Yu Mizote1, Kazumasa Wakamatsu2, Shosuke Ito2, Akiko Uenaka3, Yoshihiro Ohue4, Koji Kurose4, Midori Isobe4, Akira Ito5, Yasuaki Tamura6, Hiroyuki Honda7, Toshiharu Yamashita8, Satoshi Nohara9, Mikio Oka4, Kowichi Jimbow8, Eiichi Nakayama10. 1. Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; Department of Respiratory Medicine, Kawasaki Medical School, Kurashiki, Japan. 2. Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Japan. 3. Faculty of Health and Welfare, Kawasaki University of Medical Welfare, Kurashiki, Japan. 4. Department of Respiratory Medicine, Kawasaki Medical School, Kurashiki, Japan. 5. Department of Chemical Engineering, Faculty of Engineering, Kyusyu University, Fukuoka, Japan. 6. Department of Pathology, Sapporo Medical Univeristy School of Medicine, Sapporo, Japan. 7. Department of Biotechnology, School of Engineering, Nagoya University, Nagoya, Japan. 8. Department of Dermatology, Sapporo Medical Univeristy School of Medicine, Sapporo, Japan. 9. Meito Sangyo Co., Nagoya, Japan. 10. Faculty of Health and Welfare, Kawasaki University of Medical Welfare, Kurashiki, Japan. Electronic address: nakayama@mw.kawasaki-m.ac.jp.
Abstract
BACKGROUND: N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma. OBJECTIVE: We investigated the effect of NPCMD on innate immune responses in monocytes. METHODS: CD14⁺ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry. RESULTS: NPCMD stimulated CD14⁺ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1β in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1β. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1β secretion in treatment with NPCMD. Inhibition of IL-1β secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD. CONCLUSION: The immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.
BACKGROUND: N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma. OBJECTIVE: We investigated the effect of NPCMD on innate immune responses in monocytes. METHODS:CD14⁺ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry. RESULTS: NPCMD stimulated CD14⁺ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1β in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1β. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1β secretion in treatment with NPCMD. Inhibition of IL-1β secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD. CONCLUSION: The immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.