Fusayuki Omori

Constitutive production of granulocyte colony-stimulating factor and interleukin-6 by a human lung cancer cell line, KSNY: Gene amplification and increased mRNA stability

Abstract: A human lung squamous cell carcinoma cell line designated KSNY was established from a patient suffering from marked neutrophilia and polyclonal hyper‐γ‐globulinemia. In our previous report, we demonstrated colony‐stimulating activities in the culture supernatant of this cell line. To determine the exact molecules for the activities, we investigated the gene expression of various cytokines in KSNY cells and showed the mRNA expression of both granulocyte colony‐stimulating factor (G‐CSF) and interleukin‐6 (IL‐6). We also detected substantial amounts of G‐CSF and IL‐6 in the culture supernatant with sensitive enzyme‐linked immunosorbent assays (ELISA). The amplification of the gene locus for G‐CSF, but not for IL‐6 was shown by Southern blot analysis. Furthermore, we also showed that the mRNAs for G‐CSF and IL‐6 were relatively stable in KSNY cells. These findings are thought to be related to the constitutive production of the cytokines in KSNY cells.

Levels of human serum granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor under pathological conditions

Levels of serum granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with various leukocyte disorders were estimated by enzyme linked immunosorbent assay (ELISA). Some cases of acute myelogenous leukemia and aplastic anemia showed elevated serum levels of G-CSF and/or GM-CSF, whereas almost all of 23 healthy controls showed G-CSF and GM-CSF levels lower than 100 pg/ml. High levels of both types of CSF were noted in patients with granulocytosis due to infection. These levels became lower after resolution of the infection. Daily changes in serum CSF levels were also examined in a patient with autoimmune neutropenia, and it was found that the peripheral neutrophilic granulocyte count changed almost in parallel with the serum G-CSF level but not with GM-CSF, following the pattern with a delay of about 4–5 h, suggesting the possibility that G-CSF mainly regulates peripheral neutrophil circulation.

Measurement of human G-CSF by enzyme-linked immunosorbent assay using monoclonal antibody

SummaryAn IgG monoclonal antibody to recombinant human granulocyte colony-stimulating factor (G-CSF), designated HG1, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. This HG1 was capable of neutralizing the colony-stimulating activity of G-CSF in vitro, and it did not cross-react with human granulocyte/macrophage colony-stimulating factor (GM-CSF). An enzyme-linked immunosorbent assay (ELISA) for measurement of G-CSF was developed using HG1 and a polyclonal antibody against G-CSF raised in a rabbit. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50pg/ml of recombinant G-CSF. This assay system therefore warrants further attention.

Detection of Granulocyte-Macrophage Colony-Stimulating Factor in Cerebrospinal Fluid of Patients with Aseptic Meningitis

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the cerebrospinal fluid from 14 infants and children with meningitis and 6 patients who suffered other diseases besides meningitis was measured by our sensitive enzyme linked immunosorbent assay for GM-CSF. The minimal detection level of GM-CSF was 40 pg/ml. Six of 9 patients (67%) with aseptic meningitis had detectable GM-CSF in cerebrospinal fluid and the concentrations of GM-CSF ranged from 49 to 114 pg/ml (mean 72 pg/ml), whereas none of 5 patients with bacterial meningitis or 6 patients with other diseases besides meningitis had detectable GM-CSF levels. There was no clear correlation between the GM-CSF levels in cerebrospinal fluid and the leukocyte count in either peripheral blood or cerebrospinal fluid, or the concentration of protein or glucose in cerebrospinal fluid.

Muroctasin [MDP-Lys(18)] augments the production of granulocyte colony-stimulating factor (G-CSF) from human peripheral blood mononuclear cells in vitro

N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-Lysine (MDP-Lys(L18), muroctasin) is an immunopotentiating substance. Neutrophilia and elevated levels of colony-stimulating factor (CSF) in peripheral blood were previously found after the administration of this compound in both mice and humans. To specify the type of CSF and to elucidate the mechanisms of the neutrophilia, we cultured human peripheral blood mononuclear cells (PBMC) in the presence of muroctasin and measured the levels of granulocyte CSF (G-CSF) in the culture supernatants using our sensitive enzyme-linked immunosorbent assay. G-CSF is an active hematopoietic growth factor specific for cells of a neutrophilic lineage, and muroctasin was found to significantly augment the G-CSF production from PBMC in vitro (P less than 0.01). Furthermore, production of G-CSF from human PBMC in the presence of muroctasin was also supported by the Northern blot analysis using cDNA encoding G-CSF as a probe.

Stimulatory effects of bestatin on human B-cell colony formation

Bestatin, (2S, 3R)-3-amino-2-hydroxy-4-phenylbutyryl-L-leucine, is a small molecular immunomodifier. Effects of this compound on human immune function were studied, in vitro, using the human B-cell colony formation technique. B-cell colonies were obtained from enriched B-cell populations placed in conditioned methylcellulose medium containing stimulators and irradiated T-cells as feeders. Addition to the culture of Bestatin at concentrations of 0.1 microgram/ml and 1 microgram/ml led to a significant increase (P less than 0.05) in the number of B-cell colonies and this effect was abolished when irradiated T-cells were not added to the culture. Bestatin increased soluble factor production induced by phytohemagglutinin (PHA)-stimulated T-cells. Such findings suggest that T-cells probably mediate this stimulatory effect of Bestatin on B-cell colony formation.

A traditional Chinese herbal medicine, Ren-shen-yang-rong-tang (Japanese-name: Ninjin-yoei-to) augments the production of granulocyte-macrophage colony-stimulating factor from human peripheral blood mononuclear cells in vitro

Ren-shen-yang-rong-tang (Japanese name: Ninjin-yoei-to, NYT) is a traditional Chinese herbal medicine. Leukocytosis and elevated levels of colony-stimulating factor (CSF) in peripheral blood were found previously after the administration of this compound to mice. In this study, human peripheral blood mononuclear cells (PBMC) were cultured in the presence of NYT in vitro, and the levels of granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) in the supernatant of cultured PBMC were measured using a sensitive enzyme-linked immunosorbent assays. NYT significantly (P less than 0.01) augmented GM-CSF production but not G-CSF production by PBMC in vitro.

Serum Granulocyte Colony-Stimulating Factor Levels in Chronic Neutropenia of Infancy

Eight infants with chronic neutropenia ranging in age from 1 to 13 months were studied for serum granulocyte colony-stimulating factor (G-CSF) levels. Serum G-CSF levels were elevated, especially when peripheral blood absolute neutrophil counts (ANC) were under 500/microliter in seven patients with autoimmune neutropenia. On the other hand, in a patient with congenital agranulocytosis (Kostmann type), G-CSF levels were below the sensitivity of the assay (less than 50 pg/ml) despite severe neutropenia (less than 100/microliter).

CD7-positive acute myeloid leukemia : further evidence of cellular immaturity

SummaryAmong 63 cases of acute myeloid leukemia (AML), 14 were found to express the CD7 antigen, a cell surface marker usually found at an early stage during T lineage differentiation. The CD7-positive AML cases consisted of 5 cases of M1, 3 cases of M2, 3 cases of M4, 1 case of M5, 1 case of M6 and 1 case of M7. Among these 63 cases, the proportion of blast cells expressing the CD34 antigen was examined. The proportion of CD34-stained cells among the CD7-positive AML cases, although varying, was significantly larger than that among the CD7-negative AML cases (P<0.05). As the CD34 antigen was expressed on hematopoietic progenitor cells and was considered to reflect an early hematopoietic stage, the high proportion of cells expressing CD34 among the CD7-positive AML cases may support the notion that CD7-positive AML cells are immature.

Measurement of human granulocyte-macrophage colony-stimulating factor (GM-CSF) by enzyme-linked immunosorbent assay

SummaryAn IgG monoclonal antibody against recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), designated HGMI, was produced by fusion of immune mouse splenocytes with HAT-sensitive murine myeloma cells. A sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of human GM-CSF was developed using this HGMI and a polyclonal antibody against GM-CSF raised in a rabbit. GM-CSF in culture supernatants of phytohemagglutinin (PHA)- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) were measured by this ELISA system and the conventional CFU-GM colony formation method. The data indicated that the ELISA was highly efficient and sensitive for the detection of as little as 50 pg/ml recombinant GM-CSF. The CFU-GM colony assay may be influenced by other cytokines which can enhance or suppress colony formation, and ELISA for GM-CSF is more useful for kinetic studies of precise levels of production from PBMC.