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Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
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Purinergic P2Y2 receptors induce increased MCP-1/CCL2 synthesis and release from rat alveolar and peritoneal

Leanne Stokes1, Annmarie Surprenant

  • 1Department of Biomedical Science, University of Sheffield, Addison Building Western Bank, Sheffield, United Kingdom.

Journal of Immunology (Baltimore, Md. : 1950)
|October 20, 2007
PubMed

Insights

Extracellular ATP activates P2Y(2) receptors on macrophages, triggering the release of the inflammatory chemokine MCP-1 (monocyte chemoattractant protein-1). This occurs via phospholipase C (PLC) activation, though mRNA increases suggest a novel pathway may also be involved.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • Macrophages are crucial in inflammation, releasing cytokines and chemokines.
  • Extracellular nucleotides act as danger signals, with P2X(7) receptors known to release IL-18 and IL-1beta.
  • The role of other purinergic receptors in cytokine/chemokine release is less understood.

Purpose of the Study:

  • To investigate if extracellular ATP can induce cytokine and chemokine release from macrophages lacking P2X(7) receptors.
  • To identify the specific purinergic receptor involved in ATP-induced MCP-1 release.
  • To explore the downstream signaling pathways mediating P2Y(2) receptor-induced MCP-1 production.

Main Methods:

  • Protein arrays were used to screen for ATP-induced cytokine/chemokine release from rat NR8383 alveolar macrophages.
  • Pharmacological profiling identified the P2Y(2) receptor as responsible for MCP-1 release.
  • Quantitative real-time PCR and Western blotting were used to assess MCP-1 mRNA and protein levels, and signaling pathway activation.

Main Results:

  • Extracellular ATPgammaS significantly increased the release of the chemokine MCP-1 (monocyte chemoattractant protein-1/CCL2) from P2X(7) receptor-deficient macrophages.
  • The P2Y(2) receptor was identified as the primary receptor mediating this ATP-induced MCP-1 release.
  • Activation of P2Y(2) receptors led to increased MCP-1 mRNA within 30 minutes and release within 60 minutes in both alveolar and peritoneal macrophages.
  • While phospholipase C (PLC) inhibition blocked MCP-1 release, evidence for canonical G(q) or G(i)/G(o) pathways in P2Y(2)R-induced mRNA elevation was lacking, suggesting a novel signaling mechanism.

Conclusions:

  • The P2Y(2) receptor plays a significant role in mediating the release of MCP-1 from macrophages in response to extracellular ATP.
  • Phospholipase C activation is critical for the release of MCP-1, but a distinct pathway may regulate P2Y(2) receptor-mediated increases in MCP-1 mRNA.
  • These findings highlight a novel signaling mechanism for purinergic receptor-mediated chemokine production in macrophages, relevant to inflammatory processes.

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