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Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes
Published on: December 11, 2015
Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma
C Yang1, J L Jones, S R Barnum
1Department of Microbiology, University of Alabama at Birmingham.
Insights
This study shows that astroglioma cells express complement regulatory proteins DAF, MCP, and CD59. These proteins are also found on rat astrocytes, suggesting a role in central nervous system immune defense.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- The complement system is a crucial part of innate immunity.
- Complement regulatory proteins (CRPs) prevent self-tissue damage.
- Astroglioma cells and astrocytes are key components of the central nervous system (CNS).
Purpose of the Study:
- To investigate the expression of complement regulatory proteins (CRPs) on human D54-MG astroglioma cells.
- To determine if primary rat astrocytes also express these CRPs.
- To understand the role of CRPs in the CNS immune response.
Main Methods:
- Immunofluorescence
- Flow cytometry
- Western blotting
- Northern blot analysis
- Phosphatidylinositol-specific phospholipase C (PIPLC) treatment
Main Results:
- Human D54-MG astroglioma cells express decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), and CD59.
- These proteins are structurally and antigenically similar to those on other cell lines.
- DAF is anchored to the D54-MG cell surface via a glycophospholipid anchor.
- Primary rat astrocytes constitutively express DAF, MCP, and CD59.
- This is the first report demonstrating CD59 expression on astrocytes.
Conclusions:
- Astrocytes express all three major complement regulatory proteins.
- The presence of these proteins on astrocytes suggests their importance in regulating complement activation within the CNS.
- CRPs on astrocytes may play a role in neural host defense mechanisms.
Abstract:
In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.
