Related Experiment Videos
Plasminogen-mediated matrix invasion and degradation by macrophages is dependent on surface expression of annexin II
D J Falcone1, W Borth, K M Khan
1Departments of Pathology, Cell Biology, Pediatrics, and Medicine, Joan and Sanford I. Weill Medical College of Cornell University, New York, NY, USA. dfalcone@mail.med.cornell.edu
Abstract:
Genetic evidence demonstrates the importance of plasminogen activation in the migration of macrophages to sites of injury and inflammation, their removal of necrotic debris, and their clearance of fibrin. These studies identified the plasminogen binding protein annexin II on the surface of macrophages and determined its role in their ability to degrade and migrate through extracellular matrices. Calcium-dependent binding of annexin II to RAW264.7 macrophages was shown using flow cytometry and Western blot analysis of EGTA eluates. Ligand blots demonstrated that annexin II comigrates with one of several proteins in lysates and membranes derived from RAW264.7 macrophages that bind plasminogen. Preincubation of RAW264.7 macrophages with monoclonal anti-annexin II IgG inhibited (35%) their binding of 125I-Lys-plasminogen. Likewise, plasmin binding to human monocyte-derived macrophages and THP-1 monocytes was inhibited (50% and 35%, respectively) when cells were preincubated with anti-annexin II IgG. Inhibition of plasminogen binding to annexin II on RAW264.7 macrophages significantly impaired their ability to activate plasminogen and degrade [3H]-glucosamine-labeled extracellular matrices. The migration of THP-1 monocytes through a porous membrane, in response to monocyte chemotactic protein-1, was blocked when the membranes were coated with extracellular matrix. The addition of plasminogen to the monocytes restored their ability to migrate through the matrix-coated membrane. Preincubation of THP-1 monocytes with anti-annexin II IgG inhibited (60%) their plasminogen-dependent chemotaxis through the extracellular matrix. These studies identify annexin II as a plasminogen binding site on macrophages and indicate an important role for annexin II in their invasive and degradative phenotype.
Insights
Annexin II on macrophages binds plasminogen, crucial for cell migration and debris clearance. This protein facilitates macrophage invasion and degradation of extracellular matrices during inflammation.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Plasminogen activation is vital for macrophage functions, including migration, debris removal, and fibrin clearance at injury sites.
- Macrophage surface proteins play a key role in their interaction with extracellular matrices and inflammatory responses.
Purpose of the Study:
- To identify and characterize the plasminogen binding protein on macrophages.
- To elucidate the role of annexin II in macrophage migration, extracellular matrix degradation, and plasminogen activation.
Main Methods:
- Flow cytometry and Western blot analysis to detect calcium-dependent annexin II binding to macrophages.
- Ligand blotting to identify proteins that bind plasminogen.
- Inhibition assays using anti-annexin II IgG to assess the impact on plasminogen and plasmin binding.
- Assays measuring plasminogen activation, extracellular matrix degradation, and monocyte chemotaxis.
Main Results:
- Annexin II was identified as a plasminogen binding protein on the surface of RAW264.7 macrophages.
- Anti-annexin II antibodies inhibited plasminogen and plasmin binding to macrophages.
- Inhibition of annexin II-plasminogen binding impaired macrophage plasminogen activation and extracellular matrix degradation.
- Annexin II blockade reduced plasminogen-dependent monocyte migration through extracellular matrices.
Conclusions:
- Annexin II serves as a critical plasminogen binding site on macrophages.
- Annexin II is essential for the invasive and degradative functions of macrophages.
- Targeting annexin II may modulate macrophage-mediated inflammatory processes.