Toshie Harada

IFN-γ Induction by SCG, 1,3-β-D-Glucan from Sparassis crispa, in DBA/2 Mice In Vitro

Sparassis crispa Fr. in an edible mushroom recently cultivable in Japan. A branched β-glucan from S. crispa (SCG) is a major 6-branched 1,3-β-D-glucan showing antitumor activity. In this study, we examined interferon-γ (IFN-γ) induction by SCG from splenocytes in DBA/2 mice in vitro. In the splenocytes derived from almost all inbred strains of mice except for DBA/1 and DBA/2 mice, IFN-γ production was not induced by SCG. The breeder and genders of DBA/2 mice showed no influence on IFN-γ induction by SCG. On the other hand, the magnitude of IFN-γ induction was lower in young mice than in their older counterparts. IFN-γ was induced by SCG in adherent splenocytes, but IFN-γ production was most significantly increased by SCG in instances involving coexistence of adherent and nonadherent splenocytes. In fact, inhibition of cell-cell contact reduced IFN-γ induction by SCG. In addition, interleukin-12 p70 (IL-12p70) was induced by SCG in DBA/2 mice. It was suggested that soluble factors and cell-cell contact med...

Contribution of dectin-1 and granulocyte macrophage–colony stimulating factor (GM-CSF) to immunomodulating actions of β-glucan

beta-Glucans are major cell wall structural components in fungi. As they are not found in animals, these carbohydrates are considered to be classic pathogen-associated molecular patterns (PAMPs), and are recognized by the innate immune system. Although their immunomodulating activities have been shown to be associated with the recognition of some fungi, and with their medicinal properties in the field of cancer immunotherapy, it is still unclear how beta-glucans mediate their effects. Recent studies have started to shed some light on their cellular receptors, such as dectin-1, and their molecular mechanisms of action. We have extensively investigated the response of leukocytes to beta-glucan, focusing on cytokine induction by SCG, which is a major 6-branched 1,3-beta-d-glucan in Sparassis crispa Fr. There is a strain difference in the reactivity of mice to SCG, and DBA/1 and DBA/2 mice are highly sensitive strains. In the process of research on cytokine induction by SCG in DBA/2 mice, we found that GM-CSF plays a key biological role in this activity. Cytokine induction by SCG was completely abolished in dendritic cells from dectin-1 knockout mice. On the other hand, controlling the level of endogenous GM-CSF production and/or dectin-1 expression could regulate the reactivity to beta-glucan. These results indicate that the key factors in the responsiveness to beta-glucan are GM-CSF production and dectin-1 expression. In this review, we describe how the key molecules related to the expression of the immunomodulating activities of beta-glucan were identified, and how the response to beta-glucan is controlled.

Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Regulates Cytokine Induction by 1,3-β-D-Glucan SCG in DBA/2 Mice In Vitro

Sparassis crispa Fr. is an edible/medicinal mushroom that recently became cultivable in Japan. SCG is a major 6-branched 1,3-beta-D-glucan in S. crispa showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice strongly react with SCG to produce interferon-gamma (IFN-gamma). In this study, cytokines induced by SCG were screened and found to be IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12 (IL-12p70). The addition of recombinant murine GM-CSF (rMuGM-CSF) to spleen cell cultures from various strains of mice synergistically enhanced IFN-gamma, TNF-alpha and IL-12p70 in the presence of SCG. In contrast, neutralizing GM-CSF using anti-GM-CSF monoclonal antibody (mAb) significantly inhibited IFN-gamma, TNF-alpha, and IL-12p70 elicited by SCG. We conclude that GM-CSF is a key molecule for cytokine induction by beta-glucan, and GM-CSF induction by SCG is the specific step in DBA/2 mice in vitro.

Chondroitin sulfate isomers in normal human urine.

Abstract 1. 1. Enzymic analysis of urinary chondroitin sulfate isomers in normal subjects was carried out on the basis of unsaturated disaccharide subunits produced by the digestion of urinary chondroitin sulfates with chondroitinases and chondrosulfatases. 2. 2. By paper chromatographic and electrophoretic separations, four unsaturated disaccharides were detected as the products of degradation of urinary chondroitin sulfate isomers with chondroitinases. 3. 3. Minor constituents of unsaturated nonsulfated disaccharide and unsaturated di-sulfated disaccharide were demonstrated in addition to the major components of unsaturated 4-sulfated and 6-sulfated disaccharides in normal human urine. This finding indicates that nosulfated and/or undersulfated chondroitin sulfate, as well as oversulfated chondroitin sulfate, are present in normal urine. The distribution of the unsaturated disaccharides fractioned by NaCl elution from Dowex columns reflected the diverse spectrum of urinary chondroitin sulfate isomers.

Enhanced Cytokine Synthesis of Leukocytes by a β‐Glucan Preparation, SCG, Extracted from a Medicinal Mushroom, Sparassis crispa

Abstract Sparassis crispa is edible mushroom recently cultivable in Japan. It contains significantly high content (∼40%) of 6‐branched 1,3‐β‐D‐glucan showing antitumor activity in mice. We recently purified a β‐glucan preparation designated as “SCG.” It was considered worth while to test SCG in vitro with whole blood collected from human volunteers. The present study is focusing on the cytokine productivity of SCG in an in vitro human system. The following results were observed: (i) SCG dose dependently enhanced IL‐8 synthesis of whole blood cell culture of human peripheral blood. (ii) IL‐8 synthesis was enhanced in both PBMC and PMN cultures. (iii) IL‐8 synthesis was induced in the culture with autologous plasma, but significantly reduced after 56°C treatment. (iv) The activity was also weak in heat inactivated fetal calf serum (FCS). (v) A complement fragment, C5a, was released by SCG dependently upon dose and kinetics. (vi) Anti‐SCG natural antibody was detected in human plasma. From these facts, SCG was observed to have the capacity to activate human leukocytes and related immune system.

Mechanism of Enhanced Hematopoietic Response by Soluble β-Glucan SCG in Cyclophosphamide-Treated Mice

SCG is a major 6‐branched 1,3‐β‐D‐glucan in Sparassis crispa Fr. SCG shows antitumor activity and also enhances the hematopoietic response in cyclophosphamide (CY)‐treated mice. In the present study, the molecular mechanism of the enhancement of the hematopoietic response was investigated. The levels of interferon‐ (IFN‐) γ, tumor necrosis factor‐ (TNF‐) α, granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), interleukin‐ (IL‐) 6 and IL‐12p70 were significantly increased by SCG in CY‐treated mice. GM‐CSF production in the splenocytes from the CY‐treated mice was higher than that in normal mice regardless of SCG stimulation. Neutralizing GM‐CSF significantly inhibited the induction of IFN‐γ, TNF‐α and IL‐12p70 by SCG. The level of cytokine induction by SCG was regulated by the amount of endogenous GM‐CSF produced in response to CY treatment in a dose‐dependent manner. The expression of β‐glucan receptors, such as CR3 and dectin‐1, was up‐regulated by CY treatment. Blocking dectin‐1 significantly inhibited the induction of TNF‐α and IL‐12p70 production by SCG. Taken together, these results suggest that the key factors in the cytokine induction in CY‐treated mice were the enhanced levels of both endogenous GM‐CSF production and dectin‐1 expression.

Highly Expressed Dectin-1 on Bone Marrow-Derived Dendritic Cells Regulates the Sensitivity to β-Glucan in DBA/2 Mice

Sparassis crispa beta-glucan (SCG) is a major six-branched 1,3-beta-D-glucan in S. crispa Fr. showing antitumor activity. We previously found that DBA/1 and DBA/2 mice are highly sensitive to SCG in vivo and in vitro. In this study, we investigated the effect of SCG on bone marrow-derived dendritic cells (BMDCs) in DBA/2 mice in vitro. SCG increased the expression of CD80, MHC class I and MHC class II molecules on the cell membrane of BMDCs. SCG induced BMDCs to produce interleukin-12p70 (IL-12p70), IL-6, and tumor necrosis factor-alpha (TNF-alpha). The magnitude of cytokine induction by SCG in DBA/2 mice was higher than that in C57BL/6, BALB/c, C3H/HeN, and C3H/HeJ mice. The expression level of the beta-glucan receptor, dectin-1, on BMDCs in DBA/2 mice was also highest in DBA/2 among these mice. Blocking dectin-1 significantly inhibited the induction of TNF-alpha production by SCG. Taken together, these results suggest that the BMDCs from DBA/2 mice are highly sensitive to the induction of cytokine production by SCG in vitro, and that this sensitivity is related to the expression level of dectin-1.

Soy Isoflavone Aglycone Modulates Expression of Cell Surface Antigens in Vitro and in Vivo

Soy isoflavone aglycones (IFAs) have a wide range of biological actions. We investigated in this study the effect of IFAs on myeloid cells. The cell surface expression of both CD80 and CD86 was up-regulated by treating myeloid cells with IFAs in vitro and in vivo. The findings suggest that IFAs could modulate the myeloid cell function.