G. Hetland, Department of Immunology and Transfusion Medicine, Ulleval University Hospital, Oslo, Norway. E-mail: email@example.com
Agaricus blazei Murill (AbM) is an edible, medicinal mushroom of Brazilian origin. It is used traditionally against a range of diseases, including cancer and chronic hepatitis, and has been cultivated commercially for the health food market. AbM has recently been shown to have strong immunomodulating properties, which has led to increasing scientific interest. In this article, we review current knowledge as to the immunological properties of AbM, and its possible clinical use in connection with infections and cancer. We also present some novel findings, which point to highly different biological potency between AbM extracts of different source and manufacturing.
Immunostimulation by medicinal mushrooms generally occurs via innate immunity, and is typically mediated by phagocytic cells. These cells ingest invading pathogens or interact with pathogen components, which in either case further stimulate innate and adaptive immunity through secretion of cytokines and chemokines. Pattern-recognition receptors (PRR) on the cell surface – such as Toll-like receptors (TLR), mannose receptors and dectin-1 – trigger the response by recognizing conserved molecular patterns in the micro-organisms . There has been increasing interest in immunomodulating substances from mushrooms. Lentinan, for example, has been used extensively in the treatment of patients with cancer in Japan .
Agaricus blazei Murill (AbM) (Himematsutake) is an edible Basidiomycetes mushroom, which grows naturally in Piedade outside of São Paulo, Brazil. According to legend, older people in this region had fewer serious diseases than those in neighbouring communities, presumably due to the use of AbM as food. Besides cancer and chronic hepatitis, this mushroom has been used in folk medicine against a variety of diseases, including diabetes, arteriosclerosis and hyperlipidaemia . In the mid-1960s, spores of AbM were taken to Japan for commercial cultivation and research. Since then, a considerable number of scientific papers have appeared, focusing mainly on the effect of AbM as an immunomodulating agent, and its therapeutic effect in connection with infections and cancer. In the present article, this work is reviewed.
One important group within the immunocompetent leucocytes is the phagocytic cells, which include monocytes, monocyte-derived macrophages and polymorphonuclear neutrophils (PMN). They all bind, internalize and eradicate invading micro-organisms. These cells use their own primitive, non-specific recognition systems, which allow them to bind a multitude of microbial products, and elicit the so-called innate immune responses. In effect, the cells act as the first line of defence against infection. Natural killer (NK) cells and NK T cells also belong to the innate immune system and are, together with macrophages, in the first line of defence against tumours .
AbM is rich in biological response modulators, such as proteoglucans [5, 6] and β-glucans , which are potent stimulators of macrophages [8–10], PMN  and NK cells . These substances are main structural components in the cell wall of yeast and fungi, but are also found in some plants such as barley. The effects are mediated via the lectin-binding site for β-glucan in complement receptor 3 (CR3) (CD11b/18) [13–15], Toll-like receptor 2 (TLR2)  and dectin-1 . Stimulation of these receptors results in the release of proinflammatory cytokines , nitric oxide and hydrogen peroxide [19, 20], lysosomal enzyme  and activation of arachidonic acid metabolism . β-1,3-glucans also induce activation of another component of innate immunity, the alternative complement pathway .
As to AbM, several studies have investigated its stimulating profile in more detail. In activated macrophages, AbM has been shown to induce secretion of nitric oxide, the proinflammatory cytokines TNF-α and IL-8 , as well as the Th1 cytokine IL-12 . A dose-dependent in vitro production of proinflammatory cytokines, including IL-1β and IL-6, has been confirmed in AbM-stimulated human monocytes and umbilical vein endothelial cells ; however, neither anti-inflammatory cytokine IL-10 nor IL-12 synthesis was observed in this study. On the other hand, an AbM proteoglycan that stimulated mouse dendritic cell maturation also increased IL-12 production . Gene expression microarray analysis of promonocytic THP-1 cells revealed upregulation of genes for chemokine ligands CXCL1-3, TLR2, dectin-1 and the IL-23α subunit of the IL-12 family, in addition to genes for IL-1β, IL-8 and cyclo-oxygenase 2 (Prostaglandin-endoperoxide synthase 2), whereas the IL-10 and IL-12 genes were not upregulated .
As to in vivo effects, increased levels of cytokines MIP-2 (murine equivalent of IL-8) and TNF-α have been observed in mice receiving AbM extract . Examination of immunomodulatory effects of AbM in mice have revealed increased numbers of antibody-producing spleen cells , elevated serum IgG levels and T-cell number in spleen, as well as increased phagocytic capacity of PMN , which is induced by proinflammatory cytokines [32, 33]. On the other hand, IL-12- and IFN-γ-mediated NK cell activity by AbM has been documented both in vitro and in vivo . The latter contrasts somehow with the previous finding of AbM-suppressed PBMC production of IFN-γ, IL-2 and IL-4 . Interestingly, also the gene for regulator of G-protein signalling (RGS1), which is important for the G-protein-linked rhodopsin-like chemoattractant receptors for IL-8 (CD128), complement anaphylatoxin C5a (CD88), bacterial formyl peptide (fMLF) and leukotriene B4 , was selectively upregulated by an AbM extract in the promonocytic THP-1 cells . The receptor for interferons α and β (IFNAR1) was also upregulated in peripheral blood leucocytes from patients with chronic IFN-α-resistant hepatitis C virus (HCV) infection, who had ingested the AbM extract for 1 week .
Extracts of AbM have been used successfully as adjuvants in DNA vaccines to improve their efficacy against hepatitis B virus (HBV) infection and foot-and-mouth disease (FMDV) [38, 39]. Relative to the DNA vaccine controls alone, mice which received either HBcAg DNA vaccine or FMDV DNA vaccine plus AbM extract had significant increase in not only, respectively, HBcAg- or FDMV-specific antibody response, but also in T-cell proliferation. As has been shown for isolated β-glucans, AbM also mediates activation of complement via the alternative pathway .
Immune and inflammatory responses are critically dependent on the ability of leucocytes to migrate from the blood into surrounding tissues at sites of inflammation. Leucocyte tethering and rolling, activation and firm adhesion comprise the classic paradigm of inflammatory cell recruitment. Specific families of adhesion molecules mediate each step of this cascade. The initial tethering and rolling are predominantly mediated by the selectin family of adhesion molecules, of which CD62L (L-selectin) is constitutively expressed in high levels on all leucocytes. The β2 (or leucocyte) integrins, i.e. the CD11/CD18 complex, including CD11b (Mac-1, C3 receptor) and CD11c (p 150/95, C4 receptor) are rapidly upregulated after activation and promote strong attachment of leucocytes to the vascular endothelium and subsequent transendothelial migration. In studies with whole blood, we recently found that AbM extract down to 0.06% final concentration increased CD11b and reduced (due to shedding) CD62L expressions on leucocytes (Fig. 1) . This particular extract is the same as the one used in Fig. 2 and contained approximately 20% extracts of two other Basidiomycetes mushrooms (see below). An update of AbM-induced changes in the expression of cytokines or chemokines and related receptors and associated proteins is given in Table 1.
Table 1. Effect of Agaricus blazei Murill on cytokines/chemokines and related proteins in leucocytes
Type of signal molecule
Genes are given in italics, while names of proteins in regular typing. Arrows indicate up- or downregulation. *Upregulation and then shedding, giving a net lower signal.
The reactivity of molecular oxygen can be increased by reduction or excitations, giving rise to highly reactive oxygen species (ROS), e.g. superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen and peroxynitrite. The principal cellular source of ROS is the PMN in which ROS are produced by the NADPH oxidase-catalysed oxidative burst reaction. PMN (and monocyte)-derived ROS are produced primarily in order to destroy invading micro-organisms. This effect is normally advantageous, but inadvertent extracellular release of ROS may induce inflammatory reactions in surrounding tissues. There are contradictory findings regarding ROS production and AbM. AbM has been reported to be an excellent source of antioxidants , albeit to a somewhat lesser degree than another Basidiomycetes mushrooms . On the other hand, addition of AbM extract to whole blood induced a slight, but significant, increase in ROS production (peroxynitrite, ONOO−) in PMN, while not in monocytes .
Immunomodulating glucans are known to induce enhanced defence against infections [44–47], and, as mentioned above, such components are abundant in AbM. Fractions of AbM have been shown to inhibit the cytopathic effect of western equine encephalitis virus in vitro . As AbM traditionally has been used against chronic hepatitis, possible in vivo effect of orally taken AbM was examined in patients with IFN-α-resistant, chronic hepatitis C virus infection . The viral load was found to be slightly, but not significantly, decreased after 1 week of treatment. However, as the treatment seemed to upregulate the gene for IFN-α-receptor, a study examining AbM intake combined with regular IFN-α treatment, would be of interest.
The commercially available AbM extracts are made using different protocols, and often contain additional components. In order to evaluate possible differences, five AbM products (A–E) were compared in a mouse model for pneumococcal sepsis. The extracts were given orally by means of a gastric catheter 1 day before the inoculation with bacteria. Among the extracts, only one (A) had a significant protective effect as demonstrated by reduced bacteraemia and increased survival rate (P < 0.05) (Fig. 2). None of the control mice given PBS survived day 5, whereas 38% (3/8) of mice given extract A lived on day 6, and another two mice were killed due to illness on day 8 (Fig. 2B). One of the other extracts (D) showed a similar, but statistically non-significant, trend for both bacteraemia and survival. The most active product was an aqueous, highly purified extract, which contained 82% AbM, 15%Hericium erinaceum (Yamabushitake) and 3%Grifola frondosa (Maitake). Although the latter species is another known medicinal mushroom with immunomodulating effects, recent examination of NF-κB activation via TLR2 has revealed that the main stimulatory effect of the extract A on monocytes is contained in the AbM fraction . Note that, as extracts B–E were pure AbM extracts according to the producers, one must compare ∼80% of extract A with 100% of each of the others.
The difference in anti-infectivity between the AbM extracts may be due to the presence of additional biological components, such as extracts from other mushrooms, with possible synergistic effects . Cultivation methods may, however, be another reason for biological differences. It is well known that mushrooms and moulds can change their phenotype (e.g. colour and sporulation), and their production of secondary metabolites [such as mycotoxins and microbial volatile organic compounds (MVOC)] depending on growth medium and conditions. For example, mycotoxins are produced under suboptimal growth conditions of moulds , and MVOC during growth on certain materials in ‘sick buildings’ .
Extracts A and E were found to be equally potent in activating the alternative pathway of complement (G. Hetland & T.E. Michaelsen, unpublished data), indicating that complement activation is not the mechanism behind the antibacterial effect of extract A. Moreover, this particular extract had no direct bacteriolytic or bacteriostatic effect as revealed by the lack of inhibition zones when culturing the pneumococci in its presence . However, similar to the proinflammatory cytokine-induced phagocytosis of S. aureus and M. tuberculosis by PMN [32, 33], the increased serum levels of proinflammatory cytokines MIP-2 (IL-8) and TNF-α upon AbM treatment, could have led to enhanced phagocytosis of S. pneumoniae.
Extract A was also used in another mouse model examining Gram-negative sepsis due to aerobic exposure of faecal solutions . In this faecal peritonitis and sepsis model, three different dilutions of faeces were inoculated intraperitoneally, which led to, respectively, severe, moderate and mild infections. In these experiments, AbM treatment given per os 24 h prior to challenge, had protective effect as demonstrated by less reduction in temperature, decrease in bacteraemia, and increase in survival rate (P = 0.03). Significant impact of the treatment was observed in severe (Fig. 3), but not moderate or mild infections, probably due to smaller differences in the parameters between challenged and control animals in the latter situations.
β-glucans have known antitumour properties [7, 53, 54], so do proteoglycan [55, 56] and ergosterol , two other ingredients of AbM [6, 58]. β-1,3/1-6-glucans from yeast and mushrooms have been used in various clinical trials against cancer [59–63]. Effects of macrofungi in connection with cancer treatment have recently been reviewed in Ref. .
Ohno et al.  proposed that the apparent antitumour effects of AbM are due to β-1,3-glucan. Others have demonstrated that a β-1,6-glucan extracted from AbM induced tumour regression in mice [65, 66], and that daily supplement of β-glucan from AbM reduced spontaneous metastasis of ovarian and lung cancer cells in a mouse model . The latter authors suggested that treatment with β-glucans might be beneficial for patients either at risk for metastasis or with metastasis. This is in agreement with a report that proposes TLR2/4 agonists to be potentially survival-prolonging molecules in patients with cancer who relapse under chemotherapy , as well as with reports that find β-glucans per se  and AbM [25, 49] to bind to TLR.
In a related study, a β-glucan–protein complex isolated from AbM was shown to have inhibitory action against Meth A fibrosarcoma in a mouse model . The results were confirmed by Ebina and Fujimiya , using another proteoglucan from AbM, and the effect has been attributed to NK cell activation and apoptosis induction . An RNA–protein complex from AbM has been shown to induce apoptosis in the leukaemia cell line HL60 . Moreover, it has been reported that treatment with AbM had inhibitory effect on leukaemia cells in patients with acute non-lymphocytic leukaemia . Aqueous extracts of AbM exhibit antimutagenic and anticlastogenic effects in vitro [72, 73], and protect against X-irradiation in mice . Furthermore, another AbM water extract inhibited abnormal collagen fibre formation in human hepatocarcinoma cells .
Other Japanese groups have shown that fat-soluble ergosterol, as well as another antiangiogenetic substance from AbM, reduced tumour growth and metastasis in sarcoma- and lung carcinoma-bearing mice [58, 76]. One interesting study showed myeloma tumour suppression by intake of an AbM extract in myeloma bearing mice, and that the tumour vanished in mice given a combination of AbM extract and marine phospholipids . These results suggest improved uptake through the gut mucosa of active substances in AbM, such as β-glucans, by encapsulation in phospholipids. Recently, a toxicity study in rats that ingested AbM extract over 2 years, found no carcinogenicity or other adverse health effects of AbM . Rather, the study demonstrated significantly lower mortality among the male rats on AbM treatment, which was suggested to be due to a lower tumour incidence in this group. Table 2 shows a summary of reported AbM-induced changes in vitro and in vivo related to cancer.
Table 2. Effect of AbM on tumours and related activity.
The in vivo results are from mouse models, except for two *human studies and one †rat model. ‡Abnormal collagen fibre formation. QOL, quality of life.
Microarray examination of peripheral leucocytes from the mentioned AbM extract-intake study in patients with HCV, revealed in addition to increased expression of IFN-α,β receptor, an upregulation of genes involved in cell signalling and cycling, as well as in transcriptional regulation . These genes may be important in antitumour defence. There are anecdotal indications  of patients with haematological cancer who have been cured, or experienced less side effects of chemotherapy, when supplementing the prescribed hospital treatment with AbM extract. This is in line with a clinical report from South Korea where AbM extract treatment reduced the chemotherapeutic side effects in patients with gynaecological cancer . In addition to enhanced quality of life during chemotherapy, supplemental AbM treatment increased NK cell activity significantly in these patients. This agrees with the reported increased NK cell activity and increased infiltration of NK cells into tumour sites secondary to AbM treatment [6, 65]. We are planning a clinical trial with AbM extract as adjuvant supplement to chemotherapy for patients with haematological cancer.
There are on-going clinical trials at Memorial Sloan-Kettering Cancer Center in New York with β-glucan from yeast and cereals in patients with neuroblastoma and leukaemia/lymphoma (protocol 05-073 and 03-095, http://www.mskcc.org). A β-glucan from G. frondosa has been shown to increase bone marrow cell haematopoiesis in mice and induce haematopoietic stem cell proliferation in umbilical vein blood cells [80, 81]. Such effects would speed up recuperation of patients with cancer after chemo- or radiotherapy. Currently, we are undertaking a phase I study with AbM extract in healthy volunteers in order to gain more data as to general immunological effects and toxicity. It should be noted that Kerrigan  has suggested that AbM is actually Agaricus subrufescens Peck, which was already described in 1893 in contrast to AbM’s description in 1947.
AbM has reasonably well-characterized immunomodulatory properties. The mushroom appears to activate the branch of the immune system that elicits both anti-infection and antitumour effects, at least in mouse models, but presumably also in humans. It prolongs life in rats and protects against side effects of chemotherapy in patients with cancer on supplemental AbM therapy. In vivo AbM extract causes upregulation of genes involved in cell signalling, cycling and transcriptional regulation, which are related to anticancer effects. However, there is a huge difference in magnitude of biological effects between different AbM extracts, which probably is dependent upon source of AbM and manufacturing procedure for the extract. We believe the evidence is sufficient to warrant further testing of AbM, both as supplementary, adjuvant cancer therapy; and in relation to treatment of patients with serious multi-antibiotic-resistant bacterial infections.
We thank Linda K. Ellertsen for the collaboration in publications reviewed here, and Lisbeth Sætre, Center for Clinical Research, Ulleval University Hospital and Else-Carin Groeng and Åse Eikeset, Department of Environmental Immunology, and Marc Gayorfar, Division of Infectious Disease Control, and personnel at Animal facilities, Norwegian Institute of Public Health, Oslo, for excellent technical assistance.