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We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8(+) T or CD4(+) T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-gamma-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-gamma plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18.

Interleukin-18 (IL-18) enhances interferon gamma (IFNgamma) production and natural killer (NK) cell activity, and elicits protective antitumor effects in vivo. IL-18 and IL-12 synergistically augment IFNgamma production reportedly because IL-12 enhances IL-18 receptor (IL-18R) expression. We now show that IL-18 also synergizes with IL-10 to augment murine splenic NK activity against Yac-1 cells in a standard 4-h chromium-release assay, but IFNgamma production is only slightly enhanced. This pattern of NK activity was also observed with severe combined immunodeficient (SCID) mouse spleen cells indicating that the cytokines were not acting on T or B cells. The cytokines had no priming activity on the spleen cells and, when cells were left unstimulated for 24 h in culture, little NK activity was induced when IL-18 was added for the next 24 h. The reverse transcriptase/polymerase chain reaction revealed that IL-18 receptor (IL-18R) mRNA was expressed early during in vitro spleen cell culture but none was expressed after culture for 24 h regardless of the stimulus. Binding of 125I-labeled IL-18 revealed that exposure to IL-10 only slightly increased IL-18R expression. Expression of perforin mRNA was constitutive and was unaffected by the cytokines; however, Fas ligand (FasL) mRNA expression was strong in cultures with IL-18 alone or combined with IL-10. When Fas-expressing cells and their parental cells were used as targets, weak Fas-mediated cytolytic activity was observed after exposure to IL-18, and this was further enhanced by combination with IL-10. Finally, the augmentation of NK activity was abrogated by the inhibitor concanamycin A, indicating that the enhanced NK activity is perforin-dependent.

Interferon-gamma-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 microg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon gamma. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4+ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4+ cells.