RAW264 macrophages stably transfected with an HIV-1 LTR reporter gene provide a sensitive bioassay for analysis of signalling pathways in macrophages stimulated with lipopolysaccharide, TNF-alpha or taxol

J Inflamm. 1995;45(2):126-35.


Bacterial lipopolysaccharide (LPS) modulates expression of a variety of genes in macrophages, and additionally activates viral promoters including the HIV-1 LTR. The HIV-1 LTR driving the luciferase reporter gene was stably transfected into the murine macrophage cell line, RAW264. In stably transfected cells, luciferase activity was LPS-dependent. As little as 0.01 ng/ml LPS was sufficient to increase luciferase activity over basal levels with maximal stimulation resulting in a 10- to 20-fold response. The cells also responded to human and murine tumour necrosis factor (TNF alpha). Endogenous TNF alpha was not involved in LPS responses, since pretreatment with alpha-TNF alpha antibody did not affect activation. Induction of HIV-1 LTR activity by LPS occurred independently of phorbol myristate acetate (PMA) sensitive protein kinase C (PKC), since depletion of PKC by prolonged exposure to PMA blocked TNF alpha and PMA responses but was not able to abolish LPS action on these cells. Taxol (5-20 micrograms/ml), a chemotherapeutic agent which mimics LPS action on macrophages, was also able to increase expression of the reporter gene driven by the HIV-1 LTR. However, lower doses of taxol that were not sufficient to trans-activate the LTR or to induce TNF alpha expression were cytotoxic to RAW264 cells suggesting that the cytotoxic and LPS-like activities of taxol were not linked. This cell line provides a convenient method for detecting LPS-like activity and is a useful tool for examining LPS and TNF alpha signalling pathways.

MeSH terms

  • Animals
  • Cell Line
  • Gene Expression
  • Genes, Reporter
  • HIV Long Terminal Repeat / genetics*
  • HIV-1 / genetics*
  • Humans
  • Lipopolysaccharides / pharmacology*
  • Luciferases / genetics
  • Luciferases / metabolism
  • Macrophages / metabolism*
  • Mice
  • Paclitaxel / pharmacology*
  • Protein Kinase C / metabolism
  • Signal Transduction
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transfection
  • Tumor Necrosis Factor-alpha / pharmacology*


  • Lipopolysaccharides
  • Tumor Necrosis Factor-alpha
  • Luciferases
  • Protein Kinase C
  • Tetradecanoylphorbol Acetate
  • Paclitaxel