An Extract of Agaricus blazei Murill Administered Orally Promotes Immune Responses in Murine Leukemia BALB/c Mice In Vivo

Abstract

Purpose. The edible mushroom (fungus) Agaricus blazei Murill (ABM) is a health food in many countries. Importantly, it has been shown to have antitumor and immune effects. There is no available information on ABM-affected immune responses in leukemia mice in vivo. Experimental Design. In this study, the authors investigated the immunopotentiating activities of boiled water–soluble extracts from desiccated ABM in WEHI-3 leukemia mice. The major characteristic of WEHI-3 leukemia mice are enlarged spleens and livers after intraperitoneal injection with murine leukemia WEHI-3 cells. Isolated T cells from spleens of ABM-treated mice resulted in increased T-cell proliferation compared with the untreated control with concanavalin A stimulation. Results. ABM decreased the spleen and liver weights when compared with WEHI-3 leukemia mice and this effect was a dose-dependent response. ABM promoted natural killer cell activity and phagocytosis by macrophage/monocytes in leukemia mice in a dose-dependent manner. ABM also enhanced cytokines such as interleukin (IL)-1β, IL-6, and interferon-γ levels but reduced the level of IL-4 in WEHI-3 leukemia mice. Moreover, ABM increased the levels of CD3 and CD19 but decreased the levels of Mac-3 and CD11b in leukemia mice. Conclusions. The ABM extract is likely to stimulate immunocytes and regulate immune response in leukemia mice in vivo.

Keywords

immune responses WEHI-3 cells NK cells phagocytosis leukemia BALB/c mice

Background and Introduction

In the United States, approximately 3.7 individuals per 100 000 die every year of leukemia.¹ In Taiwan, about 4 per 100 000 die of leukemia each year based on 2009 reports from the Department of Health, Taiwan. Treatment of leukemia, however, is not satisfactory. Recently, greater attention has been given to the use of natural products that have fewer side effects when compared with chemotherapeutic drugs or synthetic compounds.² For example, different types of mushrooms have been used as immune potentiators, and polysaccharides and peptides with antitumor activity have been isolated from such mushrooms (eg, black mushroom shiitake, Hericium erinaceum, and Calvatia caelata).^3-6

The fungus Agaricus blazei Murill (ABM) has been traditionally used as a medicine. ABM contains polysaccharides, and it has been effectively used in treating and preventing cancer.^7-9 ABM promotes the production of interferon (IFN) and interleukin (IL) for preventing viruses and other external factors from entering the tissue.¹⁰ ABM has been shown to have antitumor activity in rodents.^9,11-13 It has been reported that the antimutagenic activity of ABM was present in aqueous extracts, fractions, and substances.^14-16 ABM contains compounds such as (1 → 3),(1 → 6)-β-glucans, (1 → 3)-α-glucans, and polysaccharide–protein complexes, which can enhance in vivo and in vitro cell-mediated immune responses and act as biological response modifiers.^4,17

ABM extracts possess antitumor activity, but it has not been reported that ABM enhances immune responses in leukemia mice in vivo. In the present study, we determined if the ABM extract would enhance immune responses in leukemia mice in vivo. Our results indicated that the ABM extract enhanced immune responses, stimulated cytokine secretions, and promoted the survival rate in WEHI-3 leukemia mice in vivo. The ABM extract may have potential to be used in the treatment of leukemia.

Materials and Methods

Materials and Reagents

RPMI-1640 medium, fetal bovine serum (FBS), penicillin–streptomycin, and L-glutamine were obtained from Invitrogen Life Technologies (Carlsbad, CA).

Preparation of ABM Extract

Agaricus blazei strain was planted in the facility of a contracted mushroom producer located in Wufeng, Taichung, Taiwan. After these mushrooms were cultivated, the fresh fruit bodies were desiccated and ground to 50 mesh powder. To obtain aqueous (hot water) extract of the basidiocarp, about 50 g of the dry basidiocarp was resuspended in 100 mL of distilled water, at 25°C under agitation for 1 hour.¹⁸ Then the crude extract of ABM was prepared as follows: hot water extract was used in the form of aqueous extract (2.5%) prepared at 100°C for 30 minutes (hot water) and then resuspended 3 times. All the solutions were filtered through cellulose ester membrane with a 0.22-µm pore and then dehydrated to powder at ultralow temperature. The prepared ABM extract was a greenish-brown powder that was subsequently dissolved in phosphate buffered saline (PBS), filtered through a 0.22-µm filter, made up to 100 mg/mL, and stored at −20°C in a freezer.^19-21

Murine WEHI-3 Leukemia Cells

The WEHI-3 murine myelomonocytic leukemia cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were grown in plastic culture flasks (75 cm²) in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin grown at 37°C under a humidified 5% CO₂ atmosphere.²² The cells were cultivated for 2 complete cycles in an incubator.

Male BALB/c Mice

Male BALB/c mice 8 weeks of age and approximately 22 to 28 g in weight were purchased from the Laboratory Animal Center, College of Medicine, National Taiwan University (Taipei, Taiwan).

Establishing Leukemia Mice and ABM Treatment

Fifty BALB/c mice were randomly divided into 5 groups receiving different treatments. Thirty mice were intraperitoneally injected with 1 × 10⁵ WEHI-3 cells for 2 weeks and then were randomly separated into 3 groups as a model of leukemia. Group I served as a control (10 animals). Group II mice were treated with deionized and distilled water (DDW, as vehicle; 10 animals). Group III animals were treated with DDW (10 animals) after intraperitoneal injection WEHI-3 cells. Group IV mice were treated with ABM (3 mg/kg) in DDW (10 animals) after intraperitoneal injection of WEHI-3 cells. Group V animals were treated with ABM (6 mg/kg) in DDW (10 animals) after intraperitoneal injection of WEHI-3 cells. ABM was administered by oral gavage to the treatment groups at the aforementioned dose daily for up to 3 weeks before being weighed.^23,24

Spleen and Liver Tissues Analysis

All animals were weighed and blood withdrawn. Spleen and liver samples were isolated and weighed individually.^25,26

Hematoxylin–Eosin Stain and Histopathology

Tissue samples from spleen were fixed in 4% formaldehyde and embedded in paraffin. Sections of 5 mm were stained with hematoxylin–eosin according to standard procedures.^24,26

Assessment of T-Cell Proliferation

The splenocytes (1 × 10⁵ cells/well) were isolated from the spleens of each mouse and 100 µL of RPMI-1640 medium was added. Splenocytes were placed in 96-well plates and stimulated with Con-A (5 µg/mL) for 72 hours. The cells were collected by centrifugation at 1500 rpm for 5 minutes and T-cell proliferation determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) as previously described.^26,27

Quantification of NK Cell Cytotoxic Activity

Approximately 1 × 10⁵ leukocytes from the spleens in 1 mL of RPMI-1640 medium from each group were cultured in each well of 24-well culture plates for 24 hours. YAC-1 cells (2.5 × 10⁷) were cultured in 15-mL tubes with serum-free RPMI-1640 medium, and PKH-67/Dilunt C buffer (Sigma-Aldrich, St Louis, MO) was added to the cells, mixed thoroughly for 2 minutes at 25°C, and then 2 mL PBS was added for 1 minute. About 4 mL of RPMI-1640 medium was added for a 10-minute incubation period followed by centrifugation at 1200 rpm at 25°C. YAC-1 cells in 100 µL were placed on 96-well plates before the addition of the leukocytes to the wells for 12 hours and NK cell activity was determined by flow cytometry (FACS Calibur, Becton Dickinson, NJ) as previously described.^28,29

Quantification of Phagocytic Activity of Macrophages

Phagocytosis was detected by using the PHAGOTEST kit (Glycotope Biotechnology GmbH, Heidelberg, Germany) as previously described.^25,30 Approximately 1 × 10⁵ leukocytes in 100 µL of whole blood from each group was incubated for 1 hour at 37°C with fluorescence in isothiocyanate-labeled Escherichia coli (20 µL). The reaction then was stopped by the addition of quenching solution (100 µL) according to the manufacturer’s instruction. After the completion of phagocytosis by monocytes/macrophages, DNA was stained according to the manufacturer’s protocol. Cells were analyzed by flow cytometry as previously described.²⁵ Fluorescence data were collected on 10 000 cells and analyzed using the CELLQUEST software.

Examination of Cytokines Levels

Approximately 1 × 10⁵ leukocytes from the spleens in 1 mL of RPMI-1640 medium from each group were cultured in 24-well culture plates for 24 hour with or without Con-A (5 µg/mL) cotreatment and stimulation. Cells were then centrifuged at 1500 × g for 5 minutes to remove the cells and debris, and the supernatants were used in the assays. The levels of IL-1β, IL-4, IL-6, and IFN-γ were quantified using the following kits: Quantikine mouse IL-1beta Immunoassay kit, Quantikine mouse IL-4 Immunoassay kit, Quantikine mouse IL-6 Immunoassay kit, and Quantikine mouse IFN-gamma Immunoassay kit (R&D Systems Inc, Minneapolis, MN). The assays were performed according to the manufacturer’s recommended procedures.^29,31

Whole Blood Samples and Immunofluorescence Staining for Surface Markers

At the end of the experiments, 1 mL blood samples ml from each animal were collected before mice were sacrificed. Blood was immediately exposed to 1× Pharm Lyse lysing buffer (BD Biosciences, San Jose, CA) for lysing of the red blood cells and centrifuged for 15 minutes at 1500 rpm at 4°C. The isolated white blood cells were stained by the FITC antimouse CD3, PE antimouse CD19, PE antimouse Mac-3, and FITC antimouse CD11b antibodies (BD Pharmingen Inc, San Diego, CA) before being analyzed for the determination of the cell marker levels by flow cytometry as previously described.^22,32

Statistics Analysis

The results were expressed as mean ± standard deviation and the difference between groups was analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test and Student’s t test. P values of less or equal to .05 were taken as significant.

Results

ABM Affected the Histopathology of Spleen Tissue, Spleen and Liver Weight, and T-Cell Proliferation in WEHI-3 Leukemia BALB/c Mice

Spleen or liver tissues were isolated from animals and were weighed and histopathologically examined. The histopathological examination results after treatment with ABM are presented in Figure 1A. The spleens had markedly decreased numbers of neoplastic cells and the number of megakaryocytes increased (Figure 1A). The spleens from ABM-treated mice showed marked expansion of red pulp, whereas the white pulp showed relatively little change. The neoplastic cells contained large irregular nuclei with clumped chromatin, prominent nucleoli, and abundant clear and light eosinophilic cytoplasm. Frequent mitotic figures were also noted. ABM reduced weights of spleen (Figure 1B) and liver (Figure 1C) and increased T-cell proliferation (Figure 1D) when compared with the leukemia mice group.

Figure 1.

Agaricus blazei Murill (ABM) extract affected the histopathology, spleen and liver weight of mice, and T-cell proliferation of Con-A-stimulated splenocytes from leukemia BALB/c mice

Effects of ABM Extract on NK Cell Activity of Splenocytes in WEHI-3 Leukemia BALB/c Mice

To investigate whether ABM is able to act on NK cell activity, leukocytes from the spleens of mice with or without ABM treatment were isolated and NK cell activity was determined. The results presented in Figure 2 show that the YAC-1 target cells were killed by NK cells from the mice after treatment with ABM extract at 3 or 6 mg/kg/d at target cells ratios of 25:1 and 50:1. This dose was effective at both target ratios and increased the activity of NK cells in a dose-dependent manner (3 mg/kg/d: 40%, 6 mg/kg/d, 160% at target cells ratio of 25:1; 3 mg/kg/d: 14%, 6 mg/kg/d: 157% at target cells ratio of 50:1).

Figure 2.

Agaricus blazei Murill (ABM) affected activity of natural killer (NK) cells in leukemia BALB/c mice

ABM Affected the Phagocytosis by Macrophage From Peripheral Blood Mononuclear Cell (PBMC) in WEHI-3 Leukemia BALB/c Mice

To investigate whether ABM affected phagocytosis, the leukocytes from ABM-treated or untreated groups were isolated and phagocytic activity was examined. The data from Figure 3 demonstrate that ABM (3 and 6 mg/kg/d) promoted the activity of phagocytosis (3 mg/kg/d: 11.31%; 6 mg/kg/d: 14.27%) in leukemia mice.

Figure 3.

Agaricus blazei Murill (ABM) extract stimulated phagocytotic activity of peripheral blood mononuclear cell (PBMC) in leukemia BALB/c mice

Effects of ABM Extract on the Levels of IL-1β, IL-4, IL-6, and IFN-γ in ABM-Treated WEHI-3 BALB/c Mice

To investigate whether ABM can affect cytokine levels, the supernatants from the spleens of mice treated with or without ABM were isolated and the levels of IL-1β, IL-4, IL-6, and IFN-γ were determined. The results presented in Figure 4 indicate that the ABM extract significantly reduced the levels of IL-4 (IL-4: 3 mg/kg/d, reduced 23.8%; 6 mg/kg/d, reduced 40%; Figure 4B). However, the ABM extract significantly increased levels of IL-1β (Figure 4A), IL-6 (Figure 4C), and IFN-γ (Figure 4D) when compared with the WEHI-3 leukemia mice (IL-1β: 3 mg/kg/d, 300%; 6 mg/kg/d, 700%; IL-6: 3 mg/kg/d, 9.52%; 6 mg/kg/d, 47.61%; IFN-γ: 3 mg/kg/d, 1.1%; 6 mg/kg/d, 40%).

Figure 4.

Agaricus blazei Murill (ABM) extract affected the levels of IL-1β, IL-4, IL-6, and IFN-γ after Con-A-stimulated splenocytes from leukemia BALB/c mice

Effects of ABM on Whole Blood Cell Surface Markers From WEHI-3 Leukemia BALB/c Mice

To investigate whether ABM affected the levels of cell surface markers, leukocytes from ABM-treated or untreated groups were isolated and levels of CD3, CD19, Mac-3, and CD11b were determined. The data for cell markers of white blood cells are presented in Figure 5A-D. The results indicated that ABM increased the levels of CD3 (3 mg/kg/d, 36.36%; 6 mg/kg/d, 52%; Figure 5A) and CD19 (3 mg/kg/d, 41.66%; 6 mg/kg/d, 91.66%; Figure 5B) but decreased the levels of Mac-3 (3 mg/kg/d, decreased 18.81%; 6 mg/kg/d, decreased 45.45%; Figure 5C) and CD11b (3 mg/kg/d, decreased 14.89%; 6 mg/kg/d, decreased 25.53%; Figure 5D) when compared with the WEHI-3-only treated groups.

Figure 5.

Agaricus blazei Murill (ABM) affected the levels of cell markers in white blood cells from leukemia BALB/c mice

Discussion

Although reports have shown that ABM contains polysaccharides that are associated with antitumor activity, there are no available reports addressing its immune responses in leukemia mice in vivo. In this study, we established leukemia mice through the injection of WEHI-3 cells and then chronically treated mice with ABM. Results showed that ABM can promote immune responses in leukemia mice in vivo. ABM also promoted T-cell proliferation (Figure 1). To determine whether the T-cell proliferation response to Con-A may be due to the ABM extract, splenocytes from leukemia mice were examined for T-cell proliferation. ABM extract elicited significant proliferative responses in the leukemia mice and increased the T-cell proliferation index by 2- or 3-fold compared with the leukemia mice (Figure 1C). Nagaokaa et al demonstrated that agaritine (β-N-(γ-L(+)-glutamyl)-4-(hydroxymethyl)phenylhydrazine; AGT), CPH (4-hydrazinylbenzylalcohol (HMPH), 4-hydrazinylbenzoic acid), MPH (4-methylphenylhydrazine), and PH (phenylhydrazine) are major ABM constituents by HPLC analysis.³³ Endo et al pointed out that AGT purified from ABM exerts antitumor activity against leukemic cells in vitro such as U937, MOLT4, HL-60, and K562 with IC₅₀ values of 2.7, 9.4, 13.0, and 16.0 µg/mL, respectively. The suppressive activity was concentrated in the heat-stable and dialyzable low-molecular-weight fractions of ABM. Their crude extract of ABM was obtained from hot water just as was our hot water extract of ABM.³⁴ Other studies also showed that ABM also contains β-glucan, and its anticancer activity might be due to either a direct or indirect effect by β-glucan.^35-37 Based on these reports, we may suggest that AGT, CPH, MPH, PH, and β-glucan are major ingredients for antileukemia activity. Thus, ABM extract could not only increase the humoral immune response but also the cellular immune response when the leukemia mice were treated with ABM.

The ABM extract significantly enhanced both NK cell activities (Figure 2) and phagocytosis of macrophages (Figure 3). It is well documented that NK cell activity and phagocytosis by macrophages both play major roles for immune responses after animals are exposed to antigen.^38-40 Immune responses involved not only leukocytes but also the interaction of leukocytes with several different cytokines.^41,42 Therefore, we examined whether specific cytokines would be affected by ABM in leukemia mice. ABM extract acted as an effective stimulator for T cells and macrophages to release IL-1β (Figure 4A), IL-6 (Figure 4C), and IFN-γ (Figure 4D). These results suggested that the ABM extract increased the immune response and might increase the antibody response against leukemia in general. Herein, we showed that ABM activated macrophages and immunocytes to induce cytokines such as interferon, and it is possible to prolong the biological life through its immunological effects. Those effects may be due to enhancement of IL-1β and IL-6 from activated macrophages and T cells, resulting in B-cell differentiation. This is in agreement with Nakajima et al, who indicated that ABM plays an important role in improving immune response against leukemia cells.¹⁰ These observations are in agreement with an earlier study in normal mice showing that dietary ABM also promoted immune responses.⁴³ Other investigators also reported that the ABM extract augmented the expression of IL-6 and IL-1β RNA in peritoneal macrophages.¹⁰

We also examined the cell markers from leukemia mice after dietary treatment with ABM. The results indicated that the percentages of CD3 and CD19 positive cells significantly increased in ABM-treated leukemia mice but that the population of CD11b and Mac-3 positive cells significantly decreased. CD3 and CD19 positive cells represented the percentage of T and B cells increase from ABM-treated leukemia mice, which is comparable to Figure 1. The B-cell differentiation requires the interaction of various cytokines that are secreted from macrophages or T cells.⁴⁴ CD19 antigen exists on cell surface membranes of nonactivated B lymphocytes.⁴⁵ IL-6 has been shown to facilitate terminal differentiation of B cell to antibody-secreting cells and its major producers are monocytes and T cells.⁴⁶ GM-CSF had been shown to have an effect on mature functional APC and causes an augmented response of the immune system in vitro by increasing the secretion of IL-1 and expression of Ig antigens.⁴⁷

Since the ABM extract could increase B cells, the augmenting effect of the ABM extract on the antibody responses resulted from expansion and differentiation of mainly monocytes and macrophages. The results demonstrated that the ABM extract inhibited leukemia-related spleen growth. In our study, the notable characteristic of the leukemia model is the elevation of peripheral monocytes and granulocytes with immature morphology as well as enlarged and infiltrated spleens compared with the normal counterpart.⁴⁸ This observation is in agreement with Mizuno, who showed that the percentage of Th1, Th2, CD4, and CD8 positive cells were significantly increased in mice orally administered a hot water–soluble fraction from ABM when compared with control mice.¹³ In the present study, we also found that the ABM extract promoted immune responses in BALB/c leukemia mice in vivo. The ABM extract can act as a potent immunological adjuvant in vivo, and its application provides an effective strategy to improve the efficacy of immune responses. Further investigations are needed to verify the generality of immune response effects of the ABM extract. Nevertheless, our data provided evidence that the ABM extract is a potential modulator of immune responses to WEHI-3 leukemia mice in vivo.

Footnotes

The author(s) declared no potential conflicts of interests with respect to the authorship and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article:

This study was supported by Grant CMU-96-073 from China Medical University, Taichung, Taiwan, and Grant DOH99-TDC-111-005 from Department of Health, Executive Yuan, ROC (Taiwan).

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