Noriko N. Miura

Dectin-2 Recognition of α-Mannans and Induction of Th17 Cell Differentiation Is Essential for Host Defense against Candida albicans

Dectin-2 (gene symbol Clec4n) is a C-type lectin expressed by dendritic cells (DCs) and macrophages. However, its functional roles and signaling mechanisms remain to be elucidated. Here, we generated Clec4n(-/-) mice and showed that this molecule is important for host defense against Candida albicans (C. albicans). Clec4n(-/-) DCs had virtually no fungal alpha-mannan-induced cytokine production. Dectin-2 signaling induced cytokines through an FcRgamma chain and Syk-CARD9-NF-kappaB-dependent signaling pathway without involvement of MAP kinases. The yeast form of C. albicans induced interleukin-1beta (IL-1beta) and IL-23 secretion in a Dectin-2-dependent manner. In contrast, cytokine production induced by the hyphal form was only partially dependent on this lectin. Both yeast and hyphae induced Th17 cell differentiation, in which Dectin-2, but not Dectin-1, was mainly involved. Because IL-17A-deficient mice were highly susceptible to systemic candida infection, this study suggests that Dectin-2 is important in host defense against C. albicans by inducing Th17 cell differentiation.

Solubilization of yeast cell-wall β-(1→3)-d-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction

The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a beta-(1-->3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The beta-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of beta-(1-->3)- and beta-(1-->6)-D-glucosidic linkages. The specific activity of the beta-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.

Relationship between Solubility of Grifolan, a Fungal 1,3-β-D-Glucan, and Production of Tumor Necrosis Factor by Macrophages in Vitro

Grifolan, GRN, is a fungal antitumor β-glucan isolated from Grifola frondosa. Various studies suggested that the underlying mechanism of the antitumor activity of GRN is strongly related to immune modulation. In the previous publication (Adachi et al., 1994; Okazaki et al., 1995), we have shown that GRN activates macrophages to produce tumor necrosis factor (TNF) in vitro. In this study, the structural unit essential to produce TNF was examined by chemical modifications of GRN. GRN suspended in distilled water was treated at 150°C for up to 3 h. Addition of the resulting turbid solution to the RAW 264.7 macrophage-like cell line produced TNF, and the relative activity was diminished in relation to the heat treatment period. The fractions with a heating period longer than 15 min did not show any activity. After centrifugation of the resulting solution, significant activity was shown by precipitate fractions, suggesting that the insoluble form of GRN is important for TNF production. Interestingly, the precipitate fraction obtained from 9 min of treatment also had significant activity. In addition, admixing the soluble fraction with the particles significantly inhibited the TNF production. In contrast to these observations, the high-molecular-mass subfraction of the soluble fraction prepared by ultrafiltration produced significant amounts of TNF. Similar phenomena were shown with sodium hydroxide treatment and dimethylsulfoxide treatment. These facts strongly suggested that insoluble as well as a high molecular mass soluble form of GRN are required for TNF production by macrophages.

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...

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.

Relationship between the physical properties of Candida albicans cell well β-glucan and activation of leukocytes in vitro

We previously reported that the fungal particle 1,3-beta-D-glucan derived from Candida albicans, a pathogenic fungus, was obtained by oxidation of the cell wall with sodium hypochlorite (NaClO). It could be solubilized by treatment with dimethylsulfoxide (DMSO). In the present study, we prepared Candida 1,3-beta-D-glucan having different physical properties, and examined the relationship between leukocyte activation and the physicochemical properties. Beta-glucan activated leukocytes significantly more effectively in a particulate than solubilized form in terms of TNF-alpha production by RAW 264.7 cells, hydrogen peroxide production by murine PEC and IL-8 production by human PBMC. Furthermore, we compared the biological activity of the glucan particles oxidized under various conditions. Interestingly, inactive and antagonistic particles were obtained under strong oxidation conditions. However, the inactive particles showed significant agonistic activity on dissolution in DMSO and following lyophilization. These facts strongly suggested that the solubility and assembly of the components influence the immunopharmacological activities of 1,3-beta-D-glucans.

Structure and biological activities of hypochlorite oxidized zymosan

Abstract Zymosan (ZYM), a strong complement activating yeast cell preparation, is also a potent inflammatory substance, which shows immunopharmacological activity. Major component of ZYM is β-glucan but contains other constituents, such as mannan, protein, and nucleic acid. We applied sodium hypochlorite (NaClO) treatment to ZYM to reduce impurities and compared the activity with native/parent ZYM. Oxidized ZYM (OX-ZYM) became a nitrogen-free agent. By NMR analysis of native OX-ZYM and zymolyase (endo-1,3-β-glucanase) digest, OX-ZYM was found to contain 1,3-β-linked and 1,6-β-linked glucan moieties, while the latter degraded by sodium metaperiodate treatment. OX-ZYM also contained small amounts of anionic groups, partly reducible by sodium borohydride. Degree of polymerization (DP) of 1,6-β-glucan moiety was estimated to be about DP10–DP50 by MALDI-TOF-MS analysis. In comparison with ZYM activities, OX-ZYM and derivatives showed strong antitumor activity to solid form of Sarcoma 180 in mice, and showed strong activity on alternative pathway of complement, but lost secondary response to ZYM-immune mice. These facts strongly suggested that a particulate form of β-glucan was prepared by NaClO treatment of ZYM and at least a part of ZYM-mediated biological activity was found unmediated by β-glucan moiety.

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.

Activation of toll-like receptor-mediated NF-κβ by zymosan-derived water-soluble fraction : Possible contribution of endotoxin-like substances

Zymosan is a well-known reagent for the examination of inflammatory response and is prepared from yeast, Saccharomyces cerevisiae. In the activation process, Toll-like receptor (TLR) 2 and TLR6 act as functional receptors for NF-κB activation. Although zymosan is primarily composed of β-glucans, little is known about the active component of zymosan-mediated biological activities. The active moiety of zymosan was fractionated by its solubility in water, and its biological activity on macrophages and TLRs-transfectants examined. The macrophage cell line, RAW264.7, was treated with zymosan-derived preparations, and tumor necrosis factor alpha (TNF-α) produced in the culture supernatant was measured by ELISA. Increased TNF-α production was observed by stimulation with water-soluble (ZWS) or water-insoluble fraction (ZWIS). ZWS showed higher activity in TNF-α production. NF-κB activation via TLR2, TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 also was enhanced by stimulation with ZWS and ZWIS. In particular, ZWS showed higher activity via TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 than other preparations. ZWS activity was decreased by treatment with polymyxin B, but not with lysozyme and zymolyase. Furthermore, ZWS contained significant more endotoxin than any other preparations. Therefore, we suggest that the active moiety of ZWS for the NF-κB activation has an endotoxin-like substance, that is abundantly observed in Gram-negative bacteria. These results imply that the inflammatory activity of zymosan is induced not only by β-glucans, but also by other endotoxin-like water-soluble substances.

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.