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

J Immunol Methods. 2008 Jun 1;335(1-2):71-8. doi: 10.1016/j.jim.2008.02.019. Epub 2008 Mar 26.

Abstract

Drugs that target tumor necrosis factor-alpha (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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipocytes / metabolism*
  • Animals
  • Anti-Inflammatory Agents / pharmacology
  • Apoptosis* / drug effects
  • Biological Assay*
  • Cell Membrane / metabolism
  • Cell Survival
  • Cells, Cultured
  • Drug Evaluation, Preclinical / methods*
  • Fas-Associated Death Domain Protein / metabolism
  • Genetic Vectors
  • Humans
  • Lentivirus / genetics
  • Mice
  • 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
  • Transfection
  • Tumor Necrosis Factor-alpha / metabolism*
  • fas Receptor / genetics
  • fas Receptor / metabolism*

Substances

  • Anti-Inflammatory Agents
  • Fadd protein, mouse
  • Fas protein, mouse
  • Fas-Associated Death Domain Protein
  • Receptors, Tumor Necrosis Factor, Type I
  • Receptors, Tumor Necrosis Factor, Type II
  • Recombinant Fusion Proteins
  • TNFRSF1B protein, human
  • Tnfrsf1a protein, mouse
  • Tumor Necrosis Factor-alpha
  • fas Receptor