Abstract
Agaricus blazei Murill (ABM) has shown particularly strong results in treating and preventing cancer and has also traditionally been used as a food source in Brazil. However, the exact immune responses regarding the phagocytosis of macrophage and, the activity of natural killer (NK) cells in normal mice after exposure to ABM extract was unclear. The goal of this study was to investigate whether or not ABM extract can promote immune responses in normal BALB/c mice. BALB/c mice were treated with different doses of ABM extract for different time periods. The results indicated that ABM extract significantly promoted the proliferation of splenocytes both in vitro and in vivo. ABM extract promoted the levels of interleukein-6 (IL-6) and, interferon-γ (IFN-γ) but reduced the levels of IL-4 in vitro and in vivo. The percentage of macrophages with phagocytosis after ABM extract treatment increased and these effects were of dose-dependent manners, both in vitro and in vivo. YAC-1 target cells were killed by NK cells from the mice after treatment with ABM extract at 3 and 6 mg/kg/day for up to 14 days at target cell ratios of 25:1 and 50:1. Taken together, these results show that ABM extract promoted immunomodulations in normal BALB/c mice in vitro and in vivo.
Many mushrooms (e.g. black mushroom shiitake, Hericium erinaceum, Calvatia caelata) have been recognized as immune potentiators and their components such as polysaccharides and peptides also been shown to have antitumor activity (1-4). Agaricus blazei Murill (ABM) has been reported to have antitumor activity (5-8). (1-4)-a-D-Glucan with (1-6)-h branching from ABM extract has been reported to have antitumor activity via natural killer cell (NK) activation and induced apoptosis (6). The peptide glucan from ABM induced direct cytotoxicity in Meth A tumor cells and had an indirect immunopotentiating action on tumor-bearing mice in a double-grafted tumor system (7).
Many reports have shown that ABM possesses antitumor activity (7, 9) but there are few reports regarding the immunopotentiating activities, particular in normal BALB/c mice. It is unclear how ABM extracts promote activities in the immune system. Therefore, in this report, we studied whether ABM extract promoted immune responses in normal BALB/c mice.
Materials and Methods
Materials and reagents. RPMI-1640 medium, L-glutamine, penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). The crude extracts of A. blazei Murill (ABM) extract were kindly offered by Dr. Chang (National Taiwan University, Taipei, Taiwan, PRC).
Male BALB/c mice. Mice at the age of 8 weeks and 22-26 g in weight were obtained from the Laboratory Animal Center, College of Medicine, National Taiwan University, (Taipei, Taiwan, PRC). BALB/c mice were maintained in cage housing in a pathogen-free isolation facility with a 12/12-h light/dark cycle. These animals were provided rodent chow and water and all experiments were conducted in accordance with the guidelines of the China Medical University Animal Ethics Research Board.
In vitro experiments. The spleens of six normal BALB/c mice were excised under anesthesia by CO2. Then the splenocytes were isolated and counted under a phase-contrast microscope and were used for further investigations as below:
For cell proliferation studies. Approximately 1×105/100 μl splenocytes in RPMI-1640 medium were placed on 96-well plates and 0, 31.5, 62.5, 125 or 250 μg/ml ABM extract were added. Cell were then stimulated with Con A (5 μM) for 72 h followed by centrifugation. Cell proliferation was determined by CellTiter 96 assay kit (Promega, Madison, WI, USA) as described elsewhere (10, 11).
For phagocytosis studies. Total phagocytes were isolated from spleen of normal BALB/c mice. Splenocytes were maintained on 12-well plates then 0, 25, 50 100 or 200 μg/ml of ABM extract were added. Approximately 1×105 cells/well were added to 50 μl of RPMI-1640 medium with FBS then mixed with 50 μl of Escherichia coli-FITC according to the manufacturer's instructions (PHAGOTEST kit; ORPEGEN Pharma Gesellschaft für biotechnologische, Heidelberg, Germany) and shaken in a shaker bath for 30 min at 37°C. Cells then were centrifuged at 1,500 rpm for 5 min. The supernatant was discarded and DNA was stained by DNA staining. Each sample was analyzed by flow cytometer (FACSCalibur; Becton Dickinson). Fluorescence data were collected on 10,000 cells and analyzed as described previously (12, 13).
IL-4, IL-6 and IFN-γ determination by cytometric bead array (CBA) method and flow cytometric analysis. Approximately 2.5×105/ml splenocytes in RPMI-1640 medium with 0, 125, 250 or 500 μg/ml ABM extract were then stimulated with Con A (5 μM) for 72 h followed by centrifugation and were cultured onto 24-well culture plates. Collected media were centrifuged, and supernatants were subjected to assays for levels of cytokines as described elsewhere (14, 15); analysis of IL-4, IL-6 and IFN-γ from the supernatants was conducted using a Mouse Th1/Th2 Cytokine Cytometric Bead Array Kit (BD Biosciences, San Diego, CA, USA) and was analyzed by flow cytometry.
In vivo experiments. Six normal BALB/c mice were individually treated with 0, 3 or 6 mg/kg/day of ABM extract in their diet for 0, 4, 9 or 14 days then their spleens were excised under anesthesia by CO2. The splenocytes were isolated and counted under phase-contrast microscope and these splenocytes were used for further investigations as below:
For proliferation studies. Approximately 1×105/100 μl splenocytes in RPMI-1640 medium on 96-well plates were stimulated with Con A (5 μM) for 72 h followed by centrifugation. Cell proliferation was determined by CellTiter 96 assay kit (Promega) as described elsewhere (10, 11).
For phagocytosis studies. Total phagocytes were isolated from peripheral blood mononuclear cell (PBMCs) of control and AMB-treated BALB/c mice. Approximately 50 μl of 1×105 cells/ml were added to 50 μl of RPMI-1640 medium with FBS then mixed with 50 μl of E. coli-FITC and arrayed as described above.
Natural killer cell activity. Approximately 1×107 cells/ml splenocytes were isolated from the spleen in control and AMB-treated BALB/c mice. 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. St. Louis, MO, USA) was added to the cells which were then 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 1200 rpm and 25°C. Approximately 1×105 YAC-1 cells were placed onto 96-well plates for 100 μl medium then 5x106 (50:1) and 2.5×106 (25:1) splenocytes were added to each well and determination of the NK cell activation after 12 h was determined by flow cytometry as described elsewhere (12, 13, 16).
IL-4, IL-6 and IFN-γ determination by CBA method and flow cytometric analysis. Approximately 2.5×105 splenocytes in RPMI-1640 medium from BALB/c mice after exposure to 6 mg/kg/day ABM extract for 0, 4, 9 and 14 days were then stimulated with Con A (5 μM) for 72 h followed by centrifugation and were cultured on 24-well culture plates. Collected media were centrifuged, and supernatants were subjected to assays for levels of cytokines as described above.
Statistical analysis. The results were expressed as mean±SD and the difference between groups was analyzed by one-way ANOVA. Values of p<0.05 were taken as being significant.
Results
In vitro studies
ABM extract affects the cell proliferation of splenocytes from BALB/c mice. The results presented in Figure 1 indicate that with Con A stimulation, all doses of ABM extract significantly promoted the proliferation of splenocytes.
ABM extract affects phagocytosis. As shown in Figure 2, the percentage of phagocytozed with phagocyted green fluorescent particles increased with ABM extract treatment and these effects were dose dependent. The results indicated that ABM extract significantly promoted phagocytosis.
Effects of ABM extract on IL-4, IL-6 and IFN-γ levels of splenocytes from normal BALB/c mice. Figure 3 indicates that ABM extract significantly and dose-dependently reduced the level of IL-4 (Figure 3A). Figure 3B indicates that ABM extract significantly promoted the level of IL-6 and this effect was dose dependent, however, ABM extract also promoted the level of IFN-γ at high doses (250 and 500 μg/ml) but had no significant effect at the low dose (Figure 3C).
In Vivo studies
ABM extract affects the cell proliferation of splenocytes in BALB/c mice. Figure 4 indicates that with Con A stimulation, both doses of ABM extract promoted significant proliferation of splenocytes. At 6 mg/kg/day of ABM, this effect was time dependent.
ABM extract affects NK activity of splenocytes in BALB/c mice. The results presented in Figure 5, show that the YAC-1 target cells were killed by NK cells from the mice after treatment with ABM extract at 6 mg/kg/day at target cells ratio of 25:1 and 50:1. This dose was effective at both target ratios and increased the activity of NK cells in a time-dependent manner.
ABM extract affects phagocytosis in BALB/c mice. The results presented in Figure 6 show that the percentage of phagocytes with phagocytozed green fluorescent particles was only significantly higher after 14 days' treatment with ABM at both doses (3 and 6 mg/kg/day).
Effects of ABM extract on IL-4, IL-6 and IFN-γ levels in ABM-treated BALB/c mice. ABM extract significantly reduced the levels of IL-4 at days 9 and 14 of examination (Figure 7A). Figure 7 indicates that ABM extract significantly promoted the levels of IL-6 (Figure 7B) and of IFN-γ (Figure 7C) at days 9 and 14.
Discussion
Several reports have shown that ABM extract has anticancer activity and causes an immune response. A protein polysaccharide complex of ABM was reported to be the cause of growth inhibition of sarcoma-180 implanted in mice, through the development immunomodulation (9); other reports also pointed out that this was possibly due to immunological mechanisms involving the action of various immunocompetent cells (7, 17, 18). Therefore, it is possible that ABM-induced anticancer activity involves the promoting of immune responses. However, the effect of the immune response in normal BALB/c mice was not clear. Therefore, in the present study, we investigated whether ABM extract promoted the immune response of normal BALB/c mice in vitro and in vivo. Moreover, other investigators had shown that a hot water extract of ABM increased the antibody production in mice through an increase in the mRNA levels of IL-1β and IL-6 (19), and it was reported that ABM augmented NK cell activation through IL-12-mediated IFN-γ production (20). However, the effect on IL-4, IFN-γ, phagocytosis and the activity of NK cells from normal BALB/c mice after expose to ABM extract has not been reported.
In the present study, our results indicated that the ABM extract promoted the proliferation of splenocytes both in vitro (Figure 1) and in vivo as compared with control (Figure 4). This is in agreement with another report with demonstrated that ABM can increase the proliferation of leukocytes in mice (21). Although there is no available information to address ABM-promotion of phagocytosis and increased activity of NK cells in normal mice, our results (Figure 2 and Figure 6) showed that ABM extract significantly increased both and in a dose-dependent manners. That ABM extract reduced the level of IL-4 in a dose-dependent manner was also a novel finding (Figure 3A and 7A). IL-4 is released from helper T-cells and plays an important role for antibody class switch of B-cells.
We also showed that ABM extract promoted the levels of IL-6 and IFN-γ (Figure 3C and Figure 7C). Other investigators also showed that the expressions of IL-6 and IL-1 mRNA were augmented by ABM extract in both peritoneal macrophages and spleen cells (5). Apparently, these effects are associated with T-cells and macrophages in mice after exposed to ABM. Mizuno et al. (5) showed that the percentage of the CD4 (Th2)- and CD8 (Th1)-positive cell population was significantly increased in mice orally administered a hot water-soluble fraction from ABM as compared with the control. Other reports had shown that ABM extract might be useful as an adjuvant to improve the efficacy of DNA vaccines in vivo (22).
The data presented here established that ABM extract promoted NK cell activity (Figure 5) and phagocytosis (Figure 6), and increased the release of associated cytokines (IL-6, IFN-γ) but reduced IL-4 levels. Recently, it was reported that ABM extract was not only able to directly induce apoptosis of tumor cells (23, 24) but also to protect them from ROS-mediated toxicity (25). This is the first finding that NK cell activity can be promoted by ABM extract in vitro, however, the mechanisms responsible remains nuclear. In the macrophage activity experiments, our results showed that ABM extract increased phagocytosis because the percentage of target cells being phagocytozed was increased after the increase of ABM extract both in vitro and in vivo (Figure 2 and Figure 6). Other reports also demonstrated that different aqueous extracts of ABM are not genotoxic (23, 26, 27). Further investigation of ABM extract in SCID and nude mice cancer models will contribute to better understand of its in vivo activity for promoting immune responses.
Acknowledgements
This study was supported by grants CMU95-083 and CMU97-276 from China Medical University, Taichung, Taiwan.
- Received May 10, 2009.
- Revision received July 6, 2009.
- Accepted July 14, 2009.
- Copyright © 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved