Differential expression and secretion of gelatinases and tissue inhibitor of metalloproteinase-1 during neutrophil adhesion.
Transmigrating neutrophils secrete a 92 kDa gelatinase (MMP-9) in order to degrade type IV endothelial basement membrane collagen. A model system for neutrophil adhesion combining a short pre-adhesion time (30 min) in plastic or endothelium-coated wells, medium removal and addition of soluble stimuli (fMLP, TNF alpha), enabled us to induce the release of a basal level of gelatinase activity (> 12% total cell content) from tertiary granules, while the release of vitamin B12 binding protein from specific granules was limited to 4% total cell content. Neutrophil gelatinase activity in unfractionated supernatants from endothelium-coated wells was significantly reduced (P < 0.01) compared to levels obtained on plastic supports, even after TNF alpha treatment or when cell populations were physically separated by trans-well inserts. In contrast, gelatin zymograms of supernatants from plastic and endothelium-coated wells remained similar. These findings suggest that MMP-9 is equally secreted but differentially inhibited by the tissue inhibitor of metalloproteinase-1 originating from the neutrophils. MMP-9 RT-PCR from neutrophils, assessed after up to one hour adhesion on plastic, yielded a single 270 bp fragment which was almost undetectable in the endothelial RT-PCR counterpart, whereas the TIMP-1 PCR product was apparent in both cell types. Furthermore, neutrophil adhesion on endothelial cells and TNF alpha activation for one hour induced the disappearance of MMP-9 cDNA without changes in TIMP-1 and beta-actin PCR products. These results suggest the existence of a dual down-regulation during neutrophil-endothelial interaction, both at the level of secreted MMP-9 activity and of MMP-9 gene transcription.
Zaoui
P
P
Laboratoire d'Enzymologie (EA 2019), CHU 38043, Grenoble, France.
Barro
C
C
Morel
F
F
D002448:Cell Adhesion; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020780:Matrix Metalloproteinase 9; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D019714:Tissue Inhibitor of Metalloproteinases; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D006023:Glycoproteins; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D014409:Tumor Necrosis Factor-alpha; D009240:N-Formylmethionine Leucyl-Phenylalanine; D017364:Collagenases; D020780:Matrix Metalloproteinase 9