Related Experiment Videos
Tumor necrosis factor as effector molecule in monocyte mediated cytotoxicity
Cancer Research
|November 1, 1986
Summary
Human monocytes kill target cells via a lipopolysaccharide (LPS)-triggered molecule identified as tumor necrosis factor (TNF). This new assay system allows for precise measurement of monocyte cytotoxicity and analysis of TNF biology in humans.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Human monocyte cytotoxicity against target cells is a key immune response.
- Previous studies suggested a role for monocyte-derived factors in cytotoxicity.
- The precise mechanism and mediators of this monocyte activity required further elucidation.
Purpose of the Study:
- To develop and validate a novel assay for measuring human monocyte cytotoxicity.
- To identify the specific molecule responsible for LPS-induced monocyte cytotoxicity.
- To characterize the properties of this cytotoxic molecule and its relationship to known cytokines.
Main Methods:
- Development of a 7-hour 51Cr release assay using actinomycin D (Act D) pretreated Wehi 164 target cells.
- Confirmation of monocyte specificity using monoclonal antibody M42 and rosetting.
- Triggering cytotoxicity with lipopolysaccharide (LPS) and analyzing released factors.
- Characterization of the cytotoxic factor using recombinant tumor necrosis factor (TNF), interleukin-1, lymphotoxin, antibody neutralization, size fractionation (HPLC), and immunoblotting.
Main Results:
- A sensitive assay was established, demonstrating that cytotoxicity is restricted to human monocytes.
- Lipopolysaccharide (LPS) at low concentrations (0.1 ng/ml) triggered monocyte cytotoxicity.
- A cytotoxic factor in monocyte supernatants, triggered by LPS, was identified as tumor necrosis factor (TNF).
- Recombinant TNF showed potent lytic activity against Act D-treated target cells, similar to the endogenous cytotoxic factor.
- Antibody neutralization and molecular weight analysis confirmed the identity of the cytotoxic factor as TNF (Mr 15,500-16,500).
Conclusions:
- Human monocyte cytotoxicity against actinomycin D-treated Wehi 164 cells is mediated entirely by lipopolysaccharide (LPS)-triggered tumor necrosis factor (TNF).
- The developed assay system is a powerful tool for analyzing TNF biology and monocyte-mediated cytotoxicity in humans.
- This study provides definitive evidence for TNF as the primary mediator of LPS-induced monocyte cytotoxicity in this model.