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Soluble factor(s) released from neutrophils activates endothelial cell matrix metalloproteinase-2
J D Schwartz1, S Monea, S G Marcus
1Department of Surgery, S. Arthur Localio Surgical Research Laboratory, New York University School of Medicine, New York 10016, USA.
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.
Conclusion:
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.
Insights
Polymorphonuclear leukocytes (PMNs) release a factor that activates endothelial cell matrix metalloproteinase-2 (MMP-2). This novel activation mechanism is independent of cell contact and offers insights into PMN-mediated microvascular injury.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Polymorphonuclear leukocytes (PMNs) and microvascular injury are key in multiple organ failure.
- Matrix metalloproteinases (MMPs), like MMP-2, are crucial for tissue remodeling by degrading extracellular matrix components.
- The activation mechanism of proMMP-2, the inactive form of MMP-2, is not fully understood.
Purpose of the Study:
- To investigate the effect of PMNs on the activation of proMMP-2 produced by endothelial cells.
- To elucidate the role of PMNs in the proteolytic processes underlying tissue remodeling and microvascular injury.
Main Methods:
- Human umbilical vein endothelial cells (HUVECs) and PMNs were cultured separately and together.
- Cocultures were established with a semipermeable membrane to assess cell-cell contact dependence.
- PMN-conditioned medium was applied to HUVEC cultures with or without various proteinase inhibitors.
- Gelatin zymography was used to analyze gelatinase activity in culture supernatants.
Main Results:
- HUVECs produce inactive 72 kDa MMP-2, while PMNs produce MMP-9 but not MMP-2.
- Coculture with PMNs or addition of PMN-conditioned medium induced active 62 kDa MMP-2 production by HUVECs.
- PMN-conditioned medium activated proMMP-2 independently of cell-cell contact.
Conclusions:
- PMNs secrete a soluble factor that activates endothelial MMP-2 via a novel mechanism.
- This activation pathway is independent of cell-cell contact and not mediated by plasmin, cysteine-, acid-, or metalloproteinases.
- Findings provide insights into PMN-driven microvascular injury and associated tissue remodeling.