Terumi Kimoto

Essential Role of Interleukin-12 (IL-12) and IL-18 for Gamma Interferon Production Induced by Listeriolysin O in Mouse Spleen Cells

ABSTRACT The mechanism of gamma interferon (IFN-γ) production induced by listeriolysin O (LLO), a cytolytic virulence factor of Listeria monocytogenes, was analyzed with special reference to the involvement of macrophage-derived cytokines in spleen cells of mice. LLO purified from the culture supernatant of L. monocytogenes was capable of inducing a high level of IFN-γ when its cytolytic activity was blocked by cholesterol treatment. The IFN-γ-inducing ability of LLO was not dependent on possibly contaminating lipopolysaccharide. Depletion of CD11b+ cells resulted in a profound decrease in IFN-γ production in response to LLO stimulation. Negative selection also suggested the contribution of DX5+ cells in IFN-γ production. Reverse transcription-PCR revealed that expression of interleukin-12 (IL-12) p35 and p40 was induced by LLO but that the IL-18 mRNA level in the CD11b+ fraction of spleen cells was unchanged. There was no change in the expression of the IFN-γ-inducing cytokine genes in the CD11b− fraction. Neutralization of IL-12 and IL-18 in culture abolished the IFN-γ production almost completely. Spleen cells from IL-12- or IL-18-deficient mice never produced IFN-γ after stimulation with LLO. These results clearly indicated that LLO, a well-known virulence factor of L. monocytogenes, is capable of inducing IFN-γ from NK cells through induction of IL-12 and IL-18 from macrophages. LLO appeared to play essential roles, not only as a bacterial virulence factor but also as a bacterial modulin in the immune response of the host.

Dissociated Linkage of Cytokine-Inducing Activity and Cytotoxicity to Different Domains of Listeriolysin O from Listeria monocytogenes

ABSTRACT Listeriolysin O (LLO), a cholesterol-binding cytolysin of Listeria monocytogenes, exhibits cytokine-inducing and cytolytic activities. Because the cytolytic activity was abolished by cholesterol treatment but the cytokine-inducing activity was not, these activities appeared to be linked to different domains of the LLO molecule. In this study, we constructed recombinant full-length LLO (rLLO529) and various truncated derivatives and examined their cytolytic, cholesterol-binding, and gamma interferon (IFN-γ)-inducing activities. rLLO529 exhibited both IFN-γ-inducing and cytolytic activities. Four truncated rLLOs possessing different C termini, which did not exert either cytolytic or cholesterol-binding activity, stimulated IFN-γ production in normal spleen cells. However, a truncated rLLO corresponding to domain 4 (rLLO416-529) did not exhibit IFN-γ-inducing activity, whereas it did bind to immobilized cholesterol. In addition, though the hemolysis induced by rLLO529 was inhibited by rLLO416-529, such inhibition was not detected upon rLLO529-induced IFN-γ production. These data indicated that domain 4 was responsible for binding of LLO to membrane cholesterol followed by oligomerization and pore formation by the entire LLO molecule. In contrast, the other part of LLO, corresponding to domain 1-3, was essential for IFN-γ-inducing activity. These findings implied a novel aspect of the function of LLO as a bacterial modulin.

Induction of gamma interferon and nitric oxide by truncated pneumolysin that lacks pore-forming activity.

ABSTRACT Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is known to exert various effects on the host immune cells, including cytokine induction, in addition to its known cytolytic activity as a member of the thiol-activated cytolysins. It is of interest to determine whether cytolytic activity is involved in triggering the cytokine production. In this study, we constructed full-length recombinant PLY and noncytolytic truncated PLYs with C-terminal deletions to examine the response of spleen cells to these PLY preparations. When cytolytic activity was blocked by treatment with cholesterol, full-length PLY was capable of inducing gamma interferon (IFN-γ) production. Truncated PLYs that originally exhibited no cytolytic activity were also active in IFN-γ induction. Therefore, the IFN-γ-inducing ability of PLY appeared to be independent of the cytolytic activity. Furthermore, IFN-γ-inducing preparations were also capable of inducing nitric oxide synthase expression and nitric oxide (NO) production, and the addition of neutralizing antibody to IFN-γ abolished the NO production. These results clearly demonstrated that PLY is capable of inducing IFN-γ production in spleen cells by a mechanism different from pore formation and that the induced IFN-γ stimulates NO production. These findings were discussed with reference to the contribution of PLY to the virulence of S. pneumoniae in vivo.

Differences in gamma interferon production induced by listeriolysin O and ivanolysin O result in different levels of protective immunity in mice infected with Listeria monocytogenes and Listeria ivanovii.

ABSTRACT Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-γ) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD50) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD50 was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-γ in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-γ-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-γ-inducing activity than LLO. By comparing the IFN-γ-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-γ-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-γ production is responsible for the failure of L. ivanovii to generate effective protective immunity.

Seeligeriolysin O, a cholesterol-dependent cytolysin of Listeria seeligeri, induces gamma interferon from spleen cells of mice

ABSTRACT Seeligeriolysin O (LSO), one of the cholesterol-dependent cytolysins produced by Listeria seeligeri, shows 80% homology to listeriolysin O (LLO) produced by Listeria monocytogenes at the amino acid sequence level. In addition to cytolytic activity, LLO has been shown to exhibit cytokine-inducing activity. In order to determine whether LSO is also capable of exhibiting these two different activities, we constructed a recombinant full-length LSO (rLSO530) and a noncytolytic truncated derivative with a C-terminal deletion (rLSO483) and compared these molecules with recombinant LLO. The cytolytic rLSO530 molecule could induce gamma interferon (IFN-γ) production in spleen cells when the cytolytic activity was blocked by treatment with cholesterol. The noncytolytic truncated rLSO483 molecule also induced IFN-γ production. Anti-LLO polyclonal antibody inhibited not only LLO-induced IFN-γ production but also LSO-induced IFN-γ production. Both NK cells and CD11b+ cells were required for LSO-induced IFN-γ production. Among the various cytokines expressed in CD11b+ cells, interleukin-12 (IL-12) and IL-18 appeared to be essential. We concluded that LSO exhibits the same biological activity as LLO.