Lactic acid bacterium potently induces the production of interleukin-12 and interferon-γ by mouse splenocytes

Abstract

We previously have reported that the lactic acid bacterium, Lactobacillus casei strain Shirota (LcS), shows marked antitumor activities and an ability to modify immune responses. In this study, we examined whether LcS can induce the production of interleukin (IL)-12 and interferon-gamma (IFN-γ), which are important cytokines for antitumor and antimicrobial immunity, from murine splenocytes in vitro in order to clarify the mechanisms of its immune modification.

Stimulation by LcS induced a marked production of IL-12 by X-ray-irradiated splenocytes (X-irr-Spl). The production of IL-12 by X-irr-Spl was independent of the presence of nylon wool column-passed splenocytes (NW-Spl). IFN-γ was produced by splenocytes by the stimulation with concanavalin A (Con A). LcS showed a synergistic stimulatory effect on the ConA-induced production of IFN-γ. In addition, X-irr-Spl were required for the IFN-γ production by NW-Spl treated with LcS. The IFN-γ production was reduced by anti-IL-12 antibody treatment. NW-Spl produced IFN-γ following treatment with recombinant IL-12. Thus, we confirmed that IFN-γ production by splenocytes was the result of the production of IL-12 from X-irr-Spl stimulated by LcS. Furthermore, in BALB/c mice, the oral administration of viable LcS augmented the production of IFN-γ but not that of IL-4 or IL-5 by splenocytes.

Thus, we suggested that LcS primarily activated X-irr-Spl, probably macrophages, and these cells secreted IL-12. The IL-12 induced by LcS stimulated the production of IFN-γ.

Introduction

Interleukin (IL)-12 is an important cytokine for inducing cellular immunity. IL-12 potently stimulates cytotoxic T cells and natural killer (NK) cells, and enhances the production of Th1 cytokines (IL-2 and interferon-gamma (IFN-γ)) and the proliferation of Th1 cells. IL-12 is secreted by B cells, dendritic cells, and macrophages. IL-12 has potent in vivo antitumor and anti-metastatic effects against murine tumors via cytotoxicity of CD8⁺ T cells and IFN-γ production from helper T cells. Some microorganisms (Song et al., 1996; Chong et al., 1996; Fujimoto et al., 1997) and their cellular components (DAndrea et al., 1992; Wysocka et al., 1995) induce IL-12 production. The production of cytokines from Th1 T cells was shown to be coincident with the activation of tumor-specific T cells (Goedegebuure et al., 1997).

IFN-γ has multiple functions in a host. It mainly augments cellular immunities, namely antitumor and anti-infection responses. IFN-γ also inhibits the production of Th2 cytokines such as IL-4 and IL-5. The inhibition of Th2 cytokines leads to the diminution of specific humoral immune responses to antigens.

It is well known that lactic acid bacteria and their products have beneficial effects on the health of animals and humans by reports from many investigators (Shahani et al., 1983; Davidkova et al., 1992; Popova et al., 1993; Perdigon et al., 1993; Pereyra and Lemonnier, 1993). The lactic acid bacterium, Lactobacillus casei strain Shirota (LcS), has immunomodulatory effects in hosts, and has shown marked antitumor activity in experimental animals and humans (Masuno et al., 1991; Aso et al., 1992; Aso et al., 1995;Okawa et al., 1989; Kato, Kobayashi, Yokokura and Mutai, 1981; Matsuzaki et al., 1987; Matsuzaki et al., 1988). We also demonstrated that the immune response mediated by T cells were suppressed in tumor-bearing mice: the levels of splenic T-cell responses (i.e. cell proliferation responses against IL-1, IL-2, phytohemaggulutinin and Con A) in tumor-bearing mice was restored to the normal level by the oral administration of LcS (Kato et al., 1994). Shimizu et al., 1981demonstrated that LcS did not show the direct mitogenic effect on T cells. LcS augments NK activity and T-cell-mediated tumor immunity and directly activates macrophage functions (Kato et al., 1983, Kato et al., 1984, Kato et al., 1988; Yasutake et al., 1984). Generally, cellular immunity plays an important role in the antitumor activity and is augmented by Th1 cytokines (e.g. IFN-γ). And IL-12 augment the production of Th1 cytokines, and restrict the production of Th2 cytokines. IL-12 and IFN-γ are key cytokines in the acquisition of non-specific and antigen-specific cellular immunity. Thus, in the present study, we examined the effect of LcS on the production of IL-12 and IFN-γ.