Original Contribution
Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation

https://doi.org/10.1016/j.freeradbiomed.2007.02.010Get rights and content

Abstract

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X7 agonists ATP and 3′-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X7-mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ERK2, p38, and JNK1/JNK2) phosphorylation and that the antioxidants N-acetylcysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.

Section snippets

Reagents

The nucleotides BzATP, ATP, UTP, α, β-methylene-ATP were obtained from Sigma (St. Louis, MO, USA). Lipopolysaccharide (Escherichia coli, serotype 0111:B4), phorbol 12-myristate 13-acetate (PMA), anisomycin, N-acetylcysteine (NAC), ascorbic acid, and methylthiazole tetrazolium (MTT) were also purchased from Sigma. The ROS indicator 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) was obtained from Molecular Probes (Eugene, OR, USA). The cell-permeable peptide JNK inhibitor (L)-JNKI1 and JNK

Extracellular nucleotide-stimulated ROS production

Although P2-receptor-stimulated ROS production has been reported for some cell types such as neutrophils, the participation of P2X7 in nucleotide-dependent ROS production in macrophages is less clear [23], [52]. Because the production of ROS is important for various macrophage functions including bacterial killing, we first sought to establish whether P2X7 agonists can stimulate the production of ROS in murine RAW 264.7 macrophages. In this regard, treatment of these cells with the selective P2X

Discussion

High levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation, and cellular damage/lysis [59]. These extracellular nucleotides activate receptors on various immune cells including monocytes and macrophages [1], [54]. This activation process results in the increased production and release of multiple inflammatory mediators, and in the current study we provide evidence that the nucleotide receptor P2X7 can promote the generation of ROS in murine

Acknowledgments

This work was supported by National Institutes of Health Grants HL56396 and AI50500. Z.A.P. was supported by Biotechnology Training Program NIH 5 T32 GM08349, University of Wisconsin. A.N.G. was supported by Hematology Training Program NIH 5 T32 HL07899, University of Wisconsin.

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    Current address: Department of Chemistry, Lawrence University, Appleton, WI 54912, USA.

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