Immunomodulatory Activities of Î²-Glucan in Mushroom - ACS

Chapter 34

Immunomodulatory Activities of β-Glucan in Mushroom Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch034

Masashi Mizuno

Graduate School of Agricultural Science, Kobe University, Nada-ku, Kobe 657-8501, Japan

Hot water extractsfromAgaricus brasiliensis S. Wasser et al. (ABSW) were dissolved in distilled water and fed to 8-weeks male ICR mice as drinking water for 14 days. The cumulative number of scratching behavior monitored for 20 min in salinetreated mice (control) was approximately 2,300 after intradermal injection of compound 48/80 which is a pruritogenic agent, whereas those in ABSW decreased to be approximately 960. To determine the effects of ABSW on degranulation in mast cell activation, histamine contents in blood were measured. ABSW treatment suppressed histamine release to 36% compared to control. When splenocytes were incubated with concanavalin A (Con A; Τ cell mitogen), contents of IFN-γ and IL-4 significantly increased compared with non-treatments of Con A, but IL-12 did not show any difference with/without Con A. Moreover, ABSW significantly induced IFN-γ production from splenocytes incubated with Con A. These results indicate that ABSW possess immunomodulating property that might be involved in the development of Th1 cells, culminating in an inhibition of immediate type allergy caused by compound 48/80.

© 2008 American Chemical Society In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch034

400 Allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis and food allergies, are steadily increasing especially in the industrialized countries. Key factors driving these rising trends are increased exposure to sensitizing allergens and reduced stimulation of the immune system during critical periods of development. Allergic responses involving IgE-dependent mast cell degranulation and eosinophil accumulation in the sites of inflammation are considered to be due to the development and activation of Th2 cells (7). The production of two distinct cytokine patterns recognized in subsets of helper T cells (Th) is especially important. The set designated as Thl is characterized by interleukin (IL)-12, interferon (IFN)-y and IL-2 production and activates macrophages. The Thl cytokines augment cell-mediated immunity. The other set, designated as Th2, is characterized by IL-4, -5, -6, -10, and -13 syntheses and Th2 cytokines promote humoral immunity (2). The representative cytokines of Th2 cells are IL-4 and IL-5. IL-4 is the major inducer of classswitching to IgE biosynthesis in B lymphocytes. IL-5 is the principal eosinophilactivating factor. On the other hand, IFN- y, which is a representative cytokine of Thl cells, is known to suppress the development of Th2 cells (5). Since it is suggested that Thl and Th2 types of reactions are reciprocally regulated in vivo (5), the modulation of Thl/Th2 balance, namely shifting the balance from Th2 to Thl dominance, should be a strategy for the therapy of allergic diseases involving Th2 cells. Allergic reactions, especially immediate type allergy, are genetically determined disorders characterized by an increased ability of B-lymphocytes to synthesize IgE antibodies towards ubiquitous antigens (allergens), able to activate the immune system after inhalation or ingestion and after penetration through the skin. IgE antibodies are able to bind to high affinity Fee receptors (FcєRI) present on the surface of mast cells/basophils (2). The mast cells, which are constituents of virtually all organs and tissue, are thought to play a major role in the development of many physiologic changes during allergic responses. Among the preformed and newly synthesized inflammatory substances released on degranulation of mast cells, histamine remains the best-characterized and most potent vasoactive mediator implicated in the acute phase of immediate type allergy (4). Compound 48/80 has been used as a direct and convenient reagent to study the mechanism of the anaphylactic reaction (5). Interest in the medicinal characters of natural products has increased due to their popular use in traditional medicine. Many available substances have been found in the foods, particularly in mushrooms. Agaricus brasiliensis S. Wasser et al (ABSW) is well-known among these mushrooms. It occurs naturally in a mountain region near Sao Paulo in Brazil and has become popularly known as "Himematsutake" in Japan and it has been traditionally used in food and folk medicine. It is reported that ABSW extract might be an effective stimulator for T cell and macrophage (6). Moreover, the expression of interleukin (IL)-12 (a

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


401 key role in Thl differentiation) and IL-18 (a proinflammatory cytokine in enhancing Thl immune response) mRNA in macrophage-like cell line, RAW264.7, stimulated with an polysaccharide purified in ABSW were investigated by RT-PCR in a previous study (7). It is reported that within 12 h there was no drastic changes mRNA expression in IL-18 during stimulation with polysaccharide purified in ABSW. However, mRNA expression in IL-18 levels rapidly increased to be 5.6-fold 24 h post treatment. The level of IL-12 p40 mRNA expression was different from those of IL-18, which started to show increase only 12 h and through 24 h post treatment. Moreover, polysaccharides from ABSW changed the percentage of splenic Thy 1.2- and L3T4 (CD4)positive cells in the T cell subsets in ABSW-treated mice (6). These results led to the presumption that this mushroom possessed the ability stimulate differentiation of naive T cells into T-helper typel (Thl), resulting in antiallergic activity. The objectives of the present study were to examine the immunomodulating effect on immunocompetent cell and mast cell in 8-weeks male ICR mice orally feed hot water extract from ABSW.

Materials and Methods Mice Seven week-old male ICR mice were purchased from Japan SLC (Sizuoka, Japan). The mice were housed in cages under specific pathogen-free conditions (air temperature at 25 °C, 12h/12h light-dark cycle). They were given a laboratory chow (Nihon Nosan, Yokohama, Japan) and water ad libitum. Care and handling of the animals were in accordance with Kobe university animal experiment guidelines.

Preparation ABSW ABSW was provided by Iwade Mushroom Institute (Mie, Japan). Mycelia were extracted with distilled water. Thefruitingbodies were extracted with hotwater. Moreover, the residue after hot-water extraction was extracted with ammonium oxalate and NaOH according to the method of Kawagishi et al. (#). After alkali extraction, the solution was neutralized and desalted. Three fractions were mixed and lyophilized. For administration to ICR mice, ABSW were suspended in sterilized ion exchanged water at concentrations of 3.6 mg/ml.


402 Compound 48/80-Induced Systemic Anaphylactic Reaction Compound 48/80 (Sigma, St Louis, MO, U.S.A.) was dissolved in saline at a concentration of 10 mg/ml. Compound 48/80 (10 //l) was intradermally injected on the back between the scapulae (P).


Observation of Scratching Behavior Scratching behavior was observed according to the method described by Inagaki et al. (5). After elicitation of the cutaneous reactions, mice were placed in an observation chamber. The behavior was recorded in the absence of an observer using a video camera for 20 min between 30 and 50 min after elicitation. Mice generally scratched several times for about one second and a series of scratchings is indicated cumulatively.

Measurement of Histamine Contents in Plasma The blood was collected from the heart to obtain plasma for histamine contents in 1 h after compound 48/80 inductions. The blood was centrifuged at 12,000 rpm for lmin at 4 °C with EDTA (20 ju\ in 1.5ml tube concentration of 50 mg/ml). Supernatant fluid is collected and plasma samples were stored at -80 °C until assay. The histamine levels in plasma were determined using an immunoassay kit (IBL, Hamburg, Germany) following the manufacturer's specifications.

Histopathological Examinations Biopsy specimens were taken from the nape skin, in which the most prominent lesions usually appear, processed by the conventional method and stained with toluidine blue.

Cytokine Assays of Splenocytes To investigate the immunoregulatory effect of ABSW on ICR mice, cytokine production from splenocyte stimulated with or without ConA in vitro was examined. Splenocytes were prepared for flow cytometry by crushing freshly dissected tissues between flat forceps in PBS (137 mM NaCl, 8.1 mM Na HP0 , 2.68 mM KC1, and 1.47 mM K H P 0 ) . Cells were passed through 75 2

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403 pmesh to get single cell suspension. Red blood cells were removed by adding hemolytic agent [0.16M NH C1 and 0.17M Tris-HCl (pH 7.65) are mixed by 9:1] for 3 min at room temperature. After washing with PBS for three times, cells (lxlO cells/ml) were cultured in a 24-well culture plate with RPMI 1640 (GIBCO BRL, Grand Island, NY) medium, supplemented with 10% heatinactivated fetal bovine serum (GIBCO BRL, Grand Island, NY) and 1.0 mg/ml NaHC0 . To determine antigen-nonspecific T-cell responses, spleen cells were stimulated with/without concanavalinA (ConA: 5 pg/ml, Wako Pure Chemical Industries, Osaka, Japan) at 37 °C for 3 days. The levels of IL-4, IL-12 (p70) and IFN-y in the culture medium were measured with an OptEIA mouse cytokine ELISA set (BD Pharmingen, San Diego, CA) according to the manufacture's protocol using by EPICS XL/XL-MC (Beckman Coulter Inc., Fillerton, CA). 4

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Results Effects of Oral Administration of ABSW on Scratching Behavior Oral administration of ABSW did result in no deference in body weight compared with the control, suggesting that mice grew normally. The cumulative number of scratching behavior for 20 min in saline-treated mice (control) was approximately 2,300 after intradermal injection of compound 48/80, whereas those in ABSW decreased to be approximately 960 (Figure 1).

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Figure 1. Scratching behavior caused by compound 48/80 in ICR mice. Each value represented the mean ± S.E. for 6 mice. Each error bar hides behind symbol. The asterisk indicated significant different from the control group (p

Immunomodulatory Activities of Î²-Glucan in Mushroom - ACS

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