The results in the current study clearly indicate that oral administration of MAK5 effectively enhances both macrophage and lymphocyte functions in aged mice. A recent study suggested involvement of the immune response in chemically induced carcinogenesis [33
]. In that study, the number of immune cells positive for dendritic cell and macrophage common markers was significantly greater in a N
-nitrosoguanidine-resistant rat strain (Buffalo) than that in a carcinogen-sensitive rat strain (ACI). Thus, the findings in this study suggest that immunomodulation by MAK5 may be partially responsible for inhibiting chemically induced carcinogenesis.
It is now known that the immunosuppression linked to aging is due to a decline in the response of lymphocytes, but not to a decline in the non-specific function of macrophages. Indeed, phagocyte function of macrophage could somewhat counteract the decreased specific immune response in old age [34
Macrophages are known to play an important role in host defense mechanisms for protection from microbial invaders [4
]. Macrophage glucose consumption increases as a result of the activation of macrophages [35
]. In the present study, glucose consumption capacity of peritoneal macrophages from old mice treated with MAK5 at all doses and incubated for up to 72 h was significantly higher compared with the control group. However, glucose consumption of peritoneal macrophages from young mice without treatment incubated for 48 and 72 h were significantly greater than those in the old mice treated with MAK5. These results suggest that MAK5 activates peritoneal macrophages from old mice, though it was not as young mice. Since glucose consumption by macrophages is related to the pentose phosphate pathway of glycolysis [35
], it is likely that MAK5 activates the pentose phosphate pathway in the peritoneal macrophages.
Some researchers have suggested that NO is a cytotoxic effector molecule of macrophage-mediated tumorcidal actions [36
]. Murine peritoneal macrophages in culture synthesize significant quantities of NO in response to LPS [31
]. Dileepan et al.
] reported that production of NO by LPS-activated macrophages from MAK5 treated mice (3 months old) was significantly higher than those from control. In the present study, we found that NO2-
production of peritoneal macrophages stimulated by LPS in old mice treated with MAK5 at the doses of 50, 100 and 200 mg/kg were significantly increased (about two fold) compared with that in the old control group. Therefore, it is speculated that the immunosuppression linked to ageing is not due to a decline in the non-specific function of macrophages [34
] and MAK5 ingestion enhances the stored capacity of macrophage function even in old mice.
In this study, MAK5 treatment alone did not cause spontaneous elevation in NO2-
production of peritoneal macrophages. These findings suggest that MAK5 treated causes priming of macrophages for enhanced sensitivity to other activating triggers, such as LPS and IFN-γ. Various cytokines such as IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor-α either independently or in combination can prime macrophages for enhanced cytolysis [39
]. It has generally been found that production of NO by macrophages is an important mediator of tumoricidal and microbicidal activity [40
]. Other researchers reported that supplementation of a similar Ayurvedic herbal food supplement (Maharishi Amrit Kalash 4) in experimental animals rodents resulted in decreased incidence of tumor growth and proliferation and decrease metastases [7
]. The mechanism(s) in which antitumor activity in made to increase by MAK is not well documented yet. Although previous studies [7
] did not examine any immune functions, the ability of MAK 5 to induce NO2-
production from macrophages may be one of the mechanisms of cancer chemoprevention.
Lectins are known to possess mitogenic activity after binding to lectin receptors [41
]. T lymphocyte mitogens such as Con A are thought to act through several subsequent steps, initially inducing IL-1 secretion in macrophages. Also, it is known that mitogenic activity, which reflects an early stage in the immune response, has been measured as a first screening of immunomodulatory activity [42
]. We [28
] reported that the stimulation index of spleen cells by Con A was increased significantly by the treatment with MAK5 at the doses of 50, 100 and 200 mg/kg in young mice. As shown in the present experiment, MAK5 at the doses of 50, 100 and 200 mg/kg exerted an augmentative effect on spleen cell proliferative responses to Con A in old mice, though it was not as young mice.
It is accepted that cytokines are major factors involved in the regulation of the immune response to antigens and infectious agents. Recently, T helper cells are divided into Th1 and Th2 cells from the profiles of cytokine secretion [43
]. It is known that Th1 cells are able to produce IL-2 and IFN-γ, whereas Th2 cells can produce IL-4 and IL-10. Th1 cells upregulates mainly cell-mediated immunity and downregulate humoral immunity, whereas Th2 cells act oppositely [44
]. A Th1/Th2 imbalance is found in cancer patients [45
]. In this study, we observed that the amounts of IL-2 and IFN-γ, but not the amount of IL-4, stimulated by Con A in young mice without treatment were significantly high when compared with those in the old mice. These results support the hypothesis that the function of Th-1 cells declines in aged mice [47
]. In the current study, MAK5 enhanced the production of IL-2 at doses of 50, 100 and 200 mg/kg, IFN-γ at doses of 100 and 200 mg/kg and IL-4 at doses of 50 and 200 mg/kg. We previously reported that the macrophage functions (glucose consumption, enzyme activity) and lymphocyte proliferation in young mice (10 weeks old) is significantly higher in groups treated with MAK at the doses of over 50 mg/kg than in controls [28
]. The result of the aged mice in the present study is in good agreement with the findings of the previous studies [28
In the present study, we could not demonstrate dose-response relationship by the applied MAK5 dosages (50, 100 and 200 mg/kg). Further research with different MAK5 dosages should be undertaken to possibly overcome this failure. Our results suggest that oral administration of MAK5 may affect the production of cytokines not only from Th1 cells, such as IL-2 and IFN-γ but also from Th2 cells, such as IL-4 in aged mice.