Modulation of interleukin-1 beta and tumor necrosis factor alpha signalingby P2 purinergic receptors in human fetal astrocytes

In human astrocytes, interleukin-1 beta (IL-1 beta) is a potent inducer of genes associated with inflammation. In this study, we tested the hypothesis that in primary cultures of human fetal astrocytes signaling by the P2 pur inergic nucleotide receptor pathway contributes to, or modulates, cytokine- mediated signal transduction. Calcium imaging studies indicated that most c ells in culture responded to ATP, whereas only a subpopulation responded to UTP. Pretreatment of astrocytes with P2 receptor antagonists, including su ramin and periodate oxidized ATP (oATP), resulted in a significant downregu lation of IL-1 beta-stimulated expression of nitric oxide, tumor necrosis f actor (TNF alpha), and IL-6 at both the protein and mRNA levels, without af fecting cell viability. In cells transiently transfected with reporter cons tructs, IL-1 beta demonstrated more potent activation of the transcription factors nuclear factor -kappa B (NF-kappa B) and activator protein-1 (AP-1) than TNF alpha. However, pretreatment with oATP downregulated activation o f NF-kappa B and AP-1 by IL-1 beta or TNF alpha. Electromobility shift assa ys using oligonucleotides containing specific NF-kappa B binding sequences confirmed that pretreatment with oATP or apyrase attenuated cytokine-mediat ed induction of this transcription factor. From these data, we conclude tha t P2 receptor-mediated signaling intersects with that of IL-1 beta and TNF alpha to regulate responses to cytokines in the CNS. Because inflammation, trauma, and stress all lead to the release of high levels of extracellular nucleotides, such as ATP and UTP, signaling via P2 receptors may provide a mechanism whereby cells can sense and respond to events occurring in the ex tracellular environment and can fine tune the transcription of genes involv ed in the inflammatory response.