Effect of GM-CSF on cytokine induction by soluble beta-glucan SCG in vitro in beta-glucan-treated mice.

SCG is a 6-branched 1,3-beta-D-glucan, which are major cell wall structural components in fungi. Leukocytes from DBA/1 and DBA/2 mice are highly sensitive to SCG, producing cytokines such as GM-CSF, IFN-gamma, TNF-alpha and IL-12p70, but not IL-6. GM-CSF plays a key biological role in this activity. In the present study, we examined the effect of giving i.p. SCG to DBA/2 mice on cytokine production in vitro. SCG was given i.p. to DBA/2 mice on day 0. Splenocytes were prepared on day 7 and cultured in the presence of SCG in vitro. The levels of cytokine production induced by SCG in vitro were lower in the cells from SCG-treated mice than in control mice. Expression of the beta-glucan receptor, dectin-1, in SCG-treated mice was comparable with that shown in control mice. However, the consumption of exogenously added rmGM-CSF in vitro was observed in SCG-treated mice. The addition of a large amount of rmGM-CSF to the culture medium resulted in larger amounts of TNF-alpha and IL-6 in SCG-treated mice than in normal mice. These results suggested that GM-CSF was closely related with the reactivity of beta-glucan. Giving SCG increased the number of macrophages and granulocytes in the spleen. These results suggested that in SCG-treated mice, a change of cell population would be related to modulation of the profile of cytokine production induced by SCG in vitro.

Highly expressed dectin-1 on bone marrow-derived dendritic cells regulates the sensitivity to beta-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.

NMR characterization of the structure of a beta-(1-->3)-D-glucan isolate from cultured fruit bodies of Sparassis crispa.

SCG, a purified beta-d-glucan, obtained from Sparassis crispa, exhibits various biological activities including an antitumor effect, enhancement of the hematopoietic response in cyclophosphamide-induced leukopenic mice, and induction of the production of cytokines. The mechanisms of these effects have been extensively investigated; however, an unambiguous structural characterization of SCG is yet to be achieved. It is well accepted that the biological effects of beta-glucan depend on its primary structures, conformation, and molecular weight. In the present study, we examine the difference of biological effects among beta-glucans, elucidate the primary structure of SCG, and compare with SPG from Schizophyllum commune using NMR spectroscopy. Our data reveal that SCG but not SPG induce cytokine production from bone marrow-derived dendritic cells (BMDCs) and their major structural units are a beta-(1-->3)-d-glucan backbone with single beta-(1-->6)-d-glucosyl side branching units every three residues.

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.

Mechanism of enhanced hematopoietic response by soluble beta-glucan SCG in cyclophosphamide-treated mice.

SCG is a major 6-branched 1,3-beta-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-)gamma, tumor necrosis factor-(TNF-)alpha, 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-gamma, TNF-alpha 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 beta-glucan receptors, such as CR3 and dectin-1, was up-regulated by CY treatment. Blocking dectin-1 significantly inhibited the induction of TNF-alpha 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.

Cell to cell contact through ICAM-1-LFA-1 and TNF-alpha synergistically contributes to GM-CSF and subsequent cytokine synthesis in DBA/2 mice induced by 1,3-beta-D-Glucan SCG.

SCG is a major 6-branched 1,3-beta-D-glucan in Sparassis crispa Fr. showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice are potently induced by SCG to produce interferon- gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12p70 (IL-12p70), and that GM-CSF plays a key biologic role among these cytokines. In this study, we investigated the contribution of cell-cell contact and soluble factors to cytokine induction by SCG in DBA/2 mice. Cell-cell contact involving intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) was an essential step for the induction of GM-CSF and IFN-gamma by SCG but not for the induction of TNF-alpha or IL-12p70 by SCG. SCG directly induced adherent splenocytes to produce TNF-alpha and IL-12p70. GM-CSF was required for the induction of TNF-alpha by SCG, and in turn, TNF-alpha enhanced the release of GM-CSF and thereby augmented the induction of IL-12p70 and IFN-gamma by SCG. Neutralization of IL-12 significantly inhibited the induction of IFN-gamma by SCG. We concluded that induction of GM-CSF production by SCG was mediated through ICAM-1 and LFA-1 interaction, GM-CSF subsequently contributed to further cytokine induction by SCG, and reciprocal actions of the cytokines were essential for enhancement of the overall response to SCG in DBA/2 mice.

Soy isoflavone aglycone modulates a hematopoietic response in combination with soluble beta-glucan: SCG.

Soy isoflavone aglycones (IFAs) have a wide range of biological actions that suggest they may be of use in cancer prevention. On the other hand, a branched beta-glucan from Sparassis crispa (SCG) is a major 6-branched 1,3-beta-D-glucan in an edible/medicinal mushroom: Sparassis crispa showing antitumor activity. We have previously reported that both oral and intraperitoneal administration of SCG enhanced the hematopoietic response in cyclophosphamide (CY)-induced leukopenic mice. In this study, we investigated the hematopoietic response due to IFA in combination with SCG in CY-induced leukopenic mice. The oral administration of IFA in combination with SCG synergistically enhanced the number of white blood cells, and increased spleen weight. Analyzing the leukocyte population by flow cytometry, the combination of IFA and SCG increased the number of monocytes and granulocytes in the spleen. Taken together, the combination of IFA and SCG synergistically provides the hematopoietic responses that are enhanced over IFA or SCG alone.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates cytokine induction by 1,3-beta-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.

Antibody to soluble 1,3/1,6-beta-D-glucan, SCG in sera of naive DBA/2 mice.

A branched beta-glucan from Sparassis crispa (SCG) is a major 6-branched 1,3-beta-D-glucan showing antitumor activity. In the present study, we examined the anti-SCG antibody in naive mice by ELISA. Using SCG coated plate, sera of naive DBA/1 and DBA/2 mice contained significantly higher titers of antibody than other strains of mice. Anti-SCG Ab titers of each DBA/1 and DBA/2 mice were significantly varied. Using various polysaccharide-coated plate, sera of DBA/2 mice also reacted with a beta-glucan from Candida spp. (CSBG) having 1,3-beta and 1,6-beta-glucosidic linkages. The SCG specific immunoglobulin (Ig) M but G was detected in sera. The reactivity of sera to coated SCG was neutralized by adding soluble SCG and CSBG as competitor. These results suggested that DBA/1 and DBA/2 strains carry specific and unique immunological characteristics to branched 1,3-/1,6-beta-glucan.

Structure and biological activities of beta-glucans from yeast and mycelial forms of Candida albicans.

We have achieved the extraction of cell wall beta-glucan from the mycelial form of Candida albicans (C. albicans) IFO 0579 (M-CSBG) by using acetic acid, sodium hypochlorite (NaClO), and dimethylsulfoxide (DMSO) treatments. The yield of M-CSBG was significantly lower (7.5% from dried mycelial cells) than that of the yeast form from C. albicans IFO 1385 (Y-CSBG, 25.9% from dried yeast cells). The properties of M-CSBG were similar to those of Y-CSBG in terms of nuclear magnetic resonance (NMR) spectra and limulus reactivity. Molecular weight (Mw) of M-CSBG was slightly higher than that of Y-CSBG. Both Y-CSBG and M-CSBG induced the production of comparable amounts of macrophage inflammatory protein-2 (MIP-2), a chemotactic factor, from mouse peritoneal exudate cells (PEC) in vitro. These findings suggest that the structure and properties of CSBG from yeast and mycelial cells are similar to each other.

Effect of CAWS, a mannoprotein-beta-glucan complex of Candida albicans, on leukocyte, endothelial cell, and platelet functions in vitro.

Candida albicans is a medically important fungus which induces a disseminated candidasis and candidemia in immunocompromised hosts, and releases a polysaccharide fraction into the blood. We recently found that C. albicans released a water-soluble polysaccharide fraction (CAWS) into synthetic medium and demonstrated that CAWS was mainly composed of a complex of mannan and beta-glucan. In the murine system, CAWS showed a lethality resembling anaphylactic shock when administered i.v., and induced coronary arteritis similar to Kawasaki Disease (KD) when given i.p. In the present study, we examined the biological activity of CAWS in the cell culture and found the following: i) CAWS slightly induced production of IFN-gamma and IL-6 by splenocytes at lower dose (ca. 10 micro g/ml), but at a higher dose strongly inhibited the proliferation of splenocytes induced by a B cell mitogen, lipopolysaccharide (LPS) and a T cell mitogen, concanavalin A. ii) The viability of these splenocytes monitored by propidium iodide staining was significantly reduced. iii) The addition of CAWS to a culture of monophage RAW264.7 cells significantly reduced cellular growth rate dose dependently. iv) The LPS-mediated synthesis of cytokines by RAW264.7 cells was significantly inhibited by CAWS. v) CAWS induced an aggregation of platelets in human platelet-rich plasma, and vi) CAWS inhibited the production of thrombomodulin by human umbilical endothelial cells and acted synergistically with TNF-alpha. Thus, CAWS strongly inhibited the cellular functions of leukocytes in vitro, partly through direct cytotoxicity. The enhanced production in injured cells of the vascular endothelium would be related to the local inflammatory response in the coronary artery.

Enhanced cytokine synthesis of leukocytes by a beta-glucan preparation, SCG, extracted from a medicinal mushroom, Sparassis crispa.

Sparassis crispa is edible mushroom recently cultivable in Japan. It contains significantly high content (approximately 40%) of 6-branched 1,3-beta-D-glucan showing antitumor activity in mice. We recently purified a beta-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 degrees 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.

Effect of SCG, 1,3-beta-D-glucan from Sparassis crispa on the hematopoietic response in cyclophosphamide induced leukopenic mice.

Sparassis crispa Fr. is an edible mushroom recently cultivable in Japan. It contains a remarkably high content of 6-branched 1,3-beta-D-glucan showing antitumor activity. Using ion-exchange chromatography, a purified beta-glucan preparation, SCG, was prepared. In this study, we examined the hematopoietic response by SCG in cyclophosphamide (CY)-induced leukopenic mice. SCG enhanced the hematopoietic response in CY induced leukopenic mice by intraperitoneal routes over a wide range of concentrations. SCG enhanced the hematopoietic response in CY-treated mice by prior or post administration. Analyzing the leukocyte population by flow cytometry, monocytes and granulocytes in the peritoneal cavity, liver, spleen and bone marrow (BM) recovered faster than in the control group. The ratio of natural killer cells and gammadelta T cells in the liver, spleen and peritoneal cavity was also increased. In contrast, CD4+ CD8+ cells in the thymus were temporarily significantly decreased by the administration of SCG. Interleukin-6 (IL-6) production of CY+SCG-treated peritoneal exdated cells (PECs), spleen cells and bone marrow cells (BMCs) were higher than that of the CY-treated group. By in vitro culture of CY-treated PEC and spleen cells, IL-6 production was enhanced by the addition of SCG. These facts suggested the possibility that IL-6 might be a key cytokine for the enhanced hematopoietic response by SCG.

Synergistic action of beta-glucan and platelets on interleukin-8 production by human peripheral blood leukocytes.

The effects of human platelets on interleukin (IL)-8 production from human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) stimulated with the fungal (1-->3)-beta-D-glucan schizophyllan (SPG) were examined using ELISA. PBMCs/PMNs in the presence of platelets and SPG enhanced IL-8 production in comparison with those in the presence of either platelets or SPG. IL-8 production was dependent on the concentration of platelets and incubation time, and the activity reached the maximal level at 18 h of incubation. These activities were also observed with the addition of platelets prestimulated with SPG to PBMCs. Addition of SPG directly enhanced expression of P-selectin on platelet membrane surfaces. These results suggest that platelets play a key role in the cytokine production of leukocytes induced by fungal (1-->3)-beta-D-glucans and might be mediated, at least in part, by P-selectin.

Application of Candida solubilized cell wall beta-glucan in antitumor immunotherapy against P815 mastocytoma in mice.

This study was designed to evaluate the antitumor activity of CSBG, purified from the cell wall of Candida albicans IFO1385. First, as an effect of CSBG on P815 mastocytoma, significant prolonged survival and suppression of the tumor growth were observed. Second, the transfer of spleen cells from CSBG-sensitized BALB/c mice to CDF1 mice led to further suppression of tumor growth as well as P815-immunized spleen cells. Third, CSBG enhanced antitumor immunity in gene therapy using B7-1-transfected P815 cells. These results strongly suggest that CSBG enhances the host defense response to tumor due in part to an adjuvant effect.

IFN-gamma induction by SCG, 1,3-beta-D-glucan from Sparassis crispa, in DBA/2 mice in vitro.

Sparassis crispa Fr. in an edible mushroom recently cultivable in Japan. A branched beta-glucan from S. crispa (SCG) is a major 6-branched 1,3-beta-D-glucan showing antitumor activity. In this study, we examined interferon-gamma (IFN-gamma) 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-gamma production was not induced by SCG. The breeder and genders of DBA/2 mice showed no influence on IFN-gamma induction by SCG. On the other hand, the magnitude of IFN-gamma induction was lower in young mice than in their older counterparts. IFN-gamma was induced by SCG in adherent splenocytes, but IFN-gamma 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-gamma 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 mediate synergistic effects on SCG-induced IFN-gamma production.