Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils.
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.
Eijkman-Winkler Laboratory of Medical Microbiology, University of Utrecht, School of Medicine, The Netherlands.
K P
KP
van Kessel
J A
JA
van Strijp
J
J
Verhoef
D002374:Catalase / Q000378:metabolism; D015550:Cell Degranulation; D003571:Cytochalasin B / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D005609:Free Radicals; D006801:Humans; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology* / Q000502:physiology; D010100:Oxygen / Q000737:chemistry; D011994:Recombinant Proteins; D016897:Respiratory Burst; D013482:Superoxide Dismutase / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005609:Free Radicals; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002374:Catalase; D013482:Superoxide Dismutase; D010450:Endopeptidases; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen