Extract of Medicinal Mushroom Agaricus blazei Murill Enhances the Non-specific and Adaptive Immune Activities in BALB/c Mice

Materials and Methods

Reagents and chemicals. Reagents were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. Enzyme-linked immunosorbent assay (ELISA) kits for cytokine measurements were purchased from BD Biosciences (San Diego, CA, USA). Fetal bovine serum (FBS) and cell culture supplies were purchased from Hyclone (Logan, UT, USA).

Preparation of AbM. AbM powder obtained from Chang Gung Biotechnology Corporation, Ltd. (Taipei, Taiwan, R.O.C.) was thoroughly mixed with distilled water at 60°C for 10 min, then cooled to room temperature and left for 5 h with stirring at 200×g to form solutions of low (136.5 mg/kg), medium (273 mg/kg) and high (819 mg/kg) concentrations which were 1-, 2- and 6-fold that of human daily intake, respectively. The AbM supernatant solution was filtered and freeze-dried. This AbM was stored at −50°C until use (18).

Animals and treatment doses. A total of 40 female BALB/c mice of six weeks of age were used. Animals were housed singly in cages. Mice were maintained under standard pathogen-free conditions with a 12-hour light/dark cycle at a temperature and relative humidity range of 20±2°C and 75±15%, respectively. They were fed orally with Laboratory Rodent Diet 5001 manufactured by PMI Nutrition International (St. Louis, MO, USA) throughout the study. Animals used in the present study were maintained in accordance with the guidelines approved by the National Science Council of the Republic of China and the Committee for the Purpose of Control and Supervision of Experiments on Animals. Experiments were performed according to law, regulations and guidelines for animal experiments in Taiwan, which are in agreement with the declaration of Helsinki. For the non-specific immune response experiments, female mice were randomized and divided into control (group 1) and experimental (groups 2-4) of 10 animals per group. Groups 2, 3 and 4 were orally-administered high, medium and low doses of AbM daily by oral gavage for six weeks. Control mice received distilled water (vehicle) by oral gavage. All mice were examined weekly as a check on health status. For the adaptive immune response experiments, 40 female mice of six weeks of age were similarly divided into four groups, each consisting of ten mice. Groups 2, 3 and 4 were administered by oral gavage high, medium and low doses of AbM, daily for six weeks as described above. After six weeks of treatment, animals were immunized by intraperitoneally injections with 2 μg of OVA w/w Complete Freunds' Adjuvant (Sigma-Aldrich, St. Louis, MO, USA). The boosting injection (6 μg of OVA w/w incomplete Freunds' Adjuvant) was given two weeks later. Splenocytes and sera were collected two weeks after the secondary immunization for measurement of OVA-specific antibody.

Determination of serum immunoglobulin G and M levels for non-specific immune response. Effects of AbM on serum IgG and IgM levels were determined in 10 mice from each group receiving distilled water or different dose treatments of AbM orally for six weeks, as described above. Whole blood was collected via orbital puncture with heparinized microhematocrit glass capillaries at the beginning, the end of the third week and the end of the experiments (the sixth week). The samples were diluted with PBS and IgG and IgM levels were quantified by an ELISA kit (Bethyl Lab, Montgomery, TX, USA).

Preparation of splenocytes. Murine splenocytes were prepared as previously described (22) with minor modifications. Mice were sacrificed and spleens were surgically removed and minced at room temperature in RPMI-1640 medium. The suspension was filtered through a nylon mesh and centrifuged at 1500 ×g for 5 min. Erythrocytes were then lysed by the addition of ACK buffer (150 mM NH₄Cl, 1.0 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.2). The splenocytes were grown in RPMI-1640 medium supplemented with 10 mM HEPES (pH 7.0), non-essential amino acids, 50 μM β-mercaptoethanol, 0.03% L-glutamine, 50 μg/ml gentamycin and 10% heat-inactivated (56°C, 30 min) fetal bovine serum. A splenocyte suspension at 5×10⁶ cells/ml was then prepared.

ELISA assays for quantitative determination of cytokines for non-specific immune response. Approximately 2×10⁶ cells/ml splenocytes isolated from the spleen of control and AbB treated mice were incubated with Concanavalin A (Con A) (final concentration 5 μg/ml) in 24-well culture plates at 37°C in 5% CO₂. After 48 h, plates were centrifuged at 1400×g for 5 min and supernatants were collected for the determination of INF-γ, IL-2, IL-4 levels using an ELISA kit (Rapidbio Lab., West Hills, CA, USA) according to the manufacturer's instructions. The optical density was measured using an ELISA reader with a wavelength of 450 nm.

Splenocyte proliferation assay for non-specific immune response. Following six weeks of treatments, spleens were collected from mice under aseptic conditions in Hank's balanced salt solution (HBSS; Sigma), minced using a pair of scissors and passed through a fine steel mesh to obtain a homogeneous cell suspension, and the erythrocytes were then lysed with ammonium chloride (0.8%, w/v). After centrifugation (380×g at 4°C for 10 min), the pelleted cells were washed three times in PBS, and resuspended in complete medium. Cell numbers were counted with a haemocytometer by the trypan blue dye exclusion technique. Cell viability exceeded 95%. Splenocyte proliferation was assayed as previously described (23). Briefly, splenocytes from each mouse were seeded into four wells of a 96-well flat-bottom microtiter plate at 5×10⁶ cell/ml in 100 μl of complete medium. Con A (final concentration 5 μg/ml) or lipopolysaccharide (LPS; final concentration 10 μg/ml) were added, and then medium was added, to a final volume of 200 μl. The plates were incubated at 37°C in a humid atmosphere with 5% CO₂. After 44 h, 50 μl of 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (2 mg/ml), Promega's CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit, was added to each well and incubated for 4 h. The plates were centrifuged (1400×g, 5 min) and the untransformed MTT was removed carefully by pipetting. Subsequently, 150 μl of a DMSO working solution (192 μl DMSO with 8 μl 1 N HCl) was added to each well, and the absorbance was evaluated in an ELISA reader at 570 nm with a 630 nm reference after 15 min. The stimulation index (SI) was calculated based on the following formula: SI=the absorbance value for mitogen-stimulated-cultures divided by the absorbance value for non-stimulated cultures.

Natural killer (NK) cell cytotoxicity assay. Approximately 1×10⁷ splenocytes/ml were isolated from the spleen of control and BALB/c mice administered AbM. YAC-1 cells (1×10⁵ cells/ml) in 15-ml tubes were washed twice with serum-free RPMI-1640 medium then PKH-67/Dil.C buffer (Sigma-Aldrich Corp.) was added to the cells, which were mixed thoroughly for 2 min at 25°C before adding phosphate-buffered saline (PBS). After 1 min, 4 ml RPMI-1640 medium were added and the cells were centrifuged for 10 min at 1200 ×g and 25°C. Approximately 1×10⁵ YAC-1 cells were placed onto 96-well plates in 100 μl medium then 1×10⁷ cells/ml (100:1), 5×10⁶ (50:1) and 2.5×10⁶ (25:1) splenocytes were added to each well and mixed with 3,3’-Dioctadecyloxacarbocyanine perchlorate (DiOC18)-labeled target cells at effector-to-target (E:T) ratios of 100:1, 50:1, and 25:1. Propidium iodide (PI) was added to the culture medium to allow for the determination of YAC-1 cell death after 3 h incubation at 37°C in 5% CO₂. Determination of the NK cell cytotoxicity after 12 h was measured using the LIVE/DEAD® cell-mediated cytotoxicity kit for animal cells (Molecular Probes, Carlsbad, CA, USA) (24). The percent age cytotoxicity was assessed by flow cytometric analysis. Lysed (PI⁺, DiOC18⁺) and viable (DiOC18⁺ and PI⁻) YAC-1 cells were identified by their dual- or single-positive staining. Total cytotoxicity was determined as: [%experimental cytotoxicity] − [%background cytotoxicity in control YAC-1 cultures].

Phagocytosis assay of peritoneal macrophages for non-specific immune response. Peritoneal macrophages were isolated from mice at the end of six weeks of AbM treatment. Briefly, the mouse peritoneal cavity was carefully exposed without disrupting blood vessels and 2 to 3 ml of RPMI was slowly injected. The lavage was sucked back and cultured in tissue culture flasks for 2 h at 37°C under 5% CO₂ to allow adherence of macrophages. The non-adherent cells were removed and flasks were washed three times with HBSS. The adherent cells were harvested from the flask using a rubber policeman and were resuspended in 10% fetal calf serum (FCS)-RPMI-1640 medium. Approximately 1×10⁵ cells/well were added to 50 μl of RPMI-1640 medium supplemented with 50 μg gentamicin sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% heat-inactivated fetal bovine serum, then mixed with 50 μl of Escherichia coli-Fluorescein isothiocyanate (FITC), according to the manufacturer's instructions (PHAGOTEST kit; ORPEGEN Pharma Gesellschaft fur biotechnologische, Heidelberg, Germany) and shaken in a shaker bath for 30 min at 37°C. Cells then were centrifuged at 1500 ×g for 5 min. Flow cytometric analysis was conducted for each sample (FACS Calibur; Becton Dickinson, USA). Fluorescence data were collected on 10⁵ cells and analyzed as described previously (25, 26).

Flow cytometric determination of various cell types in spleen for non-specific immune response. Leukocytes from spleen of AbM-treated mice and normal control groups were isolated and processed for immunofluorescence (IF) staining of cell surface antigens for flow cytometric analysis. Briefly, single-cell suspensions of harvested leukocytes were prepared at 2×10⁷ cells/ml in staining buffer (10% FCS in PBS) and pre-incubated with 1 μg of the 2.4 G2 antibody for 5 to 10 min on ice prior to staining. Fifty microliters of cell suspension (equal to 10⁶ cells) were dispensed into each tube along with a previously determined optimal concentration of cell surface-specific antibody in 50 μl of staining buffer. Each test tube was mixed gently and incubated for 30 min in the dark in an ice bath. Cells were stained with fluorochrome-labeled antibodies of the appropriate isotype control for non-specific binding. Cells stained in the absence of primary antibodies served as negative controls. After the incubation period, cells were washed by adding 2 ml of staining buffer, followed by centrifugation for 7 min (at 300 to 400×g) at 4°C. The washing was repeated twice. Cell pellets were suspended in 500 μl of staining buffer and analyzed on a BD LSR II flow cytometer (BD Biosciences).

Measurement of OVA-specific IgG, IgM and IgE. Whole-blood samples (0.1 to 0.2 ml) were collected by retro-orbital puncture of immunized mice at four weeks after booster injection of OVA. OVA-specific IgG, IgM and IgE antibodies in sera were detected by an indirect ELISA. Microtiter plate wells (Nunc, USA) were coated with 100 μl OVA solution (25 μg/ml in 50 mM carbonate–bicarbonate buffer, pH 9.6) for 24 h at 4°C. The wells were washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS/Tween) and then blocked with 5% FCS/PBS at 37°C for 2 h. After three washings, 100 μl of a series of diluted sera samples (initial dilution 1:50) or 0.5% FCS/PBS as control were added to wells. The plates were then incubated for 2 h at 37°C, and then washed three times. Aliquots of 100 μl of rabbit horseradish peroxidase conjugated anti-mouse IgG, IgM or IgE diluted 1:10000, were added to each plate. The plates were further incubated for 2 h at 37°C. After washing, the peroxidase activity was assayed as follows: 100 μl of substrate solution (10 mg of O-phenylenediamine and 37.5 μl of 30% H₂O₂ in 25 ml of 0.1 M citrate–phosphate buffer, pH 5.0) was added to each well. The plate was incubated for 10 min at 37°C, and the enzyme reaction was terminated by adding 50 μl/well of 2 N H₂SO₄. The optical density was measured in an ELISA reader at 490 nm, and where sets of sera samples were subjected to within and between group comparisons, ELISA assays were performed on the same day for all of the samples.

Splenocyte proliferation assay for adaptive immune response. Spleens were collected from the OVA-immunized mice. All the protocols were the same for proliferation as the non-specific response procedures described above.

Cytokine analysis of cultured supernatants of splenocytes for determination of the adaptive immune response. Splenocytes (2×10⁶ cells/well) from immunized mice prepared as described above were incubated with Con A (final concentration 5 μg/ml), LPS (final concentration 10 μg/ml) or OVA (final concentration 30 μg/ml), in 24-well culture plates at 37°C in 5% CO₂. After 48 h, the plates were centrifuged at 1400 ×g for 5 min and culture supernatants were collected for the determination of INF-γ, IL-2, IL-4 levels. The presence of INF-γ, IL-2, IL-4 in the cultured supernatants of splenocytes were determined using a mouse ELISA kit (Rapidbio Lab.) according to the manufacturer's instructions. The optical density was measured using an ELISA reader with wavelength of 450 nm.

Flow cytometric analysis of various cell types in spleen for determination of adaptive immune response. Leukocytes from spleens of AbM-treated mice and normal control groups were isolated and processed for IF staining of cell surface antigens for flow cytometric analysis. The methods were the same for flow cytometric analysis of various cell types in spleen tissue for determination of non-specific immune response as discussed above.

Statistical analysis. Statistical analysis for comparison between two groups was performed using the Student's t-test. Data are expressed as the mean±standard deviations. Values of p<0.05 were considered as being significant.