Plasma G-CSF and GM-CSF Concentration and Amount of Their Receptors on the Granulocyte in Kawasaki Disease

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.

Plasma G-CSF and GM-CSF Concentrations and Expression of their Receptors on the Granulocyte in Children with Leukocytosis

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.

Expression of Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) and Clinical Correlates in Acute Leukemia / 대한혈액학회지

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) is commonly used to reduce leukopenic period during treatment of malignancy including acute leukemia. Leukemic blasts expressing granulocyte colony-stimulating factor receptor (G-CSFR) were reported and also may proliferate in response to therapeutic administration of G-CSF. However, it is not clear whether G-CSFR expression on leukemic blasts is related to clinical outcome such as leukocyte recovery or leukemia relapse. Current study evaluated expression of G-CSFR in acute leukemia and correlated with hematologic and clinical parameters. METHODS: Peripheral blood or bone marrow aspirate was evaluated from 20 patients with acute myelogenous leukemia (AML) and 10 with acute lymphoblastic leukemia (ALL), 2 with acute undifferentiated leukemia (AUL), 1 with acute biphenotypic leukemia (ABL), 1 with acute mixed-lineage leukemia (AMLL). G-CSFR expression was analyzed using flow cytometry and was correlated with immunophenotype and response for chemotherapy. RESULTS: More than 20% of blasts were positive for G-CSFR in 65% (13/20) of AML, 40% (4/10) of ALL, and all negative in ABL, AMLL, and AUL. Except that all 6 monocytic lineage leukemias (M4, M5) and all three cases of ALL with CD33 expression were positive, no consistent correlation was observed among G-CSFR expression pattern, type of acute leukemia, response to induction therapy and relapse (P>0.05). CONCLUSION: Current study revealed G-CSFR was expressed on not only myelogenous leukemic cells but also lymphoid ones. Although our data suggest G-CSFR expression does not affect therapeutic outcome, it remains to be determined whether G-CSF therapy is safe in G-CSFR-positive acute leukemia.

Quantities of Receptor Molecules for Colony Stimulating Factors on Leukocytes in Measles

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.

Transcriptional activation function of intracellular domain of EPOR and G-CSFR protein in yeast: a comparative study / 解放军医学杂志

Objective To investigate the feasibility of searching for proteins which interact with intracellular domain of hematopoietic growth factor receptors using yeast two-hybrid system. Methods RT-PCR method was performed to amplify the genes of intracellular domains of G-CSF receptor and EPO receptor in NFS-60 and BET-2 cells of mice. The genes were cloned into yeast expression plasmid pGBKT7 vector,and then transformed into yeast AH109. The yeast proteins were isolated and analyzed with Western blotting. Transcriptional activation was analyzed by the ?-galactosidase colony-lift filter assay. Results The intracellular domains of G-CSF receptor and EPO receptor genes were successfully cloned into pGBKT7 vector. The results of Western blotting assay showed that both proteins were expressed in the yeast cells. The ?-galactosidase colony-lift filter assay demonstrated that G-CSF receptor alone had no activity of transcriptional activation,while the EPO receptor alone could activate transcription. Conclusion The findings suggested that intracellular domain of G-CSF receptor could be used as a bait to find interacting proteins using yeast two-hybrid system,while that of the EPO receptor could not. Therefore the system could not be applied to all hematopoietic factor receptor.

Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes

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.