors. Although it is known that the level of serum G-CSF and GM-CSF, and presentation of the receptors are increased in infectious diseases, there have been no studies to find the correlation between the granulopoiesis and leukocytosis. This study was designed to measure G-CSF and GM-CSF in leukocytosis and in control and to demonstrate the possible pathogenesis of granulopoiesis in leukocytosis using quantitative analysis of G- CSF, GM-CSF and their CSFr. METHODS: The plasma levels of G-CSF, GM-CSF of 13 children without leukocytosis and 14 children with leukocytosis were measured. Counts of cell surface G-CSFr and GM-CSFr were measured by combining anti G-CSFr and anti GM-CSFr monoclonal antibodies to their respective receptors by using quantitative flow cytometric assay. RESULTS: There was no significant difference betweeen the plasma concentration of G-CSF and GM-CSF in acute leukocytosis and in the control group. However, levels of G-CSFr in acute leukocytosis decreased significantly compared to the control(P=0.012) and the levels of GM-CSFr in both groups revealed no significant difference. CONCLUSION: Increase in the number of leukocyte in leukocytosis was mediated by increasing the number of neutrophil, and increased plasma concentration of G-CSF may be the cause of neutrophilia. But GM-CSF did not have any influence on leukocytosis.
Antibodies, Monoclonal ; Child* ; Communicable Diseases ; Cytokines ; Granulocyte Colony-Stimulating Factor* ; Granulocyte-Macrophage Colony-Stimulating Factor* ; Granulocytes* ; Humans ; Leukocytes ; Leukocytosis* ; Neutrophils ; Plasma* ; Receptors, Cell Surface ; Receptors, Granulocyte Colony-Stimulating Factor ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor

PURPOSE: This study aimed to demonstrate the possible pathogenesis of granulopoiesis in patients of Kawasaki disease(KD) using quantitative analysis of G-CSF, GM-CSF and their CSFr. METHODS: The plasma levels of G-CSF, GM-CSF, G-CSFr and GM-CSFr were studied in 14 patients in the acute phase of KD; 13 children with normal peripheral white blood cell counts were used as the normal control group. The plasma concentration of G-CSF, GM-CSF were analyzed by ELISA. The G-CSFr and GM-CSFr on the peripheral granulocytes were analyzed by a quantitative flow cytometric assay and QuantiBRITE, and the quantitative changes of receptors which did not combine with G-CSF and GM-CSF were measured. RESULTS: The total number of leukocytes in KD was similar to normal control group, but the leukocytes increased according to the number of neutrophils. The plasma concentration of G-CSF were decreased similar to normal control group(P=0.133), but that of GM-CSF decreased more than the normal control group(P=0.227). The quantity of G-CSFr, GM-CSFr were revealed to be no less than the normal control(P=0.721, P=0.912). After incubation with excessive G-CSF, the expressed G-CSFr on the neutrophils were decreased in both groups(P=0.554). The quantities of expressions of GM- CSFr on the neutrophil after incubation with the excessive GM-CSF were always increased in both groups(P=0.255). The amount of GM-CSFr of neutrophils are in proportion to total white blood cells (r=0.788, P=0.035), but it wasn't in the case of KD(P=0.644). CONCLUSION: The leukocytosis in KD that mediated by increasing neutrophil was not correlated with the plasma concentrations of G-CSF and GM-CSF, and the amount of expression of G-CSFr and GM-CSFr on granulocyte. It is possible that the reduction of concentration of GM-CSF results by increasing the active GM-CSFr.
Child ; Enzyme-Linked Immunosorbent Assay ; Granulocyte Colony-Stimulating Factor* ; Granulocyte-Macrophage Colony-Stimulating Factor* ; Granulocytes* ; Humans ; Leukocyte Count ; Leukocytes ; Leukocytosis ; Mucocutaneous Lymph Node Syndrome* ; Neutrophils ; Plasma* ; Receptors, Granulocyte Colony-Stimulating Factor ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor

In this study, we report that an acute phase reactant, serum amyloid A (SAA), strongly inhibits dendritic cell differentiation induced by GM-CSF plus IL-4. SAA markedly decreased the expression of MHCII and CD11c. Moreover, SAA decreased cell surface GM-CSF receptor expression. SAA also decreased the expression of PU.1 and C/EBPα, which play roles in the expression of GM-CSF receptor. This inhibitory response by SAA is partly mediated by the well-known SAA receptors, Toll-like receptor 2 and formyl peptide receptor 2. Taken together, we suggest a novel insight into the inhibitory role of SAA in dendritic cell differentiation.
Dendritic Cells* ; Granulocyte-Macrophage Colony-Stimulating Factor* ; Interleukin-4 ; Receptors, Formyl Peptide ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor* ; Serum Amyloid A Protein* ; Toll-Like Receptors

To evaluate soluble GM-CSF-Ralpha expression in patients with acute myeloid leukemia (AML) and its clinic significance, plasma concentration of solGM-Ralpha in de novo 66 patients with AML was detected by enzyme-linked immuno-sorbent assay, and the relationship between solGM-Ralpha levels and various clinical parameters was analyzed. The result showed that the levels of solGM-Ralpha in plasma of patients with AML were significantly higher than that in plasma of normal controls; the lowest level of solGM-Ralpha was found in plasma of patients with AML-M3 (3897.75 +/- 2651.43 pg/ml), the highest level of solGM-Ralpha was observed in plasma of patients with AML-M5 (9990.92 +/- 6325.43 pg/ml). Patients with high level of solGM-Ralpha were generally accompanied with a distinct clinical picture, including higher counts of white blood cell and myeloid precursors, as well as higher expression of CD34, CD95 and CD116 antigen. It is concluded that the high level of solGM-Ralpha in plasma of patients may suggest AML poor prognosis and play a role in pathogenesis of leukemia, the GM-CSF and its receptor solGM-Ralpha needs further study.
Adolescent ; Adult ; Aged ; Antigens, CD34 ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Leukemia, Myeloid, Acute ; blood ; Male ; Middle Aged ; Prognosis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; blood ; fas Receptor ; blood

We analyzed the comparative amounts of granulocyte-colony stimulating factor (G-CSFr) and granulocyte macrophage CSF (GM-CSFr) receptors expressed on neutrophils and monocytes in measles patients to investigate the role of these CSFrs in the development of leukopenia including neutropenia and monocytopenia in measles. EDTA-anticoagulated peripheral blood of 19 measles patients, 10 children with other infections showing leukopenia and 16 children with normal complete blood cell counts (CBC)s were analyzed using flow cytometry and QuantiBRITE. The leukocyte (5260 +/- 2030/uL vs. 9900 + 2680/uL, p=0.000), neutrophil (2580 +/- 960/uL vs. 4250 +/- 2750/uL, p=0.024) and the lymphocyte counts of measles patients (1810 +/- 1430/uL vs. 4530 +/- 3450/uL, p= 0.006) were lower than in the normal controls. The neutrophils of measles patients expressed similar amounts of G- CSFr (1858 +/- 355) as normal children (1764 +/- 477, p= 0.564) and leukopenic patients (1773 +/- 673, p=0.713), but lower levels of GM-CSFr (535 +/- 118) than normal children (957 +/- 344, p=0.000) and leukopenic patients (832 +/- 294, p=0.002). The monocytes of measles patients expressed similar amounts of G-CSFr (916 +/- 336) and GM-CSFr (3718 +/- 906) as normal children (1013 +/- 391 and 4125 (2645, p > 0.05) but less than leukopenic patients (1454 +/- 398 and 5388 +/- 806, p > 0.05). The neutrophil and monocyte counts of measles patients did not correlate with the amount of G-CSFr or GM-CSFr expressed on neutrophils or monocytes (p > 0.05), but in the normal children, the monocyte count correlated with the levels of GM-CSFr on monocytes (r=0.951, p=0.049). In conclusion, neutropenia is one of the more important characteristics of measles patients, which could be due to the decreased GM-CSFr expression on neutrophils. However, the monocytopenia found in measles patients is not due to the decreased expression of CSFr on the monocytes.
Human ; Leukocyte Count ; Measles/*blood ; Monocytes/*chemistry ; Neutropenia/etiology ; Neutrophils/*chemistry ; Receptors, Granulocyte Colony-Stimulating Factor/*blood ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/*blood

BACKGROUND AND OBJECTIVES: Airway epithelial cells contribute to the pathogenesis of air disease by their interaction with inhalant pathogenic extracts. Airborne fungi interact with nasal epithelial cell and enhance the production of inflammatory cytokines. Glucocorticosteroids (GCs) have been used therapeutically for nasal polyps and allergic disease with potent anti-inflammatory effects. The purpose of this study was to investigate the inhibitory effect of GCs on fungi induced nasal epithelial cell activation. MATERIALS AND METHODS: The epithelial cells of nasal polyps were obtained from patients and stimulated with Alternaria. To evaluate the anti-inflammatory effects of GCs, Alternaria was pretreated with GCs (triamcinolone, dexamethasone, and budesonide) and cultured with epithelial cells. Interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) were measured to determine the activation of epithelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) test for protease-activated receptors (PARs) mRNA expression in nasal epithelial cells were performed. RESULTS: Alternaria enhanced the production of IL-8 and GM-CSF from nasal epithelial cells. GCs inhibited the activation of nasal epithelial cells, but the PAR2 and PAR3 mRNA expression were not suppressed by GCs. CONCLUSION: These data suggest that GCs inhibit the production of chemical mediators by Alternaria, but anti-inflammatory effect of GCs are not associated with PARs.
Adrenal Cortex Hormones* ; Alternaria ; Colony-Stimulating Factors ; Cytokines ; Dexamethasone ; Epithelial Cells* ; Fungi ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Interleukin-8 ; Nasal Polyps* ; Receptors, Proteinase-Activated ; RNA, Messenger

Administration of G-CSF may not always respond in rise of neutrophil counts in different patient population. In order to understand a possible inter-relationship between the G-CSF and GM-CSF induced leukocyte responses and expression levels of receptors for G-CSF (G-CSFr) and GM-CSF (GM-CSFr), the levels of each receptor and CSF were measured in patients with basophilia (8), eosinophilia (14) and bacterial infection showing neutrophilia (12) in comparison with normal healthy adults (12) and children (14). G-CSFr was expressed in neutrophils in the largest amount followed by monocytes, but GM-CSFr was expressed more in monocytes than neutrophils. Lymphocytes and basophils did not express G-CSFr or GM-CSFr. The amount of GM-CSFr in neutrophils was present less in patients with infection than normal control (P = 0.031). The neutrophils expressed more G-CSFr than GM-CSFr. The quantity of G-CSFr in eosinophil showed marked interval change, higher in acute stage. The plasma concentrations of G-CSF in patients with infection were much higher than normal adults or children (117.95 +/- 181.16 pg/ml, P < 0.05). Binding assay with excess amount of CSFs could discriminate the patient who did not show any response to G-CSF or GM-CSF administration. After incubation with excess CSFs, more receptors were blocked in children than in adults (G-CSF P = 0.024, GM-CSF P = 0.006). These results indicate that the amount of CSFr in leukocyte varies in different types of leukocyte, and changes according to the patients' condition even in the same type of leukocyte, and the CSFrs of children bind to CSFs more than those of adults.
Adult ; *Bacterial Infections ; Basophils/chemistry ; Breast Neoplasms ; Child ; Colony-Stimulating Factors/*blood ; Eosinophilia ; Human ; Leukemia, Myeloid, Chronic ; *Leukocyte Disorders ; Monocytes/chemistry ; *Neoplasms ; Neutrophils/chemistry ; Receptors, Colony-Stimulating Factor/*analysis ; Receptors, Granulocyte Colony-Stimulating Factor/analysis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/analysis

This study was aimed to detect the ratio of CD34+ cells in bone marrow mononuclear cells (BMMNCs) and the expression rate of G(M)-CSFR on CD34+ cells in bone marrow of the patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The ratio of CD34+ cells in BMMNCs and the expression rate of G(M)-CSFR on cells of 27 AA patients, 45 MDS patients and 20 controls were detected by flow cytometry (FCM). The results showed that the ratio of CD34+ cells in BMMNCs of AA patients reduced and was significantly different from controls (p<0.05), the ratio of CD34+ cells in MDS patients elevated and was significantly different from controls (p<0.05). Compared with controls and MDS-RA patients, the ratio of CD34+ cells in MDS-RAEB patients significantly elevated (p<0.05), but there was no significant difference between MDS-RA patients and controls (p>0.05). The ratio of CD34+ cells in MDS-RA patients was significantly higher than that in AA patients (p<0.05). There was no significant difference in expression rate of G-CSFR on CD34+ cells between AA patients and controls, MDS patients and controls, AA patients and MDS patients, MDS-RA patients and MDS-RAEB patients (p>0.05). The expression rate of GM-CSFR in MDS patients was significantly higher than that in AA patients and controls (p<0.05), but there was no significant difference between AA patients and controls, MDS-RA patients and MDS-RAEB patients (p>0.05). In AA patients, the ratio of CD34+ cells in BMMNCs was less than 0.1% accounts for 6/8 SAA patients, compared with 2/19 in CAA (p<0.05). There was no correlation between the expression rate of either G-CSFR or GM-CSFR and neutrophil count at diagnosis (r=0.058 and r=0.044). In MDS patients, there was no correlation between bone marrow CD34+ cells ratio and peripheral neutrophil count at diagnosis (r=-0.335). And there was no correlation between the expression of either G-CSFR or GM-CSFR and neutrophil count on diagnosis (r=0.064 and r=0.051). It is concluded the detection of CD34+ cells and their surface expression rate of G(M)-CSFR in AA and MDS is useful in diagnosis and differential diagnosis of these two diseases.
Adult ; Anemia, Aplastic ; metabolism ; Antigens, CD34 ; immunology ; Bone Marrow Cells ; cytology ; immunology ; metabolism ; Case-Control Studies ; Female ; Flow Cytometry ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; metabolism ; Receptors, Granulocyte Colony-Stimulating Factor ; metabolism ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism

The purpose of the present study was to investigate the biological mechanism for modulating granulocytopoiesis by Panax ginseng. The techniques of culture of hematopoietic progenitor cells and hematopoietic stromal cells in vitro, biological assay of hematopoietic growth factor (HGF), immunocytochemistry, in situ hybridization of nucleic acid, immunoprecipitation and Western blot were used to explore the effect of total saponins of Panax ginseng (TSPG) on the expression of human granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-macrophage colony stimulating factor receptor alpha (GM-CSFRalpha). The results indicated that (1) bone marrow stromal cell (BMSC), thymocyte (TC), splenocyte (SC), endothelial cells (EC), and monocyte (MO) conditioned media prepared with TSPG (50 microg/ml) could significantly enhance the proliferation of CFU-GM; (2) the expressions of GM-CSF in protein and mRNA level in BMSC, TC, SC, EC and MO induced by TSPG (50 microg/ml) were much higher than that of the control; (3) the expression of GM-CSFRalpha protein in hematopoietic cells induced by TSPG (50 microg/ml) was stronger than that of the control; (4) TSPG (50 microg/ml) could stimulate the transient tyrosine phosphorylation of GM-CSFR and Shc protein. We speculate that TSPG may directly and/or indirectly promote the stromal cells and lymphocytes to produce GM-CSF and other cytokine and induce bone marrow hematopoietic cells to express GM-CSF receptors (GM-CSFRalpha), leading to the regulation of the GM-CSFR-mediated signals transduction pathway and the proliferation of human CFU-GM.
Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Panax ; chemistry ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Saponins ; isolation & purification ; pharmacology ; Signal Transduction ; Stromal Cells ; cytology ; metabolism