Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G.
To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation.
Heck
L W
LW
Department of Medicine, Veterans Administration Medical Center, Birmingham, Alabama.
Blackburn
W D
WD
Irwin
M H
MH
Abrahamson
D R
DR
D056649:Cathepsin G; D002403:Cathepsins / Q000032:analysis / Q000494:pharmacology*; D002462:Cell Membrane / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007797:Laminin / Q000032:analysis / Q000378:metabolism*; D009504:Neutrophils / Q000032:analysis / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000494:pharmacology*; D012697:Serine Endopeptidases
D007797:Laminin; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534096:Ctsg protein, mouse; D010196:Pancreatic Elastase