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Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human
K P van Kessel1, J A van Strijp, J Verhoef
1Eijkman-Winkler Laboratory of Medical Microbiology, University of Utrecht, School of Medicine, The Netherlands.
Abstract:
Activated human neutrophils (PMN) degrade rTNF-alpha resulting in a loss of cytotoxic activity against murine L-929 cells (L cells). This inactivation is mediated through proteases released from activated PMN. Exposure of TNF to H2O2, glucose oxidase, xanthine oxidase, or myeloper-oxidase-H2O2-halide did not affect TNF cytotoxicity for L cells. Exposure to trypsin, chymotrypsin, pronase E, or elastase, however, did diminish TNF bioactivity. FMLP-stimulated PMN in the presence, but not in the absence, of cytochalasin B reduced TNF activity, whereas PMA-stimulated PMN did not affect TNF. Stimulation of PMN with opsonized bacteria also induced TNF inactivation as well as the supernatant of FMLP-stimulated cells. Addition of protease inhibitors to the FMLP-stimulated cytochalasin B-treated PMN abrogated the inactivation of TNF cytotoxicity for L cells, whereas scavengers were not protective. In addition, PMN from a chronic granulomatous disease patient also decreased TNF bioactivity. Inactivation of TNF by activated PMN correlated with granule release and not with superoxide production. Exposure of TNF to proteases and FMLP-activated PMN also resulted in a loss of reactivity with anti-TNF antibodies, as measured by ELISA, and in the formation of an approximately 10-kDa split product from the 17-kDa rTNF molecule. Partial degradation of TNF by proteases released from activated PMN may result in a diminished TNF bioactivity and thereby contribute to the regulation of local inflammatory reactions.
Insights
Activated neutrophils degrade tumor necrosis factor-alpha (TNF) via proteases, reducing its cytotoxic activity. This protease-mediated inactivation, linked to granule release, impacts inflammatory responses.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Neutrophils are key immune cells involved in inflammation.
- Tumor necrosis factor-alpha (TNF) is a critical cytokine in inflammatory and immune responses.
- The regulation of TNF activity is essential for controlling inflammation.
Purpose of the Study:
- To investigate the mechanism by which activated neutrophils inactivate tumor necrosis factor-alpha (TNF).
- To determine if proteases released from neutrophils are responsible for TNF inactivation.
- To explore the role of neutrophil activation pathways and granule release in TNF degradation.
Main Methods:
- Exposure of recombinant TNF-alpha (rTNF) to activated human neutrophils (PMN) and purified proteases.
- Assay of TNF cytotoxicity against L-929 cells.
- ELISA to measure TNF antibody binding.
- Analysis of TNF degradation products using SDS-PAGE.
- Stimulation of PMN with FMLP, PMA, and opsonized bacteria.
Main Results:
- Activated PMN, particularly FMLP-stimulated cells, degraded rTNF-alpha, leading to loss of cytotoxic activity.
- Proteases like trypsin, chymotrypsin, pronase E, and elastase diminished TNF bioactivity.
- TNF inactivation correlated with neutrophil granule release, not superoxide production.
- Degradation resulted in loss of antibody binding and formation of a ~10-kDa TNF fragment.
- Protease inhibitors blocked TNF inactivation, while scavengers did not.
Conclusions:
- Activated human neutrophils inactivate TNF-alpha through the release of proteases.
- This degradation process involves granule release and leads to reduced TNF bioactivity.
- Neutrophil-mediated TNF inactivation may play a role in regulating local inflammatory reactions.