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J Biol Chem. 1991 Oct 5;266(28):18846-53.

Human neutrophil elastase releases a ligand-binding fragment from the 75-kDa tumor necrosis factor (TNF) receptor. Comparison with the proteolytic activity responsible for shedding of TNF receptors from stimulated neutrophils.

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  • 1Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021.


To localize the protease(s) involved in shedding of tumor necrosis factor receptors (TNF-R) from activated neutrophils (PMN) (Porteu, F., and C. Nathan (1990) J. Exp. Med. 172, 599-607), we tested subcellular fractions from PMN for their ability to cause loss of TNF-R from intact cells. Exposure of PMN to sonicated azurophil granules at 37 degrees C resulted in inhibition of 125I-TNF binding; 50% inhibition ensued when PMN were treated for approximately 1 min with azurophil granules equivalent to 2-3 PMN per indicator cell. The TNF-R-degrading activity in azurophil granules were identified as elastase by its sensitivity to diisopropyl fluorophosphate (DFP), alpha 1-antitrypsin and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MSAAPV-CK), and by the ability of purified elastase to reproduce the effect of azurophil granules. Elastase preferentially acted on the 75-kDa TNF-R, reducing by 85-96% the binding of 125I-TNF to mononuclear cells expressing predominantly this receptor, while having no effect on endothelial cells expressing almost exclusively the 55-kDa TNF-R. Elastase-treated PMN released a 32-kDa soluble fragment of p75 TNF-R that bound TNF and reacted with anti-TNF-R monoclonal antibodies. In contrast, fMet-Leu-Phe-activated PMN shed a 42-kDa fragment from p75 TNF-R, along with similar amounts of a 28-kDa fragment from p55 TNF-R. Shedding of both TNF-Rs by intact activated PMN was more extensive than shedding caused by elastase and was completely resistant to DFP and MSAAPV-CK. Thus, the TNF-R-releasing activity of azurophil granules is distinct from that operative in intact stimulated PMN and could provide an additional mechanism for the control of cellular responses to TNF at sites of inflammation.

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