A flow cytometric and immunofluorescence microscopic study of tumor necrosis factor production and localization in
Insights
Lipopolysaccharide (LPS) stimulates human monocytes to rapidly secrete tumor necrosis factor (TNF) via the secretory pathway. LPS also induces TNF to appear as a trans-membrane protein on the monocyte surface.
Area of Science:
- Immunology
- Cell Biology
Background:
- Tumor necrosis factor (TNF) is a key cytokine involved in inflammation and immunity.
- Understanding the production and localization of TNF in monocytes is crucial for comprehending immune responses.
Purpose of the Study:
- To investigate the production and cellular localization of TNF in human monocytes following lipopolysaccharide (LPS) stimulation.
- To determine the mechanism and timeline of TNF secretion and cell surface expression.
Main Methods:
- Utilized monoclonal and polyclonal antibodies against recombinant human TNF.
- Employed flow cytometry and immunofluorescence microscopy to detect TNF.
- Stimulated primary human monocytes with LPS.
Main Results:
- LPS induced rapid, transient accumulation of TNF in perinuclear vesicles within 20 minutes.
- TNF peaked in vesicles and was released into the medium by 40 minutes, indicating secretion via the secretory pathway.
- Plasma membrane-associated TNF was detected in a small monocyte population after 90 minutes of LPS exposure, increasing to approximately 50% by 24 hours.
- Cell surface TNF was not due to re-binding of released TNF to its receptor.
Conclusions:
- TNF secretion in monocytes follows the typical pathway for secretory proteins.
- LPS stimulation leads to the expression of TNF as a trans-membrane protein on the surface of monocytes.
Abstract:
The production and localization of tumor necrosis factor (TNF) in human monocytes were investigated by using monoclonal and polyclonal antibodies against recombinant human TNF together with flow cytometry and immunofluorescence microscopy. Lipopolysaccharide (LPS) induced a rapid and transient accumulation of TNF in perinuclear vesicles which was detected 20 min after the addition of LPS. The fluorescence intensity of the vesicles peaked at 40 min of LPS exposure, concomitantly with the release of TNF into the medium. Thus, our results indicate that the secretion of TNF is typical for secretory proteins as it involves passage through the secretory apparatus. Additional studies demonstrated that plasma membrane-associated TNF could not be detected in live monocytes not exposed to LPS. However, after 90 min with LPS, a small population of monocytes expressed membrane-associated TNF, and by 24 hr approximately 50% of the monocytes displayed TNF on the plasma membrane. Furthermore, our results indicate that plasma membrane-associated TNF does not represent released TNF bound back to its own receptor. Thus, our findings support the view that TNF exists as a surface trans-membrane protein in LPS-stimulated monocytes.
