Modulation of Interleukin-1β and Tumor Necrosis Factor α Signaling by P2 Purinergic Receptors in Human Fetal Astrocytes

Abstract

In human astrocytes, interleukin-1beta (IL-1beta) 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 purinergic nucleotide receptor pathway contributes to, or modulates, cytokine-mediated signal transduction. Calcium imaging studies indicated that most cells in culture responded to ATP, whereas only a subpopulation responded to UTP. Pretreatment of astrocytes with P2 receptor antagonists, including suramin and periodate oxidized ATP (oATP), resulted in a significant downregulation of IL-1beta-stimulated expression of nitric oxide, tumor necrosis factor (TNFalpha), and IL-6 at both the protein and mRNA levels, without affecting cell viability. In cells transiently transfected with reporter constructs, IL-1beta demonstrated more potent activation of the transcription factors nuclear factor -kappaB (NF-kappaB) and activator protein-1 (AP-1) than TNFalpha. However, pretreatment with oATP downregulated activation of NF-kappaB and AP-1 by IL-1beta or TNFalpha. Electromobility shift assays using oligonucleotides containing specific NF-kappaB binding sequences confirmed that pretreatment with oATP or apyrase attenuated cytokine-mediated induction of this transcription factor. From these data, we conclude that P2 receptor-mediated signaling intersects with that of IL-1beta and TNFalpha 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 extracellular environment and can fine tune the transcription of genes involved in the inflammatory response.

Extracellular Nucleotides Differentially Regulate Interleukin-1β Signaling in Primary Human Astrocytes: Implications for Inflammatory Gene Expression

“…However, activated lymphocytes, macrophages, microglia, and platelets, as well as cells undergoing necrosis or apoptosis, release high concentrations of different nucleotide diphosphates and triphosphates into the extracellular space (Dubyak and el-Moatissim, 1993). Using chemical antagonists, we recently found that autocrine or paracrine activity of extracellular nucleotides on P2 receptors is required for astrocyte activation (Liu et al, 2000), and similar findings have also been reported in murine macrophages (Hu et al, 1998;Sikora et al, 1999). In the present study, we tested the hypothesis that increased concentrations of extracellular nucleotides regulate IL-1␤ signal transduction in human astrocytes via a P2 receptormediated pathway.…”

“…Culture purity was determined by immunostaining for glial fibrillary acidic protein (astrocytes), microtubule-associated protein-2 (neurons), and CD68 (microglia). Recombinant human IL -1␤ was a gift from the Biological Response Modifiers Program at the National C ancer Institute (Frederick, MD); the dose used (10 ng /ml) was based on dose -response studies that we have performed previously on IL -1-mediated signal transduction in human fetal astrocytes (Liu et al, 2000). It is identical to that used in studies that have examined the IL -1 signaling cascade leading to N F-B and AP-1 activation in several other cell types (Delhase et al, 1999;NinomiyaTsuji et al, 1999).…”

“…L ow-level background activity was noted in cells transfected with ⌬56fosdE-L uc (empty vector control for 6AP1-L uc), but did not reach the levels observed in control cells transfected with 6AP1-L uc. Controls for transfection efficiency were performed using pGreen Lantern (Life Technologies); they demonstrated that transfection efficiency did not differ between wells from the same cases plated at the same density, as we have also described previously (Liu et al, 2000).…”

Stimulation of P2Y1 Receptors Causes Anxiolytic-like Effects in the Rat Elevated Plus-maze: Implications for the Involvement of P2Y1 Receptor-Mediated Nitric Oxide Production

“…Confirming this possibility, the blockade of the P2 receptors by PPADS may lead to a decrease of the NO release, an effect that is synergistic with the action of NOS inhibitors. Further evidence for a close relationship between P2 receptors and NO production is given by a study of Liu et al (2000), which shows that pretreatment of astrocytes with P2 receptor antagonists including PPADS results in a down regulation of interleukin-1b-stimulated NOS expression.…”

Curcumin Inhibits Activation of Vγ9Vδ2 T Cells by Phosphoantigens and Induces Apoptosis Involving Apoptosis-Inducing Factor and Large Scale DNA Fragmentation

“…V␦2 ϩ T cells (5 ϫ 10 6 ) were stimulated in 24-well plates for 1 h with medium alone or 50 U/ml IL-2, IPP2X, or IPP2X in the presence of curcumin at 30 M. Nuclear extracts were prepared by a modified Dignam method on ice, and EMSA was performed as described previously (23). Oligonucleotides containing the NF-B consensus binding sequence (5Ј-AGT TGA GGG GAC TTT CCC AGG C-3Ј) or the AP-1 consensus binding sequence (5Ј-CGC TGG ATG AGT CAG CCG GAA-3Ј) were radiolabeled with [␥-32 P]ATP using polynucleotide T4 kinase (Gel Shift Assay Core System kit; Promega, Madison, WI).…”