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. 2007 Feb 2;282(5):2871-9.
doi: 10.1074/jbc.M608083200. Epub 2006 Nov 27.

ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages

Cristiane M Cruz  1 , Alessandra Rinna, Henry Jay Forman, Ana L M Ventura, Pedro M Persechini, David M Ojcius
Affiliations

ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages

Cristiane M Cruz et al. J Biol Chem. .

Abstract

Secretion of the proinflammatory cytokines, interleukin (IL)-1beta and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), whose inactivation would shift the equilibrium in favor of PI3K. ATP-dependent ROS production and PI3K activation also stimulate transcription of genes required for an oxidative stress response. In parallel, ATP-mediated ROS-dependent PI3K is required for activation of caspase-1 and secretion of IL-1beta and IL-18. Thus, an increase in ROS levels in ATP-treated macrophages results in activation of a single pathway that promotes both adaptation to subsequent exposure to oxidants or inflammation, and processing and secretion of proinflammatory cytokines.

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Figures

FIGURE 1
FIGURE 1. ATP induces ROS production in macrophages
A, left, alveolar macrophages were preincubated with an ROS-sensitive dye, DCF (5 µm), for 5 min, before treating with 3 mm ATP for another 5 min at 37 °C. Enhancement in DCF fluorescence was measured immediately in a FACS, which showed a large increase in fluorescence in ATP-treated macrophages, compared with DCF-labeled untreated controls. A, right, DCF fluorescence was quantified as the mean fluorescence measured by FACS. Preincubation of macrophages with 25 mm NAC for 5 min before ATP treatment abrogated the increase in ROS production. As a positive control, maximal ROS production was measured when untreated macrophages were incubated with 1 mm H2O2for 5 min. p < 0.001 for cells treated with ATP, compared with cells treated with control buffer, or cells treated with ATP with NAC. B, macrophages were preincubated with DCF and then treated with control buffer (left) or 3 mm ATP (right) for 5 min, and visualized immediately with a fluorescence microscope. Phase-contrast microscopy (top panels) does not show obvious morphological changes in macrophages due to ATP treatment. Green fluorescence (bottom panels) was emitted from cells that had been treated with ATP (right, bottom) but not from untreated controls (left, bottom). C, ROS production was maximal after a 5-min incubation with 3 mm ATP, and returned to basal levels within 15 min, as measured by FACS of DCF-labeled cells. All experiments were performed in triplicate at least 3 times on separate days. The values show averages and S.D. from 3 samples of a representative experiment.
FIGURE 2
FIGURE 2. Ligation of several purinergic receptors can stimulate ROS production in macrophages
A, incubation of DCF-labeled macrophages with 3 mm ATP, 3 mm UTP, 0.4 mm ADP, or 0.1 mm UDP results in an increase in ROS levels in macrophages, as measured by FACS of DCF-stained macrophages. p < 0.01 for cells treated with ATP, compared with cells treated with control buffer. B, preincubation of macrophages with 0.3 mm of the P2X7 antagonist, oxATP, for 2 h inhibits partially ATP-mediated ROS production due to treatment with 3 mm ATP for 5 min. p < %0.05 for cells treated with oxATP and ATP, compared with cells treated with ATP. C, incubation with 1 mm of the P2X7 agonist, BzATP, for 5 min induces as much ROS production as 3 mm ATP. p < 0.001 for cells treated with BzATP, compared with cells treated with control buffer. The values show averages and S.D. from 3 samples of a representative experiment, and represent results obtained from at least three representative experiments.
FIGURE 3
FIGURE 3. Treatment of macrophages with ATP results in ROS-dependent PI3K activation and Akt phosphorylation
Macrophages were pretreated for 5 min with 25 mm NAC or 50 µM of the PI3K inhibitor, LY294002, for 5 min, and incubated for 5 min with 3 mm ATP. Akt phosphorylation was determined by Western blot, using an Ab recognizing Akt phosphorylated on residue Thr308. The Western blot is representative of three experiments performed on separate days.
FIGURE 4
FIGURE 4. ATP-dependent ROS production and PI3K activation are upstream from ERK1/2 phosphorylation
A, macrophages were incubated with 3 mm ATP, and cells were lysed at different times after the addition of ATP. ERK1/2 phosphorylation was determined by Western blot, probing with an Ab recognizing ERK1/2 phosphorylated on residues Thr202 and Tyr204. Akt phosphorylation was measured with an Ab recognizing Akt phosphorylated on residue Thr308. The Western blot is representative of three experiments performed on separate days. B, ERK1/2 phosphorylation was confirmed by FACS. Macrophages were pretreated for 5 min with control buffer (left panel), 1 µm of the PI3K inhibitor, wortmannin (middle panel), or 10 µm of the ERK1/2 inhibitor, PD98056 (right panel), for 5 min, and incubated for 5 min with 3 mm ATP. ERK1/2 phosphorylation was measured by FACS, after incubating per-meabilized cells with the Ab recognizing phosphorylated ERK1/2. C, ERK1/2 phosphorylation was quantified as the mean fluorescence measured by FACS. Preincubation of macrophages with the ERK1/2 inhibitor, PD98056, or the PI3K inhibitors, wortmannin or LY294002, for 5 min before ATP treatment decreased significantly the extent of ERK1/2 phosphorylation. p < 0.001 for cells treated with wortmannin and ATP, compared with cells treated with ATP. The results are representative of at least two experiments performed on separate days.
FIGURE 5
FIGURE 5. ATP-dependent ROS production induces glutathionylation of PTEN
Macrophages were pretreated for 5 min with control buffer or 3 mm ATP for 1 min. Cells were lysed, and the proteins were immunoprecipitated overnight with PTEN-conjugated beads. A, the extent of glutathionylation was determined by Western blot, using an Ab recognizing GSH. The anti-GSH Ab revealed the presence of immunoprecipitated protein both as a monomer corresponding to the molecular mass of PTEN (60 kDa) or a larger complex (200 kDa). The level of glutathionylation of PTEN in both monomeric and multimeric forms increases after ATP stimulation of macrophages. Treatment of the immunoprecipitate with the reducing agent dithiothreitol before electrophoresis dissociates GSH from all proteins. B, the results were confirmed by reprobing the same membrane with an Ab recognizing total PTEN. The anti-PTEN Ab also revealed the presence of PTEN in monomers and a larger complex. Macrophage stimulation with ATP causes the amount of PTEN in the larger complex to increase slightly. Treatment with the reducing agent dithiothreitol causes PTEN to dissociate from the larger complex. The Western blots are representative of two experiments performed on separate days.
FIGURE 6
FIGURE 6. Stimulation of macrophages with ATP results in PI3K- and ERK1/2-dependent up-regulation of genes involved in GSH synthesis
A, incubation of macrophages with 3 mm ATP, 3 mm UTP, or 0.4 mm ADP for 20min, followed by an additional 6 h in the absence of nucleotides results in an increased expression of gclc and gclm, as measured by real-time PCR. p < 0.001 for gclm of cells treated with ATP, compared with cells treated with control buffer; and p < 0.01 for gclm of cells treated with ADP, compared with cells treated with control buffer. B, macrophages were pretreated for 5 min with the PI3K inhibitors, LY294002 (50 µm), or the ERK1/2 inhibitor, PD98056 (10 µm), for 5 min, and then incubated for 5 min with 3 mm ATP. Expression of gclm and gclc was measured by real-time PCR. p < 0.001 for gclm of cells treated with ATP, compared with cells treated with ATP or either LY294002 or PD98056. The results are representative of at least three experiments performed on separate days.
FIGURE 7
FIGURE 7. ATP-dependent ROS production and ERK1/2 activation are upstream from caspase-1 activation
A, macrophages were stained with the fluorescent caspase substrate, FAM-VAD-fmk, pretreated for 10 min with the redox inhibitor, DPI (2 µm), and then incubated for 6.5 or 12 h with 3 mm ATP. Caspase-1 was measured by FACS. The specificity of caspase-1 activation was verified by pretreating the stained cells with the irreversible caspase-1 inhibitor, Z-YVAD-fmk. p < 0.001 for cells treated with DPI and ATP for 12 h, compared with cells treated with ATP for 12 h. B, stained macrophages were pretreated for 5 min with 10 µm of the ERK1/2 inhibitor, PD98056, for 5 min, before incubation for 5 min with 3 mm ATP. p < 0.001 for cells treated with PD98056 and ATP for 6.5 or 12 h, compared with cells treated with ATP alone. The values show averages and S.D. from experiments performed in triplicate, and represent results obtained from at least two representative experiments.
FIGURE 8
FIGURE 8. ATP stimulation of macrophages leads to IL-1β secretion through a pathway requiring ROS production and PI3K activation
Macrophages were primed with 1 µg/ml LPS for 2 h at 37 °C, before treating the macrophages with the caspase-1 inhibitor, Z-YVAD-fmk (50 µm) for 30 min, DPI (2 µm) for 10 min, or LY294002 (50 µm) for 10 min. The cells were then stimulated with 3 mm ATP for 6 h. Secretion of IL-1β was measured by ELISA. p < 0.001 for cells treated with ATP and Z-YVAD-fmk, DPI, or LY294002, compared with cells treated with ATP alone. The experiment was performed twice in duplicate, and the results represent the average and S.D.
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