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

Abstract

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X 7 . In this regard, P2X 7 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 P2X 7 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 P2X 7 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 P2X 7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X 7 -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-acetyl-cysteine 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 P2X 7 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.

Cell signaling via the P2X7 nucleotide receptor: linkage to ROS production, gene transcription, and receptor trafficking

“…Similarly, we have observed that stimulation of the MAP kinases ERK1/2, p38, and c-Jun N-terminal kinase (JNK) in the murine macrophage cell line RAW 264.7 by P2X 7 agonists is sensitive to antioxidants, suggesting that ROS may play a more global role in P2X 7 signaling [21]. Although P2 receptor potentiation of ROS production has been seen with several immune cell types, the role of P2X 7 in nucleotidedependent ROS production in macrophages is less well known.…”

“…For example, reports by our lab and others have shown that P2X 7 activation results in ROS production, which may act as a second messenger in several P2X 7 agonist-induced events [16,17,21,52,53]. However, the mechanisms by which P2X 7 promotes ROS production are less well understood, and ROS generation in response to P2X 7 agonists has not been demonstrated in primary cells.…”

“…Although P2 receptor potentiation of ROS production has been seen with several immune cell types, the role of P2X 7 in nucleotidedependent ROS production in macrophages is less well known. To this end, we have shown that P2X 7 agonists stimulate ROS production in RAW macrophages but not in a RAW cell line containing a non-functional P2X 7 receptor [21]. However, because the behavior of cell lines may differ from primary cells, we have now tested whether P2X 7 agonists can promote ROS production in human primary cells.…”

“…The generation of ROS has also been implicated in several human disorders, including hypertension, atherosclerosis, ischemia/reperfusion injury, neurodegenerative disease, chronic granulomatous disease, and the deleterious effects of aging [18,56]. The use of ROS by leukocytes in the defense against pathogens is well recognized, but the role of ROS as second messengers is sill emerging, e.g., ROS generation has been linked to the activation of transcription factors, including apurinic/apyrimidinic endonuclease 1 (APE1)/Ref1, nuclear factor κB (NFκB), and AP-1, as well as the signaling kinases p90 ribosomal S6 kinase (p90RSK) and various MAPKs [21,[57][58][59][60][61][62][63][64][65]. Although the mechanisms of ROS-induced signaling have not been fully resolved, multiple processes including inactivation of phosphatases through cysteine modification and the activation of redox sensitive systems, have been proposed [57,61,62,66].…”

“…Interestingly, P2X 7 ligation by extracellular ATP can also modulate lipopolysaccharide (LPS)-mediated macrophage activation, and P2X 7 stimulation is known to potentiate the release of several LPS-induced pro-inflammatory mediators, including interleukin-1β (IL-1β), tumor necrosis factor-α, and nitric oxide [17][18][19][20][21][22]. However, although there is a growing literature regarding several cell signaling and cytokine processing events that are initiated upon P2X 7 activation, relatively little is known about the regulation of receptor trafficking and the mechanisms by which P2X 7 activation can impact on long-term cellular processes such as the regulation of gene transcription.…”