A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites - PubMed (original) (raw)

. 2006 Jul;116(7):2033-43.

doi: 10.1172/JCI27544. Epub 2006 Jun 15.

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A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites

Pham My-Chan Dang et al. J Clin Invest. 2006 Jul.

Abstract

Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM-CSF-induced phosphorylation of Ser345, while p38 MAPK inhibitor abrogated TNF-alpha-induced phosphorylation of Ser345. Transfection of HL-60 cells with a mutated p47phox (S345A) inhibited GM-CSF- and TNF-alpha-induced priming of ROS production. This event was also inhibited in neutrophils by a cell-permeable peptide containing a TAT-p47phox-Ser345 sequence. Furthermore, ROS generation, p47phox-Ser345 phosphorylation, and ERK1/2 and p38 MAPK phosphorylation were increased in synovial neutrophils from rheumatoid arthritis (RA) patients, and TAT-Ser345 peptide inhibited ROS production by these primed neutrophils. This study therefore identifies convergent MAPK pathways on Ser345 that are involved in GM-CSF- and TNF-alpha-induced priming of neutrophils and are activated in RA. Inhibition of the point of convergence of these pathways might serve as a novel antiinflammatory strategy.

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Figures

Figure 1

Figure 1. Mass spectrometry analysis of GM-CSF–induced p47phox phosphorylation: Ser345 was phosphorylated in primed human neutrophils.

(A) LC-MS/MS analysis of an aliquot of the p47phox tryptic peptide mixture (gradient: 0–38% acetonitrile in 35 minutes). The 2 upper panels depict the base peak chromatograms of electrospray ionization MS/MS experiments performed during the analysis in data-dependent acquisition mode. The lower panel represents the base peak chromatogram of the MS survey scans only. The arrow indicates the elution time of the phosphopeptide that was sequenced by MS/MS. (B) Identification of the phosphorylated peptide QARPGPQ[pS]PGSPLEEER (amino acids 338–354). The MS/MS spectrum displayed a near-complete y-ion series that reflects the amino acid sequence and the position of the phosphate group at Ser345. The elimination of phosphoric acid from the phosphoserine residue during MS/MS generated a dehydroalanine residue at the position corresponding to Ser345 (s; residue mass, 69 Da). The spectrum also demonstrated that the Ser348 residue (S) was not phosphorylated (residue mass, 87 Da).

Figure 2

Figure 2. Use of an antibody to phospho-Ser345 demonstrates that GM-CSF and TNF-α induce phosphorylation of p47phox on Ser345 in a concentration- and time-dependent manner.

(A) Neutrophils (1 × 107 cells/ml) were incubated with various concentrations of GM-CSF for 20 minutes or were incubated with GM-CSF (12.5 ng/ml) for the times indicated. Cells were lysed, and proteins from 4 × 105 cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody (p-Ser345) or anti-p47phox antibody (p47phox). (B) Neutrophils (1 × 107 cells/ml) were incubated with various concentrations of TNF-α for 20 minutes or were incubated with TNF-α (10 ng/ml) for the times indicated. Cells were lysed, and proteins from 4 × 105 cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody or anti-p47phox antibody. Western blots from different experiments were scanned; phosphorylated and total p47phox were quantified by densitometry; and the intensity of phosphorylated p47phox was corrected for the amount of p47phox. Results are expressed as mean ± SEM (n = 3). *P < 0.05 compared with untreated neutrophils or time 0 minutes.

Figure 3

Figure 3. Effect of different neutrophil agonists on the phosphorylation of Ser345.

Neutrophils (1 × 107 cells/ml) were incubated with GM-CSF (12.5 ng/ml) for 20 minutes, TNF-α (10 ng/ml) for 20 minutes, and different concentrations of fMLP (A) or PMA (B) for different time periods. Cells were then lysed, and proteins from 4 × 105 cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody or anti-p47phox antibody. Data are representative of 3 independent experiments using cells from different donors.

Figure 4

Figure 4. Effect of genistein, a protein tyrosine kinase inhibitor, on GM-CSF– and TNF-α–induced p47phox phosphorylation.

Neutrophils were incubated without (Control) or with 100 μM genistein for 30 minutes, then with 12.5 ng/ml GM-CSF (A) or 10 ng/ml TNF-α for 20 minutes (B). p47phox from 32P-labeled neutrophils (5 × 107 cells) was immunoprecipitated with anti-p47phox antibody and analyzed by SDS-PAGE, Western blotting, and autoradiography ([32P]p47phox). Total cell lysates (4 × 105 cells) from unlabeled treated cells were also analyzed by SDS-PAGE and Western blotting using the anti–phospho-Ser345 antibody or anti-p47phox antibody. Data are representative of 4 experiments.

Figure 5

Figure 5. Effect of MAPK inhibitors on GM-CSF– and TNF-α–induced p47phox and MAPK activation.

(A) Neutrophils (1 × 107 cells/ml) were incubated with GM-CSF (12.5 ng/ml) or TNF-α (10 ng/ml) for 20 minutes and lysed, and proteins from 4 × 105 cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-ERK1/2 or anti–phospho-p38 antibody (p-p38MAPK). (B and C) Neutrophils were incubated with SB203580 (10 μM), PD98059 (50 μM), or UO126 (10 μM) for 30 minutes, then treated with GM-CSF (B) or TNF-α (C) for 20 minutes. p47phox from 32P-labeled neutrophils (5 × 107 cells) was immunoprecipitated with anti-p47phox antibody and analyzed by SDS-PAGE, Western blotting, and autoradiography. Total cell lysates (4 × 105 cells) from unlabeled cells were also analyzed by SDS-PAGE and Western blotting with anti–phospho-Ser345 antibody or anti–phospho-ERK1/2 antibody (p-ERK1/2), which reflects MEK1/2 activity. p38 MAPK activity was assessed by in vitro phosphorylation of hsp27, as described in Methods. Data are representative of 3 experiments.

Figure 6

Figure 6. Mutation of p47phox -Ser345 to Ala inhibits the priming process in HL-60 cells.

HL-60 cells were transfected with a plasmid that encodes WT p47phox or p47phox in which Ser345 was mutated to alanine (S345A) and differentiated into neutrophils by DMSO. (A) A total of 5 × 105 nondifferentiated and differentiated cells were lysed and analyzed by SDS-PAGE and Western blotting. p47phox and p67phox were detected with specific antibodies and HRP-labeled goat anti-rabbit antibody. (B) Differentiated HL-60 cells were washed, incubated for 5 hours in RPMI, washed in PBS, then treated with TNF-α (10 ng/ml) or GM-CSF (12 ng/ml) for 15 minutes in Hanks buffer. ROS generation was measured by chemiluminescence after stimulation with fMLP (10–7 M). Data are presented as mean ± SEM (n = 5). *P < 0.05 compared with WT transfected cells.

Figure 7

Figure 7. Effect of a cell-permeable peptide containing the Ser345 sequence on the priming effect of GM-CSF and TNF-α on neutrophil ROS production.

Neutrophils were incubated with a cell-permeable peptide corresponding to amino acids 339–350 (ARPGPQSPGSPL) of p47phox (TAT-peptide-Ser345) or with a scramble peptide (TAT-scramble peptide) linked to TAT peptide, for 30 minutes, then with GM-CSF (A) or TNF-α (B) for 20 minutes, before stimulation with fMLP (10–7 M). ROS production was measured using a luminol-amplified chemiluminescence technique. Data are presented as mean ± SEM (n = 4). *P < 0.05.

Figure 8

Figure 8. Priming of NADPH oxidase activity in neutrophils isolated from synovial fluid of patients with RA.

(A) Resting neutrophils (5 × 105 cells) isolated from blood or from synovial fluid of RA patients were incubated in HBSS in the presence of luminol (10 μM), and spontaneous (without stimulation) chemiluminescence was measured over time. (B) The same preparation was then stimulated with fMLP (10–7 M), and chemiluminescence was measured over time. (C) O2•– production was quantified by cytochrome c reduction assay; control (100%) basal O2•– production was 0.14 ± 0.01 nmol/min/106 cells, and control (100%) fMLP-induced O2•– production was 3.6 ± 0.5 nmol/min/106 cells. Data are presented as mean ± SEM (n = 10). *P < 0.05 compared with control.

Figure 9

Figure 9. Phosphorylation of p47phox on Ser345 and phosphorylation of ERK1/2 and p38 MAPK are increased in neutrophils isolated from synovial fluid of patients with RA.

Resting neutrophils (5 × 105 cells) isolated from blood or from synovial fluid of RA patients were lysed, and proteins were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody, anti–phospho-ERK1/2, or anti–phospho-p38 antibody. Total p47phox, ERK1/2, and p38 MAPK were detected using specific antibody. Results from 3 of 10 RA patients are shown.

Figure 10

Figure 10. The cell-permeable peptide TAT-Ser345 inhibits the basal and primed ROS production by neutrophils from patients with RA.

Neutrophils isolated from RA patients (5 × 105) were incubated with a cell-permeable peptide corresponding to TAT-peptide-Ser345 sequence or with a scramble peptide for 30 minutes, then ROS production was measured with the luminol-amplified chemiluminescence technique in the absence (A) or presence (B) of fMLP (10–7 M). Data are presented as mean ± SEM (n = 3). *P < 0.05.

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