A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia.
Increased matrix metalloproteinase (MMP) proteolytic activity contributes to the pathogenesis of many neuroinflammatory and neurodegenerative conditions in the CNS. To fully understand this process, it is important to define the MMP expression profile of specific cell types, including the CNS-resident cells astrocytes and microglia. While previous studies have characterized astrocyte MMP expression by using mixed glial cultures, these results are likely complicated by the presence of contaminating microglia within these cultures. In the current study, we sought to clarify this complexity, by taking a novel approach to prepare pure astrocyte cultures entirely devoid of microglia, by promoting neural stem cell (NSC) differentiation into astrocytes. The MMP expression profile of mixed glial cultures, neurosphere-derived astrocytes, and pure microglia was characterized by RNase protection assay. This revealed that MMP gene expression is largely cell-type specific. Astrocytes constitutively expressed MMP-11, MMP-14, and MMP-2 and showed induction of MMP-3 in response to IL-1beta but did not respond to lipopolysaccharide (LPS). In contrast, microglia constitutively expressed high levels of MMP-12 and showed strong induction of MMP-9 and MMP-14 in response to LPS. Gelatin zymography confirmed that LPS and TNF-alpha induced strong expression of MMP-9 in microglia but not astrocytes. In summary, these studies demonstrate that neurosphere-derived astrocytes represent an attractive alternative system in which to study astrocyte behavior in vitro. Using this system, we have shown that astrocytes and microglia express distinct sets of MMP genes and that microglia, not astrocytes, are the major source of MMP-9 in response to LPS or TNF-alpha.
Crocker
Stephen J
SJ
Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, California, USA.
Frausto
Ricardo F
RF
Whitton
J Lindsay
JL
Milner
Richard
R
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D020782:Matrix Metalloproteinases / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000201:enzymology*
D020782:Matrix Metalloproteinases