IL-12 Production Induced by Agaricus blazei Fraction H (ABH) Involves Toll-like Receptor (TLR)

Agaricus blazei Murill is an edible fungus used in traditional medicine, which has various well-documented medicinal properties. In the present study, we investigated the effects of hemicellulase-derived mycelia extract (Agaricus blazei fraction H: ABH) on the immune system. First, we examined the cytokine-inducing activity of ABH on human peripheral mononuclear cells (PBMC). The results indicated that ABH induced expression of IL-12, a cytokine known to be a critical regulator of cellular immune responses. Flow cytometric analysis demonstrated the induction of IL-12 production by the CD14-positive cell population, consisting of monocytes/macrophages (Mo/Mφ). Furthermore, the elimination of Mo/Mφ attenuated IL-12 production in PBMC. ABH-induced IL-12 production was inhibited by anti-CD14 and anti-TLR4 antibodies but not by anti-TLR2 antibody. The activity of ABH was not inhibited by polymyxin B, while the activity of lipopolysaccharide used as a reference was inhibited. Oral administration of ABH enhanced natural killer (NK) activity in the spleen. These findings suggest that ABH activated Mo/Mφ in a manner dependent on CD14/TLR4 and NK activity.

Microbiology Research Institute. Ltd, Yamanashi, Japan) and all ABPC products were made from this compound. Samples of 1.5 g of A.blazei compound were ground and mixed with distilled water to a final concentration of 0.1 g/ml (w/v). After centrifugation, the supernatant was collected and passed through a 0.45 m filter (Millipore Co., Bedford, MA) for use in the experiments.

Preparation of PBMCs
Heparinized human peripheral blood was obtained from healthy donors. PBMCs were isolated using the Ficoll-Hypaque density-gradient method, as described previously (13). Peripheral blood was centrifuged at 2000 r.p.m. for 10 min to remove plasma. Blood cells were diluted with phosphate buffered saline (PBS), then overlaid onto Ficoll-Hypaque solution (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) and centrifuged at 2000 r.p.m. for 30 min. The PBMC layers were collected and washed twice with PBS. The cells were resuspended to a concentration of 1 ϫ 10 6 cells/ml in RPMI-1640 medium supplemented with 10% foetal calf serum, 100 U/ml penicillin and 100 g/ml streptomycin.

RNA Preparation Quantification and RNase Protection Assay
After stimulation of human PBMC, total RNA was extracted from the cells using TRIzol (Invitrogen Corp., Carlsbad, CA). RNase protection assay was performed using the hCK-2RiboQuant Multiprobe RNase Protection Assay system (BD Pharmingen, San Jose, CA) according to the manufacturer's instructions. Aliquots of 10 g of RNA were used for each assay. Gels from three distinct experiments were analyzed using a BAS3000 and Image Gage (Fuji Film, Tokyo, Japan).

Quantification of IL-12p40 and p70 in Culture Supernatants
Isolated human PBMC were cultured in media containing ABH, 1 ml of 10% FCS/RPMI1640 with various concentrations of ABH, for 0-16 h. The levels of IL-12p40 in the supernatants were measured using an enzyme-linked immunosorbent assay (ELISA) kit, optEIA TM ELISA kit (BD Pharmingen) according to the manufacturer's protocol. The concentration of IL-12p40 and p70 was determined using the data analysis program Softmax (Molecular Devices, Menlo Park, CA).

NK Activity Assay
NK activities of human PBMC and murine spleen cells were determined by lactate dehydrogenase (LDH) assay. The NKsensitive mouse cell line YAC-1 was used as target cells. Target and effector cells were mixed at the indicated effector/target (E/T) ratios at 0.2 ml/well using 96-well round-bottomed multiwell plates (BD Labware, Lincoln Park, NJ). After incubation for 4 h, cells were centrifuged at 250 g for 4 min, and then the cell-free supernatant was collected for LDH assay using CytoTox96 (Promega, Madison, WI). The percentage of specific LDH release was calculated by the following formula: % cytotoxicity ϭ [(experimental LDH release) Ϫ (spontaneous LDH release by effector and target)/(maximum LDH release) ϫ (spontaneous LDH release)] ϫ100. For the control experiments, the target cells were incubated either in culture medium alone to determine spontaneous release or in a mixture of 2% Triton X-100 to define the maximum LDH release. The spontaneous release was always Ͻ10% of the maximum release. All assays were performed in triplicate.

Treatment of PBMC with ABH induced IL-12 Production
Expression of IL-12 messenger RNA (both p35 and p40 subunits) was determined in PBMC with or without ABH stimulation by RNase protection assay. As shown in Figure 1, IL-12 messenger RNA expression (p40 subunit) was induced within 4 h after ABH stimulation. In unstimulated controls, IL-12 messenger RNA was not induced until 16 h. We quantified the release of IL-12 p40 protein in human PBMC culture IL-12-inducing activity of Agaricus blazei extract supernatants in relation to ABH dose. As shown in Figure 2A, IL-12 p40 was induced significantly in all donors. The levels of IL-12 production were reduced at concentrations of ABH in excess of 170 ng/ml. Figure 2B shows induction of IL-12 p70 (heterodimer of p40 and p35) by ABH. These observations suggest that ABH induces IL-12 production by PMBC.

CD14-positive Cells Produced IL-12 on Stimulation with ABH
To determine the target population of ABH, we analyzed IL-12-production levels of each population by intracellular cytokine staining method and flow cytometry. After 16-hour culture, only CD14 positive population was induced IL-12p40 ( Figure 3). Neither normal media (control) nor peripheral dendritic cells (lin-/CD11c and HLA-DR+) induced production of IL-12 p40 protein (data not shown). We then added anti-CD3/CD19 and CD14 monoclonal antibody-conjugated Dynal beads to Ficoll PBMC fractions to deplete each fractions. IL-12 production was observed in PBMC from which CD3 and CD19 were depleted.
However, depletion of CD14 caused a 20% reduction in the level of IL-12p40 production ( Figure 4).

Involvement of CD14 and TLR4 in IL-12 Induction by ABH
Human TLRs have been shown to transduce intracellular signals by some mycobacterial ligands. These reports suggest that TLRs initiate human innate immune responses and pattern recognition by TLRs regulates the nature of not only innate but also adaptive immune responses (14)(15)(16).
Furthermore, flow cytometry was performed to quantify the IL-12-producing cells. In PBMC pre-treated with anti-hTLR4 and anti-hCD14 monoclonal antibodies, the levels of IL-12 production by CD14-positive cells were reduced to 1.33% and to 0.19% in samples treated with anti-hTLR4 and anti-hCD14 mAb, respectively. No effect was observed in samples treated with control antibody. There were no differences in the ratio of CD14positive cells in PBMC before and after antibody treatment (Fig. 5B). These results suggested that anti-hTLR4 and anti-hCD14 mAbs blocked signal transduction via TLR4 and CD14.

Induction of IL-12 by ABH was Not Inhibited by Polymyxin B
CD14 and TLR4 have been reported as subunits of the lipopolysaccharide (LPS) receptor complex. The CD14-TLR4-MD2 heterotrimer recognises the LPS/LBP-complex (17)(18)(19)(20). Our results indicated that CD14 and TLR4 were essential components for induction of IL-12 by ABH. To exclude the possibility that cellular activation by ABH was a result of endotoxin contamination in the extracts, the abilities of ABH and reference LPS to induce IL-12 production by human PBMC were examined in the presence and absence of polymyxin B. Polymyxin B showed little effect on IL-12 induction by ABH, IL-12-inducing activity of Agaricus blazei extract  whereas that of LPS was inhibited completely (Fig. 6). We measured the amount of endotoxin in the ABH extract using an Endospecy kit for endotoxin quantification (Seikagaku Corp., Tokyo, Japan). The ABH extract used in the present study was essentially free from endotoxin contamination (data not shown). proliferation, interferon-␥ (IFN-␥) production and cytotoxic activities by T cells and natural killer cells (21)(22)(23). To evaluate its effects in vivo, we examined the effect of oral administration of ABH on NK cytotoxic activity of murine spleen cells. ABH-treated mice showed significantly higher levels of NK cytotoxic activity than controls; the ABH-treated group showed 40% cytolysis, while the control group showed 25% cytolysis (Fig. 7). This result suggested that oral administration of ABH can induce NK activity in vivo.

Discussion
Hemicellulase-digested A.blazei extract showed a very high degree of water solubility. Almost 95% by dry weight was extracted in water, in contrast to non-digested samples, which showed only about 20% extraction (data not shown). Various methods were employed to extract the components from A.blazei, including treatment with acid and organic solvent. These methods for extraction were not suitable for evaluation and analysis of total activity of all components of A.blazei. The high degree of water solubility obtained by hemicellulase digestion is a major advantage for analysis of the effects of Agaricus. In the present study, we were able to analyze the total effect of Agaricus extract as we could extract almost all of the components.
As the first step to determine the effects of ABH, we attempted to identify inducible or suppressible cytokines. We found the IL-12-inducing activity in the water extract. The optimum concentration of ABH for induction of IL-12 was from 170 to 510 ng/ml, and higher concentrations showed lower levels of induction.
Agaricus blazei extract was reported previously to show a suppressive effect against proliferation and cytokine expression using PBMC (24). The effects of elements with the ability to suppress cell activity may appear at high doses. Suppressive elements did not have sufficient effects to suppress the induction of IL-12 to Ͻ170 ng/ml.
The results of intracellular cytokine staining and populationspecific elimination showed CD14 positive cells, monocytes/ macrophages (Mo/M) to be the target population of ABH. We detected no IL-12 production by peripheral dendritic cells (DC). Peripheral circulating Mo circulate for 1-3 days before entering tissues, where they differentiate into mature resident M or DCs (25). Our results suggested that ABH possessed the potential to activate immature Mo and promote the Th1 response in tissue. As a result of this activity, ABH maintains the Th1 response level and cellular immunity. ABH-treated mice have been reported to show higher levels of NK activity in the spleen than untreated controls. The ability of DCs to produce cytokines differs among DC subsets depending on the tissue to which they belong (26,27). Peripheral Mo heterogeneity was reported previously (28)(29)(30). Thus, we speculate that ABH may have different effects on different subsets of Mo and DC. Therefore, the effects of ABH should be analyzed from various viewpoints.
Various models to explain the mechanism of the immunomodulatory effects of many mycobacterial extracts and components have been reported. These include cell activation by intracellular signals transduced by TLRs. The results of the present study indicate that the activity of ABH to induce IL-12 production is dependent on CD14 and TLR4 (Fig. 5). LPS causes activation of Mo/M via the CD14/TLR4/MD2 receptor complex (31). The Limulus activity of ABH was 80 pg/ml, equivalent to E.coli LPS (data not shown). Therefore, the endotoxin content in the ABH used in the present study was Ͻ1 pg/ml, which was completely inactive in our system. On the other hand, polymyxin B-treated ABH also induced IL-12 production at almost the same level as untreated ABH (Fig. 6). This excluded the possibility that the activation of Mo/M was due to response to contamination of the ABH extract by LPS.
We speculate on two possible explanations for this observation: ␤-Glucan in ABH may be recognised by a different receptor complex, or other components, such as mannan, may be mainly responsible for the IL-12-inducing activity of ABH. As ABH is a mixture of various components, it is necessary to identify and isolate the elements responsible for the action of ABH.
Some studies of the anti-tumor effects of A.blazei have demonstrated direct cytotoxic effects (1). The chloroform/ methanol extracts of A.blazei were shown to have antitumor activity against solid tumors. Ergosterol in this extract inhibits angiogenesis and causes the death of tumor cells by preventing neovascularisation (5). When the particles from fruiting bodies of A.blazei Murill (ABP-F) were reacted with human serum, the formation of complement-opsonised ABP, iC3b-ABP-F complexes, and binding of the complexes to human PBMCs were observed. In addition, PBMCs incubated in the presence of iC3b-ABP-F complexes inhibited the proliferation of a human tumor cell line in vitro (38).
These reports indicated that components of A.blazei have various activities. It was reported that A.blazei extract acts mainly through modulation of the immune system, activating macrophages, neutrophils and lymphocytes (3,4,8). On the other hand, ethanol extracts of A.blazei mycelia were shown to reduce the cytopathic effects of the western equine encephalitis (WEE) virus (39). Several fungi incubated with immune cells showed activation of some cells, especially antigen-presenting cells, and induced cytokine production (40)(41)(42)(44)(45)(46)(47)(48). The ethanol extracts of A.blazei stimulated macrophages and induced expression of the cytokines IL-8 and TNF (49).
This report indicates that the extract of A.blazei mycelia induces IL-12 production. IL-12 exerts multiple biological activities, which include activation of CD8ϩ CTLs, differentiation of CD4ϩ T lymphocytes, induction of nitorogen oxide production by macrophages, induction of type 1 responses by helper T lymphocytes and NK cell activation (50,51). IL-12 has also been shown to possess potent antitumor activity in a wide variety of murine tumor models (49,(52)(53)(54)(55). Recent reports have demonstrated that the in vivo antitumor capacity of IL-12 is mediated via NK and/or NKT cells (56,57). The findings of the present study will help in furthering our understanding of the immunomodulatory and anti-viral effects of A.blazei. IL-12 promotes and sustains the cellular immune system. Oral administration of ABH was shown to enhance the NK activity of mouse spleen cells (Fig. 7). It is notable that ABH modulates immune responses not only in vitro but also in vivo. In the context of A.blazei administration as a functional food, the induction of IL-12 and enhancement of cellular immunity are important for various biological activities.
In conclusion, we have demonstrated that hemicellulasederived ABH induces IL-12 production by human peritoneal monocytes via TLR4-CD14 and enhances cytotoxic activity against tumor cells in vivo. These responses may be involved in the biological activities of ABH, such as its anti-tumor, antiinfection and anti-allergic effects. ABH is a non-proliferative and non-pathogenic microbial component that can trigger an immune response that is useful in the fight against infectious diseases and cancer. The accumulation of such data concerning the effects of components and clarification of mechanisms of their action are essential to understand features of mushroom such as the biological response modifier (BRM).