Related Experiment Videos
Regulation of tumor necrosis factor expression in a macrophage-like cell line by lipopolysaccharide and cyclic AMP
S M Taffet1, K J Singhel, J F Overholtzer
1Department of Microbiology and Immunology, SUNY Health Science Center, Syracuse 13210.
Abstract:
Bacterial lipopolysaccharide (LPS, 1 microgram/ml) induced the rapid production of tumor necrosis factor (TNF-alpha) mRNA in the RAW264 macrophage-like cell line. TNF-alpha mRNA peaked within 45 min of LPS treatment and remained high for greater than 3 hr. Transcription of TNF-alpha mRNA was increased within 15 min of LPS treatment. The quantity of TNF-alpha mRNA in LPS-stimulated cells was reduced to basal levels by treatment with cAMP, cAMP analogs, or agents which raise intracellular cAMP. This was not a general effect on all mRNA levels as the expression of a second gene, ornithine decarboxylase, was enhanced by cAMP treatment. cAMP did not have an effect on the stability of TNF-alpha mRNA. This is in contrast to the protein synthesis inhibitor, cycloheximide, which leads to a stabilization of TNF-alpha mRNA. Our results suggest that the primary regulation of tumor necrosis factor by cAMP and LPS occurs at the transcriptional level.
Insights
Bacterial lipopolysaccharide rapidly increases tumor necrosis factor-alpha (TNF-alpha) mRNA. Cyclic AMP (cAMP) reduces TNF-alpha mRNA levels by affecting transcription, not mRNA stability, in macrophages.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Bacterial lipopolysaccharide (LPS) is a potent activator of immune responses.
- Tumor necrosis factor-alpha (TNF-alpha) is a key pro-inflammatory cytokine.
- Understanding the regulation of TNF-alpha production is crucial for immune modulation.
Purpose of the Study:
- To investigate the molecular mechanisms regulating TNF-alpha mRNA production in response to LPS.
- To determine the role of cyclic AMP (cAMP) in modulating TNF-alpha mRNA levels.
- To elucidate whether cAMP affects TNF-alpha mRNA transcription or stability.
Main Methods:
- RAW264 macrophage-like cell line stimulation with LPS.
- Quantification of TNF-alpha mRNA levels using Northern blot or RT-PCR.
- Treatment with cAMP, cAMP analogs, and protein synthesis inhibitors (e.g., cycloheximide).
- Assessment of ornithine decarboxylase mRNA expression as a control.
Main Results:
- LPS induced rapid and sustained TNF-alpha mRNA production, peaking at 45 minutes.
- TNF-alpha mRNA transcription increased significantly within 15 minutes of LPS treatment.
- cAMP treatment reduced TNF-alpha mRNA levels without affecting ornithine decarboxylase mRNA.
- cAMP did not alter TNF-alpha mRNA stability, unlike cycloheximide.
Conclusions:
- LPS-induced TNF-alpha production in macrophages is primarily regulated at the transcriptional level.
- cAMP acts as a negative regulator of TNF-alpha transcription.
- These findings highlight a novel mechanism for controlling inflammatory responses via cAMP-mediated transcriptional control of TNF-alpha.