M.C. Fung

ANALYSIS OF IMMUNOMODULATING CYTOKINE mRNAs IN THE MOUSE INDUCED BY MUSHROOM POLYSACCHARIDES

The immunomodulating action of two mushroom antitumor polysaccharides, polysaccharide-protein complex (PSPC) and lentinan, was elucidated through analysing the expression profile of cytokines during a time course (0 h to 48 h) after their administration. Among the 5 cytokine genes, the induction of a marked increase in the mRNA levels of IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma and M-CSF by PSPC and lentinan was observed in the peritoneal exudate cells and splenocytes. However, the time point of their increased production was different after PSPC and lentinan administration.

Pineal indoles stimulate the gene expression of immunomodulating cytokines

Summary. Male C57 mice received 10 consecutive daily intraperitoneal injections of melatonin, 5-methoxytryptamine or 5-methoxytryptophol (5 mg/kg body weight). Control mice received the alcoholic saline vehicle. All mice were sacrificed 24 hours after the last injection. Following extraction of RNA from peritoneal exudate cells (PEC) and splenocytes, the level of gene expression was analyzed with the reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that melatonin up-regulated the level of gene expression of transforming growth factor-β (TGF-β), macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-α (TNF-α) and stem cell factor (SCF) in PEC, and the level of gene expression of interleukin-1β (IL-1β), M-CSF, TNF-α, interferon-γ (IFN-γ) and SCF in splenocytes. 5-Methoxytryptamine augmented the level of gene expression of TGF-β, M-CSF and SCF in PEC, and the level of gene expression of IL-1β, TNF-α, IFN-γ, M-CSF and SCF in splenocytes. 5-Methoxytryptophol elevated the level of gene expression of TNF-α, IL-1β, TGF-β and M-CSF in PEC, and the level of gene expression of inducible nitric oxide synthase (iNOS), IL-1β, M-CSF, TNF-α, IFN-γ and SCF in splenocytes.

Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complexes

Two antitumor polysaccharide-protein complexes, PSPC and PSK from mushrooms, were compared for their modulating effect on cytokine and cytokine receptor gene expression. RNA samples were isolated from the splenocytes and peritoneal exudate cells of the untreated or treated mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the cytokine gene expression. Nine out of 17 cytokine mRNAs and 5 out of 6 cytokine receptor mRNAs were detected in the splenocytes and peritoneal exudate cells from both untreated and treated mice. However, IL-4 was only detected in the splenocytes while IL-7 and IL-1R(typeI) were only detected in the peritoneal exudate cells. Among the 9 cytokine genes, the expression level of M-CSF was up-regulated in splenocytes and peritoneal exudate cells of the mice by PSPC and PSK. The expression level of TNF-alpha was only up-regulated in the peritoneal exudate cells by PSK, but not by PSPC.

Involvement of tumor necrosis factor (TNF-α) in arsenic trioxide induced apoptotic cell death of murine myeloid leukemia cells

Arsenic trioxide (As(2)O(3)) has recently been shown to be effective to inhibit the growth and to induce apoptosis in acute promyelocytic leukemia (APL) but not in acute myeloid leukemia (AML) cells. Recently, we have isolated an As(2)O(3) sensitive subclone JCS-16 from the murine myeloid leukemia WEHI 3B (JCS). At the concentrations of 0.3-3 microM, As(2)O(3) induces a dose-dependent cytotoxicity and growth inhibition on the JCS-16 cells. As(2)O(3) also induces apoptotic cell death, as judged by the presence of apoptotic nuclei, at 6 h after treatment. Morphological differentiation was not observed in As(2)O(3) treated JCS cells. Neutralizing anti-TNF-alpha antibody was found to reduce the As(2)O(3)-mediated apoptotic cell death of JCS-16 cells. Growth inhibitory effect of As(2)O(3) was also reduced after the addition of anti-TNF-alpha. In addition, reverse transcription polymerase chain reaction (RT-PCR) and reverse northern blot analysis demonstrated that the expression of TNF receptor (TNF-R2), IL-4, and IL-4R was down-regulate at 1 h after As(2)O(3) treatment. The expression of TNF-alpha and TNF-R1 was not affected. Our results suggest that the autocrine action of TNF-alpha might play a role in As(2)O(3)-induced apoptotic cell death of JCS-16 leukemia cells.

EFFECTS OF BIOCHANIN A ON THE GROWTH AND DIFFERENTIATION OF MYELOID LEUKEMIA WEHI-3B (JCS) CELLS

The effects of biochanin A on the growth and differentiation of a recently characterized myeloid leukemia cell line WEHI-3B (JCS) were investigated. Biochanin A not only inhibited the growth of JCS cells in a dose-dependent manner (0 - 200 microM) but also induced the morphological differentiation of JCS cells. The phagocytic activity of biochanin A-treated JCS cells was also increased. Flow cytometric analysis showed that the expression of macrophage differentiation markers Mac-1 and F4/80 was up-regulated in biochanin A-treated JCS cells. The expression level of Mac-1 was higher than that of F4/80. The expression of cytokine genes was studied by reverse transcription-polymerase chain reaction (RT-PCR) and cycle titration. mRNA levels of IL-1alpha, IL-1beta and IL-4 were found to be up-regulated at 46 hours after incubation of JCS cells with biochanin A. Although the expression of LIF was also up-regulated, the LIF receptor gene was not expressed in the uninduced or induced JCS cells. Our results suggest that IL-1alpha, IL-1beta and IL-4 may act on the later stage of biochanin A-mediated differentiation of JCS cells.

Effects of midazolam on the differentiation of murine myeloid leukemia cells.

The effects of midazolam (MID) on the in vitro growth and differentiation of two murine myeloid leukemia WEHI 3B (JCS) and M1 cells were studied. MID inhibits the proliferation of both M1 and JCS cells in a dose-dependent manner. At the concentration of 10 micrograms/ml, MID was found to induce both monocytic and granulocytic differentiation of the JCS but not M1 cells. Induction of morphological differentiation of the JCS cells was also associated with the enhanced expression of the differentiation antigens Mac-1, F4/80, and Gr-1 for the cells. Results from mRNA phenotyping experiments also indicated that the expression of tumor necrosis factor (TNF-alpha) and neutrophil-specific J11d differentiation marker was significantly upregulated in MID-treated JCS cells. In addition, the phagocytic activity of MID-treated JCS cells was increased towards opsonized yeast cells. Results from this investigation suggested that MID may be used as an inducer for further study on the mechanisms of differentiation in these myeloid leukemia cells.