Simple and highly sensitive assay system for TNFR2-mediated soluble- and transmembrane-TNF activity

• Published in: Journal of immunological methods 2008-06, Vol.335 (1), p.71-78
• Main Authors: Abe, Yasuhiro, Yoshikawa, Tomoaki, Kamada, Haruhiko, Shibata, Hiroko, Nomura, Tetsuya, Minowa, Kyoko, Kayamuro, Hiroyuki, Katayama, Kazufumi, Miyoshi, Hiroyuki, Mukai, Yohei, Yoshioka, Yasuo, Nakagawa, Shinsaku, Tsunoda, Shin-ichi, Tsutsumi, Yasuo
• Format: Article
• Language: English
• Subjects: Adipocytes - metabolism; Animals; Anti-Inflammatory Agents - pharmacology; Apoptosis - drug effects; Bioassay; Biological and medical sciences; Biological Assay; Cell Membrane - metabolism; Cell Survival; Cells, Cultured; Chimeric receptor; Drug Evaluation, Preclinical - methods; Fas; fas Receptor - genetics; fas Receptor - metabolism; Fas-Associated Death Domain Protein - metabolism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Genetic Vectors; Humans; Lentivirus - genetics; Mice; Molecular immunology; Protein Structure, Tertiary; Receptors, Tumor Necrosis Factor, Type I - deficiency; Receptors, Tumor Necrosis Factor, Type I - genetics; Receptors, Tumor Necrosis Factor, Type II - drug effects; Receptors, Tumor Necrosis Factor, Type II - genetics; Receptors, Tumor Necrosis Factor, Type II - metabolism; Recombinant Fusion Proteins - metabolism; Techniques; TNF; TNFR2; Transfection; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2008.02.019

Drugs that target tumor necrosis factor-α (TNF) are particularly important in the treatment of severe inflammatory progression in rheumatoid arthritis, Crohn's disease and psoriasis. Despite the central role of the TNF/TNF receptor (TNFR) in various disease states, there is a paucity of information concerning TNFR2 signaling. In this study, we have developed a simple and highly sensitive cell-death based assay system for analyzing TNFR2-mediated bioactivity that can be used to screen for TNFR2-selective drugs. Using a lentiviral vector, a chimeric receptor was engineered from the extracellular and transmembrane domain of human TNFR2 and the intracellular domain of mouse Fas and the recombinant protein was then expressed in TNFR1 −/−R2 −/− mouse preadipocytes. Our results demonstrate that this chimeric receptor is capable of inducing apoptosis by transmembrane- as well as soluble-TNF stimuli. Moreover, we found that our bioassay based on cell death phenotype had an approximately 80-fold higher sensitivity over existing bioassays. We believe our assay system will be an invaluable research tool for studying TNFR2 and for screening TNFR2-targeted drugs.