Soluble factor(s) released from neutrophils activates endothelial cell matrix metalloproteinase-2.
OBJECTIVE
Polymorphonuclear leukocyte (PMN) infiltration and microvascular injury are hallmarks of the tissue remodeling associated with multiple organ failure. These processes require the concerted action of various proteolytic enzymes, including serine and matrix metalloproteinases (MMPs). Matrix metalloproteinase-2 (MMP-2) plays an important role in the turnover of various ECM components, including type IV collagen, fibronectin, and gelatins. Like all MMPs, MMP-2 is secreted as an inactive zymogen (proMMP-2) and activated extracellularly by limited proteolytic cleavage. The physiologic mechanism(s) of proMMP-2 activation remains unclear. This study was designed to characterize the effect of PMNs on the activation of proMMP-2 produced by endothelial cells.


METHODS
PMNs and human umbilical vein endothelial cells (HUVECs) were grown either separately or together for 2-16 h. To evaluate the role of cell-cell contact, cocultures were also established in which the two cell types were separated by a semipermeable polycarbonate membrane. Alternatively, PMN-conditioned medium was added to HUVEC cultures with or without various proteinase inhibitors (aprotinin, 1,10-phenanthroline, Batimastat, E-64, eglin c peptide, or pepstatin A). After incubation, the culture supernatants were analyzed by gelatin zymography to characterize the gelatinases.


RESULTS
HUVECs produce MMP-2 in its inactive (72 kDa) form. PMNs produce high levels of MMP-9 (gelatinase B, 92 kDa) but no MMP-2. Coculture of PMNs with or addition of PMN-conditioned medium to HUVECs results in the production of active (62 kDa) MMP-2. ProMMP-2 activation by PMN-conditioned medium is not blocked by inhibitors of plasmin, cysteine-, acid-, or metalloproteinases.


CONCLUSIONS
PMNs release a soluble factor that activates endothelial cell MMP-2 through a novel mechanism independent of cell-cell contact and not attributable to the activities of plasmin, cysteine-, acid-, or metalloproteinases. These findings may provide insight into the tissue remodeling that accompanies PMN-mediated microvascular injury.
Department of Surgery, S. Arthur Localio Surgical Research Laboratory, New York University School of Medicine, New York 10016, USA.
J D
JD
Schwartz
S
S
Monea
S G
SG
Marcus
S
S
Patel
K
K
Eng
A C
AC
Galloway
P
P
Mignatti
P
P
Shamamian
D007611:Aprotinin / Q000494:pharmacology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D004789:Enzyme Activation / Q000276:immunology; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D018375:Neutrophil Activation / Q000502:physiology*; D009504:Neutrophils / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015843:Serpins / Q000494:pharmacology; D012995:Solubility; D013876:Thiophenes / Q000494:pharmacology; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology
D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D010436:Pepstatins; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D013876:Thiophenes; C028656:eglin proteinase inhibitors; C014195:Streptomyces pepsin inhibitor; D010649:Phenylalanine; D007611:Aprotinin; C080985:batimastat; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D007930:Leucine; C024974:E 64; C031375:pepstatin; C025205:1,10-phenanthroline