Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.
Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.
Daudi
I
I
Department of Physiology and Cell Biology, Albany Medical College, New York 12208.
Gudewicz
P W
PW
Saba
T M
TM
Cho
E
E
Vincent
P
P
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D003094:Collagen / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism; D007457:Iodine Radioisotopes; D019272:Leukocyte Elastase; D007962:Leukocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008297:Male; D003012:Microbial Collagenase / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D015054:Zymosan / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D005353:Fibronectins; D007457:Iodine Radioisotopes; D010664:Phenylmethylsulfonyl Fluoride; C026704:methoxysuccinyl-alanyl-alanyl-prolyl-valine chloromethyl ketone; D005780:Gelatin; D003094:Collagen; D015054:Zymosan; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D003012:Microbial Collagenase