Kyowa Wellness Co., Ltd. (Nihonbashi-Hakozaki-cho, Chuo-ku, Tokyo, Japan) provided the Agaricus Blazei Murill (ABM) extract. Briefly, the dried fruiting bodies of ABM were extracted with hot water by the method of Mizuno et al. [7]. Components of ABM (100 g) included water (3.4 g), protein (37.4 g), lipid (3.4 g), fiber (4.7 g), saccharide (45.0 g) and Na (6.0 mg). The ABM extract was centrifuged at 1800 × g for 10 min to remove any mushroom pieces. The supernatant was freeze dried, diluted to 100 mg/ml with PBS (−) [Ca²⁺, Mg²⁺—free Dulbecco’s phosphate buffered saline (Nissui Pharmaceutical, Tokyo, Japan)], sterilized by 0.22 µm filtration (Millipore, MA, USA) and stored at 4˚C. Before use, the ABM extract was diluted with PBS (−) to 1 mg/ml, 10 mg/ml, and 50 mg/ml. Including endotoxin dose in ABM extract was EU/ml. This concentration of LPS didn’t response immunologically. Also LPS concentration from to EU/ml didn’t show chemotactic activity for neutrophils.

Male Hartley Guinea pigs were obtained from Japan SLC (Shizuoka, Japan). They were housed in transparent plastic cages with stainless wire lids in the animal facility of Kyoto Sangyo University (Kyoto, Japan). They were maintained under standard conditions, with a dark period from 8 pm to 8 am, and water and food were provided ad libitum. This study was approved by the committee for animal care at Kyoto Sangyo University.

Blood was collected from guinea pigs, diluted and mixed twice with PBS (−). An equivalent amount of saline containing 3.5% dextran was added, and the mixture sat at room temperature for 30 min to precipitate red blood cells. The leukocyte-rich supernatant was centrifuged at 400 × g for 30 min (Ficoll-paque Plus, GE Healthcare, Tokyo, Japan). The precipitation was hemolyzed with lysis buffer and centrifuged at 185 × g for 5 min. After the supernatant was discarded, Neutrophils were washed twice with PBS (−). Isolated neutrophils were resuspended in RPMI1640 containing 0.1% delipidated bovine serum albumin, 100 U/ml penicillin and 100 µg/ml streptomycin with HEPES or R (+) (RPMI1640 containing 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin) at a concentration of 2 × 10⁶ cell/ml. The percentage of neutrophils was over than 95% pure.

Chemotactic activity for neutrophils was evaluated with EZ-TAXIScan (GE Healthcare). EZ-TAXIScan was used to detect real time horizontal chemotactic activity of neutrophil. Time-lapse images of neutrophils during migration were obtained using EZ-TAXIScan equipped with a six channel chamber. The chamber has an etched silicon substrate and a flat glass plate, both of which form two compartments with a 4 μm-deep microchannel. Neutrophils (0.75 μl of 2 × 10⁶ cells/ml adjusted with RPMI1640 containing 0.1% delipidated bovine serum albumin, 100 U/ml penicillin and 100 µg/ml streptomycin with HEPES) were put into a hole where the device is held together with a stainless holder, and 0.75 μl of PBS (−) as control, 1, 10, 50 and 100 mg/ml of ABM extract; and 10^{–6 M N-formyl-methionyl-leucyl-phenylalanine (f-MLP) as active control of chemotaxis were added into a contra-hole. The holder assembly was filled with the RPMI1640 solution mentioned above and incubated for 30 min at 37˚C. A charge-coupled device (CCD) camera was used to record the migration of neutrophils toward the high concentration of each sample. Migrated neutronphils were analyzed by TAXIScan Analyzer2. The radian (rad) and velocity (µm/sec) of migrated neutrophils were calculated by TAXIScan Analyzer2 and used as an indicator of chemotactic activity of neutrophols.}

Neutrophils (1 × 10⁶/ml) adjusted with R (+) were incubated at 37˚C for 20 min with ABM extract (final concentration 5 mg/ml) or PBS (−) (control) and FITC-labeled latex beads (Polyscinences Inc., PA, USA). Neutrophils were washed twice with PBS (−) containing 3 mM EDTA and resuspended in 300 μl of PBS (+) [(PBS (−) containing 1% fetal calf serum (FCS)]. Phagocytic activity of neutrophils toward the latex beads was measured using a fluorescence activated cell sorter (FACS) Calibur (Becton-Dickinson, CA, USA).

Neutrophils were incubated with 2’,7’-dichlorofluorescin diacetate (DCFH-DA, [Molecular Probes, Eugene, OR, USA], final concentration 2 μM) or hydroethidine (HE, [Polysciences, PA, USA], final concentration 5 μM) at 37˚C for 15 min. ABM extract was added to achieve a final concentration 5 mg/ml. The control consisted of the addition of an equivalent final concentration of PBS (−). After a shaking incubation for 20 min at 37˚C, neutrophils were washed twice with PBS (+) and resuspended in PBS (+). Fluorescent intracellular dichlorofluorescein (DCF) and ethidium, indicators of intracellular hydrogen peroxide and superoxide, respectively, were assayed by FACS.

All values are expressed as mean ± standard deviation (S.D.). Comparisons between control and ABM extract were made with the student’s t-test. p-values less than 0.05 were considered statistically significant.

Neutrophils were chemotatically attracted to AMB extract (Figure 1), and the course of migration was similar to the straight pathway taken by neutrophils exposed to f-MLP, a positive control of chemotaxis (Figure 2). Chemotactic activity toward AMB extract was indicated by increased direction and velocity of migrating neutronphils compared to control (Figure 3). Along with neutrophils exposed to f-MLP, neutrophils exposed to 1, 10, 50, and 100 mg/ml ABM extract had significantly greater migratory direction and velocity than the control (except for the direction at 1 mg/ml of AMB extract).

Neutrophils demonstrated phagocytic activity when exposed to ABM extract at final concentration of 5 mg/ml

(Figure 4). Compared to the control, neutrophils exposed to ABM extract had 2.4 times more phagocytic activity, a statistically significant increase.

Production of Reactive Oxygen Species (ROS), H₂O₂ and, was significantly increased when neutrophils were exposed to ABM extract at final concentration of 5 mg/ml (Figure 5) compared to control. Production of H₂O₂ was 1.5 times greater (Figure 5(a)) and production of was 2.4 greater (Figure 5(b)) by neutrophils exposed to ABM extract compared with control.