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P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand.

Lu YM, Tao RR, Huang JY, Li LT, Liao MH, Li XM, Fukunaga K, Hong ZH, Han F - J Neuroinflammation (2012)

Bottom Line: Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility.Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7).Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT

Background: The cerebral microvascular occlusion elicits microvascular injury which mimics the different degrees of stroke severity observed in patients, but the mechanisms underlying these embolic injuries are far from understood. The Fas ligand (FasL)-Fas system has been implicated in a number of pathogenic states. Here, we examined the contribution of microglia-derived FasL to brain inflammatory injury, with a focus on the potential to suppress the FasL increase by inhibition of the P2X(7)-FasL signaling with pharmacological or genetic approaches during ischemia.

Methods: The cerebral microvascular occlusion was induced by microsphere injection in experimental animals. Morphological changes in microglial cells were studied immunohistochemically. The biochemical analyses were used to examine the intracellular changes of P2X(7)/FasL signaling. The BV-2 cells and primary microglia from mice genetically deficient in P2X(7) were used to further establish a linkage between microglia activation and FasL overproduction.

Results: The FasL expression was continuously elevated and was spatiotemporally related to microglia activation following microsphere embolism. Notably, P2X(7) expression concomitantly increased in microglia and presented a distribution pattern that was similar to that of FasL in ED1-positive cells at pathological process of microsphere embolism. Interestingly, FasL generation in cultured microglia cells subjected to oxygen-glucose deprivation-treated neuron-conditioned medium was prevented by the silencing of P2X(7). Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility. Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7). Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

Conclusion: FasL functions as a key component of an immunoreactive response loop by recruiting microglia to the lesion sites through a P2X(7)-dependent mechanism. The specific modulation of P2X(7)/FasL signaling and aberrant microglial activation could provide therapeutic benefits in acute and subacute phase of cerebral microembolic injury.

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P2X7 signal contributes to the migration of microglia in BV-2 microglia cells. (A) P2X7-specific shRNA transfection significantly reduced the increase in FasL generation induced by the OCM from OGD-stimulated cortical neurons. **P <0.01 vs. Control; #P <0.05, ##P <0.01 vs. OCM treatment alone. (B) OCM-induced FasL protein expression was abolished by lentiviral infection of P2X7-shRNA in the BV-2 cell line. Quantified data are shown in (C). **P <0.01 vs. Control; #P <0.05 vs. OCM treatment alone. (D) Representative immunoblots (upper) and quantitative analyses (lower) for FasL from BV-2 cultures following actinomycin D or anisomycin treatment. *P <0.05 vs. Control; #P <0.05 vs. OCM treatment alone. OCM, OGD-treated cell-free conditioned medium.
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Figure 5: P2X7 signal contributes to the migration of microglia in BV-2 microglia cells. (A) P2X7-specific shRNA transfection significantly reduced the increase in FasL generation induced by the OCM from OGD-stimulated cortical neurons. **P <0.01 vs. Control; #P <0.05, ##P <0.01 vs. OCM treatment alone. (B) OCM-induced FasL protein expression was abolished by lentiviral infection of P2X7-shRNA in the BV-2 cell line. Quantified data are shown in (C). **P <0.01 vs. Control; #P <0.05 vs. OCM treatment alone. (D) Representative immunoblots (upper) and quantitative analyses (lower) for FasL from BV-2 cultures following actinomycin D or anisomycin treatment. *P <0.05 vs. Control; #P <0.05 vs. OCM treatment alone. OCM, OGD-treated cell-free conditioned medium.

Mentions: In the brain parenchyma, injured cells release purines that initiate P2X7 purinergic signals, which act as early proinflammatory signals and lead to the production of cytokines and chemokines [36-38]. Therefore, we examined whether P2X7 expression could also regulate FasL production in cultured microglia, and we investigated the potential mechanisms associated with this process. Blockade of P2X7 signaling by P2X7-specific shRNA transfection significantly suppressed OCM-induced FasL production in the culture supernatants, whereas P2X7 shRNA alone did not affect FasL levels (Figure 5A), which indicated a causative role for P2X7 in initiating microglia-mediated FasL generation. Western blotting of BV-2 microglial cell lysates showed that microglia expressed FasL 6 h after the OCM treatment (Figure 5B, C). The suppression of P2X7 activation with P2X7 shRNA significantly prevented OCM-induced FasL expression but did not affect Fas expression (Figure 5B, C). These findings further support the idea that enhanced P2X7 expression may act as an important upstream regulator of FasL formation in OCM-treated microglial cells. These results highlight the ability of neuron-conditioned culture medium from OGD-stimulated cortical neurons to maintain a controlled inflammatory state through the activation of a P2X7-FasL signal that favors the microenvironment of activated microglia.


P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand.

Lu YM, Tao RR, Huang JY, Li LT, Liao MH, Li XM, Fukunaga K, Hong ZH, Han F - J Neuroinflammation (2012)

P2X7 signal contributes to the migration of microglia in BV-2 microglia cells. (A) P2X7-specific shRNA transfection significantly reduced the increase in FasL generation induced by the OCM from OGD-stimulated cortical neurons. **P <0.01 vs. Control; #P <0.05, ##P <0.01 vs. OCM treatment alone. (B) OCM-induced FasL protein expression was abolished by lentiviral infection of P2X7-shRNA in the BV-2 cell line. Quantified data are shown in (C). **P <0.01 vs. Control; #P <0.05 vs. OCM treatment alone. (D) Representative immunoblots (upper) and quantitative analyses (lower) for FasL from BV-2 cultures following actinomycin D or anisomycin treatment. *P <0.05 vs. Control; #P <0.05 vs. OCM treatment alone. OCM, OGD-treated cell-free conditioned medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3420259&req=5

Figure 5: P2X7 signal contributes to the migration of microglia in BV-2 microglia cells. (A) P2X7-specific shRNA transfection significantly reduced the increase in FasL generation induced by the OCM from OGD-stimulated cortical neurons. **P <0.01 vs. Control; #P <0.05, ##P <0.01 vs. OCM treatment alone. (B) OCM-induced FasL protein expression was abolished by lentiviral infection of P2X7-shRNA in the BV-2 cell line. Quantified data are shown in (C). **P <0.01 vs. Control; #P <0.05 vs. OCM treatment alone. (D) Representative immunoblots (upper) and quantitative analyses (lower) for FasL from BV-2 cultures following actinomycin D or anisomycin treatment. *P <0.05 vs. Control; #P <0.05 vs. OCM treatment alone. OCM, OGD-treated cell-free conditioned medium.
Mentions: In the brain parenchyma, injured cells release purines that initiate P2X7 purinergic signals, which act as early proinflammatory signals and lead to the production of cytokines and chemokines [36-38]. Therefore, we examined whether P2X7 expression could also regulate FasL production in cultured microglia, and we investigated the potential mechanisms associated with this process. Blockade of P2X7 signaling by P2X7-specific shRNA transfection significantly suppressed OCM-induced FasL production in the culture supernatants, whereas P2X7 shRNA alone did not affect FasL levels (Figure 5A), which indicated a causative role for P2X7 in initiating microglia-mediated FasL generation. Western blotting of BV-2 microglial cell lysates showed that microglia expressed FasL 6 h after the OCM treatment (Figure 5B, C). The suppression of P2X7 activation with P2X7 shRNA significantly prevented OCM-induced FasL expression but did not affect Fas expression (Figure 5B, C). These findings further support the idea that enhanced P2X7 expression may act as an important upstream regulator of FasL formation in OCM-treated microglial cells. These results highlight the ability of neuron-conditioned culture medium from OGD-stimulated cortical neurons to maintain a controlled inflammatory state through the activation of a P2X7-FasL signal that favors the microenvironment of activated microglia.

Bottom Line: Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility.Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7).Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT

Background: The cerebral microvascular occlusion elicits microvascular injury which mimics the different degrees of stroke severity observed in patients, but the mechanisms underlying these embolic injuries are far from understood. The Fas ligand (FasL)-Fas system has been implicated in a number of pathogenic states. Here, we examined the contribution of microglia-derived FasL to brain inflammatory injury, with a focus on the potential to suppress the FasL increase by inhibition of the P2X(7)-FasL signaling with pharmacological or genetic approaches during ischemia.

Methods: The cerebral microvascular occlusion was induced by microsphere injection in experimental animals. Morphological changes in microglial cells were studied immunohistochemically. The biochemical analyses were used to examine the intracellular changes of P2X(7)/FasL signaling. The BV-2 cells and primary microglia from mice genetically deficient in P2X(7) were used to further establish a linkage between microglia activation and FasL overproduction.

Results: The FasL expression was continuously elevated and was spatiotemporally related to microglia activation following microsphere embolism. Notably, P2X(7) expression concomitantly increased in microglia and presented a distribution pattern that was similar to that of FasL in ED1-positive cells at pathological process of microsphere embolism. Interestingly, FasL generation in cultured microglia cells subjected to oxygen-glucose deprivation-treated neuron-conditioned medium was prevented by the silencing of P2X(7). Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility. Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7). Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult.

Conclusion: FasL functions as a key component of an immunoreactive response loop by recruiting microglia to the lesion sites through a P2X(7)-dependent mechanism. The specific modulation of P2X(7)/FasL signaling and aberrant microglial activation could provide therapeutic benefits in acute and subacute phase of cerebral microembolic injury.

Show MeSH
Related in: MedlinePlus