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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Microglia P2Y₆ receptors mediate nitric oxide release and astrocyte apoptosis
Clara Quintas, Diana Pinho, Clara Pereira
1Department of Drug Sciences, Laboratory of Pharmacology, REQUIMTE and Center for Drug Discovery and Innovative Medicines, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira n° 228, Porto 4050-313, Portugal. gloria@ff.up.pt.
Background:
During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis.
Methods:
Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining.
Results:
UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia.
Conclusions:
In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.
Insights
Uracil nucleotides, specifically UDP, activate microglial P2Y6 receptors, leading to nitric oxide release that induces astrocyte apoptosis. This mechanism controls astrocyte proliferation and prevents excessive astrogliosis during inflammation.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Cerebral inflammation causes uracil nucleotide leakage, activating glial pyrimidine receptors.
- Activated microglia adopt an anti-inflammatory phenotype, but their effect on astrogliosis is unclear.
Purpose of the Study:
- Investigate pyrimidine receptor involvement in microglia-astrocyte signaling.
- Determine the impact on astrogliosis in a chronic inflammation model.
Main Methods:
- Primary astrocyte-microglia co-cultures treated with lipopolysaccharide (LPS) and uracil nucleotides.
- Assessed cell proliferation, P2Y6 receptor and iNOS expression, nitric oxide (NO) release, and cell death.
Main Results:
- UDP activated P2Y6 receptors, increasing NO release from microglia.
- UDP-induced NO release triggered astrocyte apoptosis, inhibiting proliferation.
- P2Y6 receptors and inducible nitric oxide synthase (iNOS) were primarily expressed in microglia.
Conclusions:
- Microglial P2Y6 receptor activation by UDP leads to NO production.
- This NO release induces astrocyte apoptosis, controlling proliferation and preventing excessive astrogliosis.
