Related Experiment Videos
Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages
W Shurety1, J K Pagan, J B Prins
1The Institute for Molecular Bioscience, The University of Queensland, Australia.
Abstract:
Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.
Insights
Tumor necrosis factor-alpha (TNF-alpha) trafficking in macrophages involves cell surface accumulation and endocytosis of the uncleaved form. This process regulates the amount of active TNF-alpha, influencing immune responses.
Area of Science:
- Immunology
- Cell Biology
Background:
- Monocytes and macrophages secrete tumor necrosis factor-alpha (TNF-alpha), a key inflammatory cytokine.
- TNF-alpha exists as a 26 kDa transmembrane protein and a 17 kDa soluble form, both biologically active.
Purpose of the Study:
- To investigate the intracellular trafficking of membrane-associated TNF-alpha in macrophages.
- To understand the role of metalloprotease inhibitors in TNF-alpha processing and trafficking.
Main Methods:
- Utilized immunoprecipitation and immunofluorescence to study TNF-alpha expression.
- Employed surface biotinylation and intracellular tracking in cycloheximide-treated cells.
- Assessed the impact of metalloprotease inhibitor BB-3103 and post-Golgi traffic perturbation.
Main Results:
- BB-3103 treatment sustained cell-associated TNF-alpha expression.
- Uncleaved TNF-alpha accumulated on the cell surface and was subsequently endocytosed into vesicles identified as endosomes.
- Inhibition of post-Golgi traffic blocked surface expression of 26 kDa TNF-alpha.
Conclusions:
- Macrophage TNF-alpha trafficking involves transport to the cell surface followed by endocytosis.
- This trafficking pathway regulates the cell surface expression of biologically active TNF-alpha.
- Provides a mechanism for modulating paracrine and autocrine immune signaling.