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Production of macrophage inflammatory protein-2 following hypoxia/reoxygenation in glial cells
1Graduate Institute of Life Sciences and Department of Physiology, National Defense Medical Center, Taipei, Taiwan, Republic of China. jywang@ndmctsgh.edu.tw
Abstract:
Polymorphonuclear neutrophils (PMNs) are known to mediate brain inflammation following hypoxia/reoxygenation (H/R), but the precise mechanisms leading to PMN recruitment are undefined. The alpha-chemokine macrophage inflammatory protein-2 (MIP-2) has specificity for the recruitment of PMNs. In this study, we found that 8 or 12 h of hypoxia followed by 24-h reoxygenation (H8/R24 or H12/R24) induced MIP-2 secretion in cultures of enriched microglia or mixed glia, respectively. Microglia, however, could not survive longer duration (>12 h) of hypoxia. Astrocytes did not produce any significant amount of MIP-2 even though astrocytes maintained 98-99% viability following H12/R24. We also found that microglia survived the H/R treatment better (following H24) in the presence of astrocytes (mixed glial culture) than in microglia-enriched culture. Reoxygenation for prolonged periods (3 and 5 days) following H24 resulted in progressively larger increases in MIP-2 production (20- and 60-fold, respectively) in mixed glial cultures. Immunocytochemical staining revealed that the cells expressing MIP-2 in response to H/R were microglia rather than astrocytes in mixed glial cultures. Examination of MIP-2 mRNA expression showed that H/R upregulated MIP-2 gene expression. Taken together, our data suggest that microglial cells are an important source of MIP-2 production and suggest a potential injury mechanism involving brain-derived production of MIP-2 in H/R.
Insights
Microglia, not astrocytes, produce macrophage inflammatory protein-2 (MIP-2) after brain hypoxia/reoxygenation (H/R). This brain-derived MIP-2 may drive inflammation and injury during H/R events.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Polymorphonuclear neutrophils (PMNs) contribute to brain inflammation post-hypoxia/reoxygenation (H/R).
- The mechanisms of PMN recruitment in H/R-induced brain inflammation are not fully understood.
- Macrophage inflammatory protein-2 (MIP-2) is an alpha-chemokine specific for PMN recruitment.
Purpose of the Study:
- To investigate the cellular source and regulation of MIP-2 production in the brain following H/R.
- To determine the role of microglia and astrocytes in mediating H/R-induced brain inflammation via MIP-2.
Main Methods:
- Primary glial cultures (enriched microglia, enriched astrocytes, mixed glia) were subjected to varying durations of hypoxia followed by reoxygenation.
- MIP-2 secretion was measured using ELISAs.
- Cell viability was assessed.
- Immunocytochemistry and MIP-2 mRNA expression analysis were performed.
Main Results:
- Hypoxia/reoxygenation (H/R) induced MIP-2 secretion in microglia and mixed glial cultures.
- Microglia, but not astrocytes, were the primary producers of MIP-2 in response to H/R.
- Microglial survival during H/R was enhanced in the presence of astrocytes.
- Prolonged reoxygenation led to significantly increased MIP-2 production.
- H/R upregulated MIP-2 gene expression in microglia.
Conclusions:
- Microglial cells are a significant source of brain-derived MIP-2 following H/R.
- Microglia-derived MIP-2 may play a crucial role in PMN recruitment and subsequent brain injury during H/R.
- Astrocytes support microglial survival and modulate the inflammatory response during H/R.