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Updated: Aug 13, 2026
Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes
Published on: May 31, 2018
Regulation of tumor necrosis factor gene expression and protein synthesis in murine macrophages treated with
1Institute of Parasitology, McGill University, Macdonald College, Ste-Anne de Bellevue, Québec, Canada.
Abstract:
The effect of murine rTNF-alpha on c-fos and TNF mRNA accumulation and protein synthesis was investigated in bone marrow-derived macrophages to examine the mechanism(s) by which TNF modulates macrophage activity. A rapid and transient expression of the c-fos gene was induced by murine rTNF. This was blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, suggesting that the murine rTNF stimulated a protein kinase C-dependent signal transduction pathway. Although LPS induced the accumulation of one TNF mRNA species, murine rTNF induced the synthesis of two distinct TNF mRNA species. Both LPS- and murine rTNF-induced TNF mRNA accumulation was equally enhanced by pretreatment with mouse rIFN-gamma. In contrast, cycloheximide pretreatment had little effect on murine rTNF-induced TNF mRNA accumulation, whereas this treatment increased LPS-induced TNF mRNA by sevenfold. These results argue that TNF mRNA accumulation can be modulated in macrophages by distinct mechanisms. As assessed by Western blot and immunoprecipitation analysis, LPS stimulated the synthesis of both cell-associated and secreted forms of TNF protein. In comparison, newly synthesized TNF protein was not detected when macrophages were treated with murine rTNF alone or in combination with murine rIFN-gamma. This demonstrates that although murine rTNF stimulated the synthesis of two distinct TNF mRNA species, additional signal(s) are necessary for their translation into protein and that such signals are present after LPS stimulation.
Insights
Murine tumor necrosis factor-alpha (TNF) triggers c-fos gene expression via protein kinase C. While TNF induces specific TNF mRNA, it doesn't directly lead to protein synthesis, unlike lipopolysaccharide (LPS).
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Tumor necrosis factor (TNF) plays a crucial role in modulating macrophage activity.
- Understanding the precise mechanisms of TNF-mediated signaling is essential for controlling inflammatory responses.
Purpose of the Study:
- To investigate how murine recombinant TNF-alpha (rTNF) affects c-fos and TNF mRNA accumulation and protein synthesis in macrophages.
- To elucidate the signaling pathways involved in TNF's modulation of macrophage function.
Main Methods:
- Bone marrow-derived macrophages were treated with murine rTNF-alpha.
- Gene expression (c-fos, TNF mRNA) and protein synthesis were analyzed.
- Inhibitors (protein kinase C inhibitor, cycloheximide) and other agents (LPS, rIFN-gamma) were used to dissect signaling pathways.
Main Results:
- Murine rTNF rapidly induced c-fos gene expression, dependent on protein kinase C.
- Murine rTNF stimulated the synthesis of two distinct TNF mRNA species, distinct from LPS-induced mRNA.
- While rIFN-gamma enhanced TNF mRNA accumulation for both LPS and rTNF, cycloheximide differentially affected it.
- LPS induced both cell-associated and secreted TNF protein, whereas rTNF alone or with rIFN-gamma did not result in detectable TNF protein synthesis.
Conclusions:
- TNF-alpha utilizes a protein kinase C-dependent pathway to regulate immediate-early gene responses like c-fos.
- Macrophage activation by TNF-alpha involves distinct mechanisms for TNF mRNA accumulation compared to LPS.
- The translation of TNF mRNA into functional protein requires additional signals present during LPS stimulation, not solely provided by rTNF or rIFN-gamma.
