Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase

• Published in: Journal of leukocyte biology Vol. 50; no. 4; pp. 331 - 340
• Main Authors: Daudi, Imbesat, Gudewicz, Paul W., Saba, Thomas M., Cho, Eshin, Vincent, Peter
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Society for Leukocyte Biology 01.10.1991; Federation of American Societies for Experimental Biology
• Subjects: Amino Acid Chloromethyl Ketones - pharmacology; Animals; Autoradiography; Biological and medical sciences; Blotting, Western; Collagen - metabolism; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibronectins - metabolism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Gelatin - metabolism; Immunobiology; Immunological reactions in vitro; Iodine Radioisotopes; Leukocyte Elastase; Leukocytes - drug effects; Leukocytes - enzymology; Leukocytes - physiology; lung vascular injury; Male; Methods; Microbial Collagenase - metabolism; Neutrophils - drug effects; Neutrophils - enzymology; Neutrophils - physiology; Pancreatic Elastase - antagonists & inhibitors; Pancreatic Elastase - pharmacology; Pancreatic Elastase - physiology; Peptide Hydrolases - pharmacology; Phenylmethylsulfonyl Fluoride - pharmacology; polymorphonuclear leukocytes; protease inhibitor; Protein Binding; Rats; Zymosan - metabolism; Rat; Enzyme; Induction; Rodentia; Stimulation; Fibronectin; Vertebrata; Mammalia; Proteolysis; Collagen; Extracellular matrix; Elastase; Lesion; Neutrophil
• ISSN: 0741-5400; 1938-3673
• DOI: 10.1002/jlb.50.4.331

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 125l‐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 (<45 kD), derived from the artifical 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.