An Extract of Agaricus blazei Murill Administered Orally Promotes
Immune Responses in Murine Leukemia BALB/c Mice In Vivo
Jaung-Geng Lin, MD, PhD1, Ming-Jen Fan, PhD2, Nou-Ying Tang, MD, PhD1,
Jai-Sing Yang, PhD1, Te-Chun Hsia, MD1, Jen-Jyh Lin, MD1,
Kuang-Chi Lai, MD, PhD1,3, Rick Sai-Chuen Wu, MD, PhD1,
Chia-Yu Ma, PhD4, W. Gibson Wood, PhD5, and Jing-Gung Chung, PhD1,2
1China
2Asia
Medical University, Taichung, Taiwan
University, Taichung, Taiwan
3China
Medical University, Yunlin, Taiwan
4Technology
5University
and Science Institute of Northern Taiwan, Taipei, Taiwan
of Minnesota, Minneapolis, MN, USA
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)-1b, IL-6, and interferon-g 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
Agaricus blazei Murill extract, immune responses, WEHI-3 cells, NK cells, phagocytosis, leukemia BALB/c mice
Background and Introduction
used as a medicine. ABM contains polysaccharides,
In the United States, approximately 3.7 individuals per
and it has been effectively used in treating and preventing
100 000 die every year of leukemia.1 In Taiwan, about 4 per
cancer.7-9 ABM promotes the production of interferon (IFN)
100 000 die of leukemia each year based on 2009 reports
and interleukin (IL) for preventing viruses and other external
from the Department of Health, Taiwan. Treatment of leukemia,
factors from entering the tissue.10 ABM has been shown to
however, is not satisfactory. Recently, greater attention has
have antitumor activity in rodents.9,11-13 It has been reported
been given to the use of natural products that have fewer side
that the antimutagenic activity of ABM was present in aqueous
effects when compared with chemotherapeutic drugs or
extracts, fractions, and substances.14-16 ABM contains compounds
synthetic compounds.2 For example, different types of
such as (1 → 3),(1 → 6)-b-glucans, (1 → 3)-a-glucans,
mushrooms have been used as immune potentiators, and
and polysaccharide–protein complexes, which can enhance
polysaccharides and peptides with antitumor activity have
in vivo and in vitro cell-mediated immune responses and act
been isolated from such mushrooms (eg, black mushroom
as biological response modifiers.4,17
shiitake, Hericium erinaceum, and Calvatia caelata).3-6
ABM extracts possess antitumor activity, but it has not
The fungus Agaricus blazei Murill (ABM) has been traditionally
been reported that ABM enhances immune responses in leukemia
mice in vivo. In the present study, we determined if the
Fifty BALB/c mice were randomly divided into 5 groups
ABM extract would enhance immune responses in leukemia
receiving different treatments. Thirty mice were intraperitoneally
mice in vivo. Our results indicated that the ABM extract enhanced
injected with 1 × 105 WEHI-3 cells for 2 weeks and
immune responses, stimulated cytokine secretions, and promoted
then were randomly separated into 3 groups as a model of
the survival rate in WEHI-3 leukemia mice in vivo.
leukemia. Group I served as a control (10 animals). Group II
The ABM extract may have potential to be used in the treatment
mice were treated with deionized and distilled water (DDW,
of leukemia.
as vehicle; 10 animals). Group III animals were treated with
Materials and Methods
Materials and Reagents
DDW (10 animals) after intraperitoneal injection WEHI-3
RPMI-1640 medium, fetal bovine serum (FBS), penicillin–
DDW (10 animals) after intraperitoneal injection of WEHI-3
streptomycin, and L-glutamine were obtained from Invitrogen
cells. Group V animals were treated with ABM (6 mg/kg) in
Life Technologies (Carlsbad, CA).
DDW (10 animals) after intraperitoneal injection of WEHI-3
Preparation of ABM Extract
cells. ABM was administered by oral gavage to the treatment
Agaricus blazei strain was planted in the facility of a contracted
groups at the aforementioned dose daily for up to 3 weeks
mushroom producer located in Wufeng, Taichung,
before being weighed.23,24
Taiwan. After these mushrooms were cultivated, the fresh
Spleen and Liver Tissues Analysis
fruit bodies were desiccated and ground to 50 mesh powder.
All animals were weighed and blood withdrawn. Spleen and
To obtain aqueous (hot water) extract of the basidiocarp,
liver samples were isolated and weighed individually.25,26
about 50 g of the dry basidiocarp was resuspended in 100 mL
Hematoxylin–Eosin Stain and Histopathology
of distilled water, at 25°C under agitation for 1 hour.18 Then
Tissue samples from spleen were fixed in 4% formaldehyde
the crude extract of ABM was prepared as follows: hot
and embedded in paraffin. Sections of 5 mm were
water extract was used in the form of aqueous extract (2.5%)
stained with hematoxylin–eosin according to standard
prepared at 100°C for 30 minutes (hot water) and then
procedures.24,26
resuspended 3 times. All the solutions were filtered through
Assessment of T-Cell Proliferation
cellulose ester membrane with a 0.22-mm pore and then
The splenocytes (1 × 105 cells/well) were isolated from the
dehydrated to powder at ultralow temperature. The prepared
spleens of each mouse and 100 mL of RPMI-1640 medium
ABM extract was a greenish-brown powder that was subsequently
was added. Splenocytes were placed in 96-well plates and
dissolved in phosphate buffered saline (PBS), filtered
stimulated with Con-A (5 mg/mL) for 72 hours. The cells
through a 0.22-mm filter, made up to 100 mg/mL, and stored
were collected by centrifugation at 1500 rpm for 5 minutes
at -20°C in a freezer.19-21
and T-cell proliferation determined using the CellTiter
Murine WEHI-3 Leukemia Cells
96 AQueous One Solution Cell Proliferation Assay kit
The WEHI-3 murine myelomonocytic leukemia cell line was
(Promega, Madison, WI) as previously described. 26,27
purchased from the Food Industry Research and Development
Quantification of NK Cell Cytotoxic Activity
Institute (Hsinchu, Taiwan). Cells were grown in plastic culture
Approximately 1 × 105 leukocytes from the spleens in 1 mL
flasks (75 cm2) in RPMI-1640 medium supplemented
of RPMI-1640 medium from each group were cultured in
with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin,
each well of 24-well culture plates for 24 hours. YAC-1 cells
and 100 mg/mL streptomycin grown at 37°C under a humidified
(2.5 × 107) were cultured in 15-mL tubes with serum-free
5% CO2 atmosphere.22 The cells were cultivated for 2 complete
RPMI-1640 medium, and PKH-67/Dilunt C buffer (Sigma-
cycles in an incubator.
Aldrich, St Louis, MO) was added to the cells, mixed
Male BALB/c Mice
thoroughly for 2 minutes at 25°C, and then 2 mL PBS was added
Male BALB/c mice 8 weeks of age and approximately 22 to
for 1 minute. About 4 mL of RPMI-1640 medium
28 g in weight were purchased from the Laboratory Animal
was added for a 10-minute incubation period followed by
Center, College of Medicine, National Taiwan University
centrifugation
(Taipei, Taiwan).
at 1200 rpm at 25°C. YAC-1 cells in 100 mL were
Establishing Leukemia Mice and ABM Treatment
placed on 96-well plates before the addition of the leukocytes
cells. Group IV mice were treated with ABM (3 mg/kg) in
to the wells for 12 hours and NK cell activity was determined
Statistics Analysis
by flow cytometry (FACS Calibur, Becton Dickinson, NJ)
The results were expressed as mean ± standard deviation
as previously described.28,29
and the difference between groups was analyzed by one-way
Quantification of Phagocytic
ANOVA followed by Dunnett’s multiple comparison test
Activity of Macrophages
and Student’s t test. P values of less or equal to .05 were
Phagocytosis was detected by using the PHAGOTEST kit
taken as significant.
(Glycotope Biotechnology GmbH, Heidelberg, Germany) as
1 hour at 37°C with fluorescence in isothiocyanate-labeled
Results
ABM Affected the Histopathology of Spleen
Tissue, Spleen and Liver Weight, and T-Cell
Proliferation in WEHI-3 Leukemia BALB/c Mice
Escherichia coli (20 mL). The reaction then was stopped by the
Spleen or liver tissues were isolated from animals and were
addition of quenching solution (100 mL) according to the
weighed and histopathologically examined. The histopathological
manufacturer’s instruction. After the completion of phagocytosis
examination results after treatment with ABM are
by monocytes/macrophages, DNA was stained according
presented in Figure 1A. The spleens had markedly decreased
to the manufacturer’s protocol. Cells were analyzed by flow
numbers of neoplastic cells and the number of megakaryocytes
cytometry as previously described.25 Fluorescence data were
increased (Figure 1A). The spleens from ABM-treated
collected on 10 000 cells and analyzed using the CELLQUEST
mice showed marked expansion of red pulp, whereas the
software.
white pulp showed relatively little change. The neoplastic
Examination of Cytokines Levels
cells contained large irregular nuclei with clumped chromatin,
Approximately 1 × 105 leukocytes from the spleens in 1 mL
prominent nucleoli, and abundant clear and light eosinophilic
of RPMI-1640 medium from each group were cultured in
cytoplasm. Frequent mitotic figures were also noted. ABM
24-well culture plates for 24 hour with or without Con-A
reduced weights of spleen (Figure 1B) and liver (Figure 1C)
(5 mg/mL) cotreatment and stimulation. Cells were then
and increased T-cell proliferation (Figure 1D) when compared
centrifuged at 1500 × g for 5 minutes to remove the cells and
with the leukemia mice group.
debris, and the supernatants were used in the assays. The
levels of IL-1b, IL-4, IL-6, and IFN-g were quantified using
Effects of ABM Extract on NK Cell Activity of
Splenocytes in WEHI-3 Leukemia BALB/c Mice
the following kits: Quantikine mouse IL-1beta Immunoassay
To investigate whether ABM is able to act on NK cell
kit, Quantikine mouse IL-4 Immunoassay kit, Quantikine mouse
activity, leukocytes from the spleens of mice with or without
IL-6 Immunoassay kit, and Quantikine mouse IFN-gamma
ABM treatment were isolated and NK cell activity was
Immunoassay kit (R&D Systems Inc, Minneapolis, MN).
determined. The results presented in Figure 2 show that the
The assays were performed according to the manufacturer’s
YAC-1 target cells were killed by NK cells from the mice
recommended procedures.29,31
after treatment with ABM extract at 3 or 6 mg/kg/d at target
Whole Blood Samples and Immunofluorescence
Staining for Surface Markers
cells ratios of 25:1 and 50:1. This dose was effective at both
At the end of the experiments, 1 mL blood samples ml from
dosedependent
each animal were collected before mice were sacrificed.
manner (3 mg/kg/d: 40%, 6 mg/kg/d, 160% at
Blood was immediately exposed to 1× Pharm Lyse lysing
target cells ratio of 25:1; 3 mg/kg/d: 14%, 6 mg/kg/d: 157%
buffer (BD Biosciences, San Jose, CA) for lysing of the red
at target cells ratio of 50:1).
blood cells and centrifuged for 15 minutes at 1500 rpm at
antimouse CD3, PE antimouse CD19, PE antimouse Mac-3,
ABM Affected the Phagocytosis by Macrophage
From Peripheral Blood Mononuclear Cell (PBMC)
in WEHI-3 Leukemia BALB/c Mice
and FITC antimouse CD11b antibodies (BD Pharmingen Inc,
To investigate whether ABM affected phagocytosis, the leukocytes
San Diego, CA) before being analyzed for the determination
from ABM-treated or untreated groups were isolated and
of the cell marker levels by flow cytometry as previously
phagocytic activity was examined. The data from
described.22,32
Figure 3 demonstrate that ABM (3 and 6 mg/kg/d) promoted
previously described.25,30 Approximately 1 × 105 leukocytes
in 100 mL of whole blood from each group was incubated for
4°C. The isolated white blood cells were stained by the FITC
target ratios and increased the activity of NK cells in a
the activity of phagocytosis (3 mg/kg/d: 11.31%; 6 mg/kg/d:
the leukemia mice and increased the T-cell proliferation
14.27%) in leukemia mice.
index by 2- or 3-fold compared with the leukemia mice
Effects of ABM Extract on the Levels
(Figure 1C). Nagaokaa et al demonstrated that agaritine
of IL-1β, IL-4, IL-6, and IFN-γ in ABM-Treated
(b-N-(g-L(+)-glutamyl)-4-(hydroxymethyl)phenylhydrazine;
WEHI-3 BALB/c Mice
4-hydrazinylbenzoic acid), MPH (4-methylphenylhydrazine),
To investigate whether ABM can affect cytokine levels, the
and PH (phenylhydrazine) are major ABM constituents by
supernatants from the spleens of mice treated with or without
HPLC analysis.33 Endo et al pointed out that AGT purified
ABM were isolated and the levels of IL-1b, IL-4, IL-6,
from ABM exerts antitumor activity against leukemic cells
and IFN-g were determined. The results presented in Figure 4
in vitro such as U937, MOLT4, HL-60, and K562 with IC50
indicate that the ABM extract significantly reduced the
values of 2.7, 9.4, 13.0, and 16.0 mg/mL, respectively. The
levels of IL-4 (IL-4: 3 mg/kg/d, reduced 23.8%; 6 mg/kg/d, reduced
suppressive activity was concentrated in the heat-stable and
40%; Figure 4B). However, the ABM extract
dialyzable low-molecular-weight fractions of ABM. Their
significantly increased levels of IL-1b (Figure 4A), IL-6
crude extract of ABM was obtained from hot water just
(Figure 4C), and IFN-g (Figure 4D) when compared with
as was our hot water extract of ABM.34 Other studies also
the WEHI-3 leukemia mice (IL-1b: 3 mg/kg/d, 300%;
showed that ABM also contains b-glucan, and its anticancer
6 mg/kg/d, 700%; IL-6: 3 mg/kg/d, 9.52%; 6 mg/kg/d,
activity might be due to either a direct or indirect effect by
47.61%; IFN-g: 3 mg/kg/d, 1.1%; 6 mg/kg/d, 40%).
b-glucan.35-37 Based on these reports, we may suggest that
Effects of ABM on Whole Blood Cell Surface
Markers From WEHI-3 Leukemia BALB/c Mice
AGT, CPH, MPH, PH, and b-glucan are major ingredients
To investigate whether ABM affected the levels of cell surface
increase the humoral immune response but also the cellular
markers, leukocytes from ABM-treated or untreated
immune response when the leukemia mice were treated
groups were isolated and levels of CD3, CD19, Mac-3, and
with ABM.
CD11b were determined. The data for cell markers of white
The ABM extract significantly enhanced both NK cell activities
blood cells are presented in Figure 5A-D. The results indicated
(Figure 2) and phagocytosis of macrophages (Figure 3).
that ABM increased the levels of CD3 (3 mg/kg/d,
It is well documented that NK cell activity and phagocytosis
36.36%; 6 mg/kg/d, 52%; Figure 5A) and CD19 (3 mg/kg/d,
by macrophages both play major roles for immune responses
41.66%; 6 mg/kg/d, 91.66%; Figure 5B) but decreased the
after animals are exposed to antigen.38-40 Immune responses
levels of Mac-3 (3 mg/kg/d, decreased 18.81%; 6 mg/kg/d,
involved not only leukocytes but also the interaction of leukocytes
decreased 45.45%; Figure 5C) and CD11b (3 mg/kg/d,
with several different cytokines.41,42 Therefore, we
decreased 14.89%; 6 mg/kg/d, decreased 25.53%; Figure 5D)
examined whether specific cytokines would be affected by
when compared with the WEHI-3-only treated groups.
ABM in leukemia mice. ABM extract acted as an effective
Discussion
stimulator for T cells and macrophages to release IL-1b
Although reports have shown that ABM contains polysaccharides
(Figure 4A), IL-6 (Figure 4C), and IFN-g (Figure 4D). These
that are associated with antitumor activity, there
results suggested that the ABM extract increased the immune
are no available reports addressing its immune responses
response and might increase the antibody response against
in leukemia mice in vivo. In this study, we established leukemia
leukemia in general. Herein, we showed that ABM activated
mice through the injection of WEHI-3 cells and then
macrophages and immunocytes to induce cytokines such as
chronically treated mice with ABM. Results showed that
interferon, and it is possible to prolong the biological life
ABM can promote immune responses in leukemia mice in
through its immunological effects. Those effects may be due
vivo. ABM also promoted T-cell proliferation (Figure 1).
to enhancement of IL-1b and IL-6 from activated macrophages
To determine whether the T-cell proliferation response
and T cells, resulting in B-cell differentiation. This
to Con-A may be due to the ABM extract, splenocytes
is in agreement with Nakajima et al, who indicated that
from leukemia mice were examined for T-cell proliferation.
ABM plays an important role in improving immune response
ABM extract elicited significant proliferative responses in
against leukemia cells.10 These observations are in agreement
AGT), CPH (4-hydrazinylbenzylalcohol (HMPH),
for antileukemia activity. Thus, ABM extract could not only
with an earlier study in normal mice showing that
Declaration of Conflicting Interests
dietary ABM also promoted immune responses.43 Other
The author(s) declared no potential conflicts of interests with respect
investigators also reported that the ABM extract augmented
to the authorship and/or publication of this article.
the expression of IL-6 and IL-1b RNA in peritoneal
Funding
macrophages.10
The author(s) disclosed receipt of the following financial support
We also examined the cell markers from leukemia mice
for the research and/or authorship of this article:
after dietary treatment with ABM. The results indicated that
This study was supported by Grant CMU-96-073 from China
the percentages of CD3 and CD19 positive cells significantly
Medical University, Taichung, Taiwan, and Grant DOH99-TDC-
increased in ABM-treated leukemia mice but that the
111-005 from Department of Health, Executive Yuan, ROC
population of CD11b and Mac-3 positive cells significantly
(Taiwan).
decreased. CD3 and CD19 positive cells represented the percentage
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