JOURNAL OF INTERFERON & CYTOKINE RESEARCH 24:161–167 (2004) © Mary Ann Liebert, Inc. Inhibition by Interleukin-18 of the Growth of Dunn Osteosarcoma Cells TAKUYA OKAMOTO, 1,2 NAOKO YAMADA, 1 TOHRU TSUJIMURA, 1,4 AYAKO SUGIHARA, 1 YASUKO NISHIZAWA, 3 HARUYASU UEDA, 4 SHIN-ICHIRO KASHIWAMURA, 4 HIROKO TSUTSUI, 5 HIROYUKI FUTANI, 2 SOUJI MARUO, 2 HARUKI OKAMURA, 4 and NOBUYUKI TERADA 1,4 ABSTRACT To examine the usefulness of interleukin-18 (IL-18) in the treatment of osteosarcomas, the effect of IL-18 on the growth of Dunn osteosarcoma cells was investigated. Daily intraperitoneal (i.p.) injection of mouse re- combinant IL-18 (2 mg/mouse) suppressed the growth of Dunn osteosarcoma cells transplanted subcutaneously (s.c.) into syngeneic C3H mice. This IL-18-induced suppression was not affected by simultaneous treatment with anti-asialo GM1 serum, which inactivates natural killer (NK) cells. However, IL-18 failed to suppress the growth of Dunn osteosarcoma cells transplanted into BALB/c-nude mice devoid of T lymphocytes or C3H- gld/gld mice deficient in functional Fas ligand (FasL). IL-18 also failed to suppress the growth of Dunn os- teosarcoma cells in vitro, although expression of IL-18 receptor mRNA and MyD88 mRNA as well as Fas mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). On the other hand, anti- mouse Fas antibody showed cytotoxicity against Dunn osteosarcoma cells in a dose-dependent manner in vitro. In addition, treatment of C3H mice with IL-18 enhanced the cytotoxic activity of CD8 1 T lymphocytes against Dunn osteosarcoma cells. These results indicate that IL-18 inhibits the growth of Dunn osteosarcoma cells in vivo by enhancing the cytotoxic activity of CD8 1 T lymphocytes through the FasL-Fas system. 161 INTRODUCTION O STEOLYSIS ASSOCIATED WITH BONE TUMORS, whether pri- mary or metastatic, is induced largely by the action of os- teoclasts whose differentiation and activation are stimulated by factors produced by the tumor. (1–3) Osteolysis causes not only complications, such as bone pain, bone fracture, and hypercal- cemia, (1) but also the release of various growth factors embed- ded in the matrix that stimulate the growth and migration of tu- mor cells and enhancetheir stimulatoryactionson the formation and activity of osteoclasts. (4) Therefore, in the treatment of os- teolytic bone tumors, it is important to inhibit osteolysisas well as tumor growth. Interleukin-18(IL-18), previouslycalled interferon- g (IFN-g)- inducing factor (IGIF), (5–7) has been shown to inhibit osteoly- sis induced by metastatic human breast and lung cancer cells in nude mice, probably through inhibition of the activity of os- teoclasts. (8–10) IL-18 also has been shown to exert antitumor ac- tion on a variety of tumor cells transplanted in mice through activation of natural killer (NK) cells or cytotoxic T lympho- cytes (CTL), (6,7,11–14) suppression of angiogenesis, (15) or in- duction of Fas ligand (FasL) on tumor cells expressing Fas. (16) Osteosarcoma is the most frequent primary malignant bone tumor excluding multiple myeloma, occurring mostly in patients between 10 and 30 years of age. (17,18) Several re- ports (19–23) showed that osteosarcomas produce factors that stimulate osteoclast differentiation and activation, such as re- ceptor activator of nuclear factor- kB (NF-kB) ligand (RANKL) and macrophagecolony-stimulatingfactor (M-CSF), consistent with destruction of the bone and the formation of osteoclast- like multinucleatedgiant cells in osteosarcomas. (17,18) IL-18 has been shown to inhibit osteoclastic bone resorption in vivo, (8,9) but it is not known if it inhibits growth of osteosarcomas. In the present study, we investigated the effect of IL-18 on the growth of osteosarcoma in vivo using murine Dunn osteosar- coma cells transplanted into mice. Departments of 1 Pathology, 2 Orthopedic Surgery, and 5 Immunology & Zoology, and 4 Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo 663-8501, Japan. 3 Department of Pathology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-0025, Japan.