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.
Gasparini
Chiara
C
Department of Physiology and Pathology, University of Trieste, Via A. Fleming 22, Trieste, Italy.
Menegazzi
Renzo
R
Patriarca
Pierluigi
P
Dri
Pietro
P
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