Ken-ichi Ishibashi

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.

Immunomodulating Activity of Agaricus brasiliensis KA21 in Mice and in Human Volunteers

We performed studies on murine models and human volunteers to examine the immunoenhancing effects of the naturally outdoor-cultivated fruit body of Agaricus brasiliensis KA21 (i.e. Agaricus blazei). Antitumor, leukocyte-enhancing, hepatopathy-alleviating and endotoxin shock-alleviating effects were found in mice. In the human study, percentage body fat, percentage visceral fat, blood cholesterol level and blood glucose level were decreased, and natural killer cell activity was increased. Taken together, the results strongly suggest that the A. brasiliensis fruit body is useful as a health-promoting food.

Barley-derived β-d-glucan induces immunostimulation via a dectin-1-mediated pathway

Barley-derived beta-glucan, a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose polymers, binds to dectin-1. However, whether it can trigger signal transduction via dectin-1 remains unclear. In this study, we used a reporter gene assay to determine whether barley-derived beta-d-glucan can activate NF-kappaB via dectin-1-mediated signaling when dectin-1 is cotransfected with Syk, CARD9, and Bcl10 in 293T cells. We found that barley-derived beta-D-glucan can activate NF-kappaB leading to cytokine production when dectin-1, Syk, CARD9, and Bcl10 are coexpressed in the cells. We also found that barley-derived beta-D-glucan can induce the phosphorylation of Syk and production of IL-6 in thioglycolate-elicited peritoneal macrophages. These results indicated that the immunostimulatory effects of barley-derived beta-d-glucan might be exerted, at least in part, via dectin-1.

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.

An unambiguous structural elucidation of a 1,3-β-d-glucan obtained from liquid-cultured Grifola frondosa by solution NMR experiments

Grifolan LE (GRN-LE), a purified beta-D-glucan, which is obtained from liquid-cultured Grifola frondosa, exhibits various biological activities, including antitumor effects. Significant progress has been made in the study of these effects. However, an unambiguous structural characterization of GRN-LE using NMR spectroscopy has not been carried out as yet. It is well accepted that the biological effects of a beta-glucan depend on its primary structure, conformation, and molecular weight. In the present study, we unambiguously elucidate the primary structure of GRN-LE using NMR spectroscopy. The data presented here reveal that GRN-LE comprises a 1,3-beta-D-glucan backbone with a single 1,6-beta-D-glucosyl side branching unit on every third residue.

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.

An ITAM-Syk-CARD9 signalling axis triggers contact hypersensitivity by stimulating IL-1 production in dendritic cells

A variety of reactive organic compounds, called haptens, can cause allergic contact dermatitis. However, the innate immune mechanisms by which haptens stimulate dendritic cells (DCs) to sensitize T cells remain unclear. Here we show that the coupling of ITAM-Syk-CARD9 signalling to interleukin-1 (IL-1) secretion in DCs is crucial for allergic sensitization to haptens. Both MyD88 and Caspase recruitment domain-containing protein 9 (CARD9) signalling are required for contact hypersensitivity (CHS). Naïve T cells require signals received through IL-1R1-MyD88 for effector differentiation, whereas DCs require CARD9 and spleen tyrosine kinase (Syk) signalling for hapten-induced IL-1α/β secretion and their ability to prime T cells. DC-specific deletion of CARD9, DAP12, Syk or NLRP3, but not MyD88, is sufficient to abolish CHS. All tested haptens, but not irritants, can induce Syk activation, leading to both the CARD9/BCL10-dependent pro-IL-1 synthesis (signal1) and reactive oxygen species-mediated NLRP3 inflammasome activation (signal2), required for IL-1 secretion. These data unveil an innate immune mechanism crucial for allergic contact sensitization to chemical compounds.

Structural characterisation and biological activities of a unique type β- d -glucan obtained from Aureobasidium pullulans

A β-d-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of β-d-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this β-d-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-β-glucans like AP-FBG with such a high number of 1-6-β-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-β-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.

Agaricus brasiliensis-derived β-glucans exert immunoenhancing effects via a dectin-1-dependent pathway

Agaricus brasiliensis is a well-known medicinal mushroom. We have previously demonstrated that Agaricus-derived polysaccharides exhibit potent antitumor effects; however, the underlying mechanism(s) have not been elucidated yet. In this study, we examined the immunoenhancing activities of Agaricus extracts. Agaricus-derived polysaccharides were characterized as 1,6-β-glucan with a small amount of 1,3-β-glucan using anti-β-glucan antibody and nuclear magnetic resonance analysis. These polysaccharides strongly induced the production of various cytokines from both murine splenocytes and bone marrow-derived dendritic cells in the presence of exogenous granulocyte-macrophage colony-stimulating factor. Polysaccharide-induced cytokine production was significantly reduced in bone marrow-derived dendritic cells derived from dectin-1-deficient mice. Furthermore, a binding assay revealed that the Agaricus-derived polysaccharides can be recognized by dectin-1, a pivotal receptor for 1,3-β-glucan. Taken together, our results clearly indicate that the immunostimulation induced by Agaricus-derived polysaccharides is exerted, at least in part, via dectin-1 in combination with granulocyte-macrophage colony-stimulating factor.

Candida soluble cell wall β-glucan facilitates ovalbumin-induced allergic airway inflammation in mice: Possible role of antigen-presenting cells

BackgroundAlthough fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble β-glucan from Candida albicans (CSBG) (Ohno et al., 2007). In the present study, the effects of CSBG exposure on airway immunopathology in the presence or absence of other immunogenic allergen was investigated in vivo, and their cellular mechanisms were analyzed both in vivo and in vitro.MethodsIn vivo, ICR mice were divided into 4 experimental groups: vehicle, CSBG (25 μg/animal), ovalbumin (OVA: 2 μg/animal), and CSBG + OVA were repeatedly administered intratracheally. The bronchoalveolar lavage cellular profile, lung histology, levels of cytokines and chemokines in the lung homogenates, the expression pattern of antigen-presenting cell (APC)-related molecules in the lung digests, and serum immunoglobulin values were studied. In vitro, the impacts of CSBG (0–12.5 μg/ml) on the phenotype and function of immune cells such as splenocytes and bone marrow-derived dendritic cells (BMDCs) were evaluated in terms of cell proliferation, the surface expression of APC-related molecules, and OVA-mediated T-cell proliferating activity.ResultsIn vivo, repeated pulmonary exposure to CSBG induced neutrophilic airway inflammation in the absence of OVA, and markedly exacerbated OVA-related eosinophilic airway inflammation with mucus metaplasia in mice, which was concomitant with the amplified lung expression of Th2 cytokines and IL-17A and chemokines related to allergic response. Exposure to CSBG plus OVA increased the number of cells bearing MHC class II with or without CD80 in the lung compared to that of others. In vitro, CSBG significantly augmented splenocyte proliferation in the presence or absence of OVA. Further, CSBG increased the expression of APC-related molecules such as CD80, CD86, and DEC205 on BMDCs and amplified OVA-mediated T-cell proliferation through BMDCs.ConclusionCSBG potentiates allergic airway inflammation with maladaptive Th immunity, and this potentiation was associated with the enhanced activation of APCs including DC.