Evidence that elastase is the TNF-R75 shedding enzyme in resting human polymorphonuclear leukocytes.
We previously showed that a metalloprotease and a serine protease mediate shedding of the TNF-R75 (75-kDa tumor necrosis factor receptor) in neutrophils. Here we show that elastase is the TNF-R75 solubilizing serine protease. Release of the TNF-R75 by resting cells was almost totally inhibited by the serine protease inhibitor diisopropylfluorophosphate (DFP), by two synthetic, chemically unrelated, elastase-specific inhibitors and by alpha1-protease inhibitor. Release after TNF or FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) stimulation was blocked by DFP and a metalloprotease inhibitor used in combination. Supernatants from resting neutrophils contained a 28-kDa fragment of the receptor, compatible with that generated by elastase, whose appearance was inhibited by DFP. Upon FMLP stimulation, the release of 28-kDa and 40-kDa fragments was observed, which was inhibited by DFP and a metalloprotease inhibitor, respectively. We conclude that elastase is the TNF-R75 sheddase of resting neutrophils and that it contributes to shedding of this receptor in stimulated cells.
Department of Physiology and Pathology, University of Trieste, Via A. Fleming 22, Trieste, Italy.
Chiara
C
Gasparini
Renzo
R
Menegazzi
Pierluigi
P
Patriarca
Pietro
P
Dri
D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000378:metabolism*; D015153:Blotting, Western; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007531:Isoflurophate / Q000494:pharmacology; D045726:Metalloproteases / Q000037:antagonists & inhibitors; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology* / Q000502:physiology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology; D018124:Receptors, Tumor Necrosis Factor / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047889:Receptors, Tumor Necrosis Factor, Type II; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D015703:Antigens, CD; D006877:Hydroxamic Acids; C423012:KB R8301; D010446:Peptide Fragments; D011480:Protease Inhibitors; D018124:Receptors, Tumor Necrosis Factor; D047889:Receptors, Tumor Necrosis Factor, Type II; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D007531:Isoflurophate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D045726:Metalloproteases; D010196:Pancreatic Elastase