Leukocyte elastase-independent proteolysis of gelatin-bound fibronectin by inflammatory macrophages.
Fragmentation of lung matrix fibronectin by proteases released from activated phagocytic cells has been implicated in lung vascular injury. We examined whether denatured collagen (gelatin)-bound fibronectin can be degraded by peritoneal exudate mononuclear phagocytes harvested from rats 96 h after intraperitoneal casein injection. Microtiter plates were pretreated with gelatin and then supplemented with purified 125I rat plasma fibronectin, which readily bound to the gelatin. Stimulated inflammatory exudate cells were added and proteolysis of the bound fibronectin was studied by the release of [125I]fibronectin fragments into the media. Following 2 h of incubation, peritoneal exudate mononuclear macrophages stimulated with opsonized zymosan released three times more radiolabeled fibronectin into the medium as compared to background controls, and 1.5 times more radiolabeled fibronectin as compared to cells not stimulated with zymosan. Western blot analysis and autoradiography confirmed the presence of fragments of fibronectin in the culture medium. Some of these fragments were clearly derived from the radiolabeled matrix, but others that were not labeled were potentially released directly from the added stimulated macrophages. The release of radiolabeled fibronectin was inhibited by N-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin specific inhibitor, but not by methoxysuccinyl-alanine-alanine-proline-valine-chloromethyl-ketone (AAPVCK), a leukocyte elastase-specific inhibitor. These results suggest that fibronectin bound to denatured collagen is susceptible to leukocyte elastase-independent enzymatic degradation by stimulated inflammatory exudate mononuclear phagocytic cells. Such proteolysis may mimic a pathological process associated with lung vascular injury during the sequestration of activated macrophages in the lung microcirculation and interstitium.
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
Frewin
M B
MB
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D007962:Leukocytes / Q000201:enzymology*; D008264:Macrophages / Q000201:enzymology*; D008297:Male; D010196:Pancreatic Elastase / Q000097:blood*; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000097:blood*; D011485:Protein Binding; D051381:Rats
D005353:Fibronectins; D010446:Peptide Fragments; D005780:Gelatin; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase