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Protease production by cultured microglia: substrate gel analysis and immobilized matrix degradation
C A Colton1, J E Keri, W T Chen
1Department of Physiology and Biophysics, Georgetown University Medical School, Washington, DC 20007.
Abstract:
The production of collagen-degrading proteases by cultured neonatal rat microglia was examined using an immobilized fibronectin-gelatin matrix coupled to a fluorescent marker and by substrate gel analysis. When microglia were plated onto the surface of the matrix and incubated under resting (nonstimulated) conditions, a small but visible amount of immobilized matrix was degraded. Treatment with lipopolysaccharide (LPS) or interleukin-1 (IL-1) significantly increased the number of microglia demonstrating substrate degradation. Substrate-SDS polyacrylamide gel electrophoresis of samples of supernatants from untreated cultured microglia indicated the presence of a 72 and a 92 kD metalloproteinase with characteristics corresponding to collagenases. Supernatants from untreated astrocyte cultures were shown to have primarily a 72 kD metalloproteinase. Proteinase activity increased on stimulation of the microglia with LPS and IL-1 in a dose-dependent fashion. These results indicate that cultured microglia release active proteases capable of degrading the extracellular matrix in a localized region. The production of proteases by activated microglia may have important physiological and pathophysiological consequences within the restricted extracellular matrix of the CNS.
Insights
Cultured microglia release collagen-degrading proteases, particularly after stimulation with lipopolysaccharide (LPS) or interleukin-1 (IL-1). This protease production impacts the central nervous system extracellular matrix.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Microglia are key immune cells in the central nervous system (CNS).
- Extracellular matrix (ECM) degradation is implicated in CNS physiology and pathology.
- The role of microglia-derived proteases in ECM remodeling is not fully understood.
Purpose of the Study:
- To investigate the production of collagen-degrading proteases by cultured neonatal rat microglia.
- To determine the effect of inflammatory stimuli on protease release from microglia.
Main Methods:
- Utilized an immobilized fibronectin-gelatin matrix with a fluorescent marker to assess matrix degradation.
- Employed substrate gel electrophoresis (SDS-PAGE) to identify and characterize protease activity.
- Stimulated microglia cultures with lipopolysaccharide (LPS) and interleukin-1 (IL-1).
Main Results:
- Resting microglia showed minimal matrix degradation.
- LPS and IL-1 significantly increased microglia-mediated substrate degradation.
- Identified 72 and 92 kD metalloproteinases (collagenases) in microglia supernatants, with increased activity upon stimulation in a dose-dependent manner.
Conclusions:
- Cultured microglia release active proteases capable of degrading the ECM.
- Microglial activation by LPS and IL-1 enhances protease production.
- These findings suggest a significant role for microglial proteases in CNS ECM regulation and potential pathophysiological processes.