Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties.
We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain-specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three-armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophosphate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes.
Steadman
R
R
Department of Cell Biology, University of Alabama, Birmingham 35294-0019.
Irwin
M H
MH
St John
P L
PL
Blackburn
W D
WD
Heck
L W
LW
Abrahamson
D R
DR
D000328:Adult; D001672:Biocompatible Materials; D056649:Cathepsin G; D002403:Cathepsins / Q000097:blood*; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D003094:Collagen; D004338:Drug Combinations; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007457:Iodine Radioisotopes; D007531:Isoflurophate / Q000494:pharmacology; D007797:Laminin / Q000097:blood* / Q000494:pharmacology* / Q000648:ultrastructure; D019272:Leukocyte Elastase; D008854:Microscopy, Electron; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology; D011509:Proteoglycans; D012697:Serine Endopeptidases
D001672:Biocompatible Materials; D004338:Drug Combinations; D007457:Iodine Radioisotopes; D007797:Laminin; D010446:Peptide Fragments; D011509:Proteoglycans; C059485:matrigel; D007531:Isoflurophate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003094:Collagen; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase