Extracellular nucleotides differentially regulate interleukin-1β signaling in primary human astrocytes: Implications for inflammatory gene expression

The cytokine interleukin-1β (IL-1β) is a potent activator of human astrocytes, inducing or modulating expression of multiple proinflammatory genes via activation of the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In this study, we examined whether IL-1β signaling is regulated in these cells by extracellular nucleotides that are released at high concentrations under inflammatory conditions and act as ligands for members of the P2 receptor family. Using reporter constructs and electromobility shift assays, we found that cotreatment of astrocyte cultures with ATP (1-100 μM) significantly potentiated IL-1β-mediated activation of NF-κB and AP-1 and that ATP alone activated AP-1. These effects were blocked by the P2 receptor antagonists XAMR 0721, periodate-oxidized ATP, and suramin. A role for ATP in modulating IL-1β-mediated inflammatory gene expression was supported further by the observation that ATP potentiated the IL-1β-induced expression of IL-8 mRNA and protein but strongly downregulated IP-10 expression. Reverse transcription-PCR and cloning demonstrated expression of the ATP-responsive P2 receptor subtypes P2Y₁, P2Y₂, and P2X₇, as well as the ATP-insensitive receptor P2Y₄. ADP, a selective agonist for P2Y₁, produced results similar to or greater than those obtained using ATP, whereas 2′-3′-O-(4-benzoyl-benzoyl)-ATp, a selective agonist for P2X₇, was less effective than ATR In contrast, UTP, a selective agonist for P2Y2 and P2Y₄, was ineffective. These studies indicate that different P2 receptor subtypes play distinct roles in the modulation of IL1β-mediated signal transduction in human astrocytes, and that signaling via P2 receptors may fine-tune the transcription of genes involved in inflammatory responses in the human CNS.