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(The FASEB Journal. 2006;20:1504-1506.)
© 2006 FASEB

Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity

Pham My-Chan Dang1, Carole Elbim, Jean-Claude Marie, Mélanie Chiandotto, Marie-Anne Gougerot-Pocidalo and Jamel El-Benna

INSERM, U773, Centre Hospitalier Universitaire Xavier Bichat; Université Paris 7 Denis Diderot, Site Bichat; and AP-HP, CIB Phenogen, Paris, France

1Correspondence: INSERM U773, Centre Hospitalier Universitaire Xavier Bichat, Faculté de Médecine, 16 rue Henri Huchard, Paris 75018, France. E-mail: dang{at}bichat.inserm.fr

ABSTRACT

Interleukin-10 (IL-10) exerts its anti-inflammatory properties by down-regulating polymorphonuclear neutrophil (PMN) functions such as reactive oxygen species (ROS) production via NADPH oxidase. The molecular mechanisms underlying this process are unclear. Partial phosphorylation of the NADPH oxidase cytosolic component p47PHOX induced by proinflammatory cytokines, such as granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-{alpha}, is essential for priming ROS production by PMN. The aim of this study was to determine whether IL-10 inhibits GM-CSF- and TNF{alpha}-induced p47PHOX phosphorylation and to investigate the molecular mechanisms involved in this effect. We found that IL-10 selectively inhibited GM-CSF- but not TNF{alpha}-induced p47PHOX phosphorylation in a concentration-dependent manner. As GM-CSF-induced p47PHOX phosphorylation is mediated by extracellular signal-regulated kinase 1/2 (ERK1/2), we tested the effect of IL-10 on this pathway. We found that IL-10 inhibited GM-CSF-induced ERK1/2 activity in an immunocomplex kinase assay. This inhibitory effect was confirmed by analyzing the phosphorylation status of the endogenous substrate of ERK1/2, p90RSK, in intact PMN. Furthermore, IL-10 decreased ROS production by adherent GM-CSF-treated PMN in keeping with the higher ROS production observed in whole blood from IL-10 knockout mice compared to their wild-type counterparts. Together, these results suggest that IL-10 inhibits GM-CSF-induced priming of ROS production by inhibiting p47PHOX phosphorylation through a decrease in ERK1/2 activity. This IL-10 effect could contribute to the tight regulation of NADPH oxidase activity at the inflammatory site.—Dang, P.M., Elbim, C, Marie, J.-C., Chiandotto, M., Gougerot-Pocidalo, M.-A., El-Benna, J. Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity.


Key Words: IL-10 • NADPH oxidase • inflammation • ROS

PHAGOCYTE NADPH OXIDASE plays a key role in host defenses against pathogens by generating large amounts of superoxide anion (O2–) and other reactive oxygen species (ROS; ref 1 , 2 ) in a phenomenon known as the respiratory burst. The vital importance of this enzyme is illustrated by a human genetic disorder called chronic granulomatous disease (CGD), which is associated with life-threatening bacterial and fungal infections (3 , 4) . Inappropriate ROS production via NADPH oxidase can also damage host tissues, amplifying the inflammatory response in diseases such as rheumatoid arthritis (5) . This implies that ROS production and, therefore, NADPH oxidase activation must be tightly regulated to ensure ROS are only generated when and where required. In resting human polymorphonuclear neutrophils (PMN), NADPH oxidase exists in an inactive state, with its components distributed in the cytosol and membranes. On PMN exposure to soluble or particulate stimuli, the cytosolic components p47PHOX, p67PHOX, p40PHOX (phox for phagocyte oxidase), and Rac2 migrate to the outer membrane, where they associate with the membrane-bound component cytochrome b558 (which comprises gp91PHOX and p22PHOX), to assemble the active NADPH oxidase (6) . The latter catalyzes NADPH-dependent reduction of oxygen to superoxide anion, a precursor of microbicidal oxidants. Phosphorylation of p47PHOX is an essential step during the activation process. p47PHOX is phosphorylated on several serine residues within the polybasic region of the protein, and these multiple phosphorylation events induce conformational changes that permit p47PHOX to interact with the cytoplasmic tail of p22PHOX and to initiate the formation of the active oxidase (7) .

Proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF{alpha}) are able to prime NADPH oxidase activity in human PMN (8 , 9) . These cytokines induce a very weak oxidative response by themselves but strongly enhance the neutrophil respiratory burst (10 11 12) on exposure to a secondary applied stimulus such as chemotactic peptides. We have recently shown that one shared mechanism by which GM-CSF and TNF{alpha} prime the PMN respiratory burst involves partial phosphorylation of p47PHOX on specific peptides (13 , 14) . This partial phosphorylation may be one way by which these two cytokines accelerate subsequent p47PHOX phosphorylation induced by chemoattractants at sites of inflammation. Interestingly, partial phosphorylation of p47PHOX is observed in synovial fluid (SF) of patients with rheumatoid arthritis, which contains high levels of priming agents such as GM-CSF, TNF{alpha}, and interleukin (IL)-8 (15 , 16) . Furthermore, more superoxide is produced by PMN isolated from SF of arthritis patients than by circulating resting neutrophils (16) .

IL-10, a major anti-inflammatory cytokine, plays an important role in down-regulating inflammatory and immune responses. IL-10 was originally identified as a cytokine synthesis-inhibiting factor (CSIF) produced by T helper 2 (TH2) cells (17) . IL-10 exerts its anti-inflammatory effects on various cell types (TH1 cells, monocytes/macrophages) and also regulates several PMN functional responses (18 , 19) . For instance, studies have shown that IL-10 potently inhibits human neutrophil production of proinflammatory cytokines in response to LPS, LPS+INF-{gamma}, and TNF{alpha} (20 21 22) . IL-10 also augments PMN release of the IL-1 receptor antagonist (23) and reduces PMN expression of the inducible isoform of cyclooxygenase, COX-2, in response to LPS (24) . In addition, IL-10 has been shown to down-regulate polymorphonuclear neutrophils ROS production under certain experimental conditions (25 26 27 28) .

Although IL-10 possesses numerous anti-inflammatory properties, little is known of the molecular mechanisms that lay behind these properties, especially in human PMN. IL-10 binds to a high-affinity cell surface receptor (IL-10R) that is structurally related to IFN receptors (29) . IL-10R is a heterotetramer comprising of two molecules of IL-10R1 and two molecules of IL-10R2 (CFB4/cortico-releasing factor-2) (30 31 32) . Janus kinase-1 (JAK1) and TYK2 are permanently associated with IL-10R1 and IL10-R2, respectively. To date, the literature cites two main signaling cascades that may be triggered by IL-10 in various cell types, namely the JAK1-TYK2/STAT1–3 pathway and the PI-3 kinase/Akt pathway (33 , 34) . However, it is not clear yet whether or not these two pathways are activated in PMN. Circulating PMN express both IL-10R1 and IL-10R2 on their surface, but IL-10R1 is weakly expressed on resting PMN compared to monocytes and lymphocytes (35 , 36) . Resting PMNs contain an intracellular pool of IL-10 receptors located in specific granules (37) . In freshly isolated PMN, the janus-activated kinase (JAK)/STAT pathway is not activated by IL-10, even though JAK1 and TYK2, and also STAT1 and STAT3 can be detected in PMN (35 , 38) . A recent study showed that this might be due to the weak expression of IL-10R1 by freshly isolated PMN. Indeed, when PMN were cultured with LPS for 3 h, IL-10R1 cell surface expression increased, rendering PMN fully responsive to IL-10 in terms of JAK/STAT pathway activation (36 , 39) .

To further examine the pro- and anti-inflammatory dynamic equilibrium at inflammatory sites, we investigated the effects of IL-10 on GM-CSF- and TNF{alpha}- treated PMN focusing on p47PHOX phosphorylation, since this event is critical for NADPH oxidase activation and therefore ROS production by PMN. We also examined the molecular mechanisms potentially involved in this effect.

MATERIALS AND METHODS

Reagents
ATP{gamma} [32P] and 32P-orthophosphoric acid were from NEN Life Science Products (Boston, MA). Formyl-methionyl-leucyl-phenylalanine (fMLP), ATP, myelin basic protein (MBP), proteases and phosphatase inhibitors were from Sigma (St. Louis, MO). Kinases inhibitors were from Calbiochem (La Jolla, CA). Endotoxin-free buffers and salt solutions were from Life Technologies (Cergy-Pontoise, France). Reagents for SDS-PAGE were from Bio-Rad (Richmond, CA). Injection-grade water and 0.9% NaCl were endotoxin-free (<0.4 pg/ml) in the limulus test (Charles River, Charlestoe, SC). Recombinant human (rh) GM-CSF, rhIL-10, and rhTNF were from PeproTech (Rocky Hill, NJ). RhIL-4, rh IL-13, and mouse anti-human IL10-R1 monoclonal antibody (mAb) were from R&D (Minneapolis, MN). Rabbit polyclonal antibody (pAb) against p47PHOX (from Dr. B. M. Babior, Scripps Research Institute, CA) was raised against the 10 COOH-terminal residues of p47PHOX and purified as described by Babior’s lab (40) . Mouse anti-human phospho-p44/42 mitogen-activated kinase (Thr202/Tyr204) (p44/42 MAPK) mAb, rabbit antiphospho-STAT3 (Tyr705) pAb, rabbit antiphospho-STAT5 (Tyr694) pAb, rabbit anti-STAT3 pAb, and rabbit anti-human p90 ribosomal S6 kinase (p90RSK) pAb were from Cell Signaling Technology (Beverly, MA). Rabbit antip44/42 MAPK polyclonal antibodies, rabbit anti-STAT5 pAb, rabbit antiphospho-p90RSK (Thr359/Ser-363) pAb, irrelevant isotypic IgG control antibody (Ab) (mouse IgG 1), and rabbit polyclonal IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). C57BL/6 IL-10 (–/–) and C57BL/6 IL10 (+/+) mice were purchased from IFFA CREDO (Saint-Germain sur l’Arbresle, France). R-phycoerythrin (PE)-conjugated monoclonal mouse anti-human CD11b Ab was from Dakopatts (Glostrup, Denmark).

PMN preparation
PMN were obtained from whole blood of healthy human donors in agreement with EFS (Etablissement Français du Sang) guidelines (no. 03/EFS/244). PMN were isolated in LPS-free conditions by Dextran sedimentation and Ficoll-Hypaque centrifugation as described previously (13) .

32P-labeling, stimulation, and fractionation of PMN
PMN were incubated in phosphate-free buffer (20 mM HEPES pH 7.4, 140 mM NaCl, 5.7 mM KCl, 0.8 mM MgCl2, and 0.025% BSA) (13) containing 0.5 mCi of [32P]orthophosphoric acid/108 cells/ml for 60 min at 30°C. PMNs at a concentration of 25 x 106 cells/ml were then incubated at 37°C for 5 min before treatment with rhGM-CSF for 20 min in the presence or absence of rhIL-10, rhIL-4, and rhIL-13 at the indicated concentrations. The reaction was stopped by adding ice-cold buffer and by centrifugation at 400 g for 7 min at 4°C. Cells were lysed by resuspending them in lysis buffer, as described previously (13) . The suspension was sonicated on ice for 3 x 15 s. The lysate was centrifuged at 100,000 g for 20 min at 4°C in a TL100 Ultracentrifuge (Beckman). Where indicated, after 32P-loading, cells were let to adhere for 1 h 30 min on plastic before treatment with rhGM-CSF, rhIL-10, rhIL13, and rhIL4.

Immunoprecipitation and Western blotting
For p47PHOX immunoprecipitation, the supernatant obtained after the 100,000 g centrifugation step, was incubated overnight with antip47phox (1/200); protein was then immunoprecipitated using gamma-bind G-Sepharose beads (Amersham Pharmacia Biotech) and washed four times as described previously (13 , 14) . The samples were then subjected to SDS-PAGE in 10% polyacrylamide gels, using standard techniques (41) . The separated proteins were transferred to nitrocellulose with Towbin’s procedure (42) . After being blocked with 5% fat dry milk in borate-buffered saline pH 8.4 (100 mM boric acid, 25 mM borax, and 75 mM NaCl) for 1 h at room temperature, the membrane was probed with antip47phox (1/5000 dilution) and then with alkaline phosphatase-labeled goat anti-rabbit Ab before NBT/BCIP staining. The intensity of the p47PHOX band was quantified by densitometry using the Scion image analysis program from the National Insitutes of Health. For 32P-based quantification (cpm) of p47PHOX, nitrocellulose membranes were analyzed in an Instant Imager apparatus (Packard) equipped with Instant Imager software. Radioactivity counts were corrected for the actual amount of p47PHOX present on the membrane. For direct detection of phospho-Stat3 (Tyr705), phospho-Stat5 (Tyr694), and phospho-p90RSK (Thr359/Ser-363), 25 x 106 PMN/ml were incubated for 20 min at 37°C with buffer alone, rhIL-10 (100 ng/ml), rhGM-CSF (15 ng/ml), or rhGM-CSF (15 ng/ml) plus rhIL-10 at various concentrations, in a final volume of 500 µl. The reaction was stopped by adding 125 µl of 5x Laemmli’s sample buffer preheated to 100°C (312.5 mM Tris-HCl pH 6.8, 20% SDS, 25% ß-mercaptoethanol, 12.5 mM orthovanadate, 50 mM paranitrophenylphosphate, 50 µg/ml leupeptin, 50 µg/ml aprotinin, and 0.125% bromphenol blue). The samples were then boiled for 10 min to completely denature the proteins, before electrophoresis and electroblotting as described above. The membranes were blocked for 1 h at room temperature in TBS/T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) containing 5% BSA and then incubated overnight with the phospho-specific primary Ab. The following dilutions were used: phospho-Stat3 (Tyr705) 1:500, phospho-Stat5 (Tyr694) 1:500, and phospho-p90RSK (Thr359/Ser-363) 1:500. Ab binding was detected using horseradish peroxidase-conjugated antimouse or anti-rabbit IgG. Blots were visualized using enhanced chemiluminescence Western blotting reagents (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were then reprobed with anti-STAT3, anti-STAT5, and antip90RSK, and blots were visualized by NBT/BCIP staining to check that equal amounts of protein had been loaded in each well.

CD11b expression at the PMN surface
Isolated PMN were incubated at 37°C with PBS, 60 ng /ml rhIL-10, 15 ng/ml rhGM-CSF, or 15 ng/ml rhGM-CSF plus 60 ng/ml rhIL-10 for 30 min. Samples (100 µl) were stained at 4°C for 30 min with PE-anti-human CD11b Ab. After one wash in ice-cold PBS, PMN were resuspended in 2% paraformaldehyde and analyzed by flow cytometry using a Becton-Dickinson FACScalibur with a 15 mW, 488 nM argon laser. Forward and side scatter were used to identify the granulocyte population and to gate out other cells and debris; 10,000 events were counted per sample. All results were obtained with a constant photomultiplier gain. The data were analyzed using CellQuest software (BD Biosciences), and mean fluorescence intensity (MFI) was used to quantify the response.

MAP kinase activity
MAP kinase activity was measured as described by Nijhuis et al. (43) . PMN were prepared as described above and stimulated at a concentration of 25 x 106 cells/ml with 15 ng/ml rhGM-CSF in the presence or absence of various concentration of rhIL-10. PMN were then lysed in 50 mM Tris-HCl, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM ß-glycerophosphate, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM benzamidine. Where indicated, PMN were pretreated with PD98059 (50 µM) for 30 min before stimulation. An aliquot of the lysate was saved for Western blotting with anti-human phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibodies (1:1000) and rabbit antip44/42 MAPK polyclonal antibodies (1:1000) as described above. The remaining lysate was precleared for 30 min with protein A agarose (Pierce Biotechnology, Rockford, IL),and MAP kinase was then immunoprecipitated with 1 µg of p44/42MAPK antibodies or an irrelevant isotypic IgG control for 1 h at 4°C on a rotating wheel. Protein A agarose was then added and immunoprecipitation was allowed to proceed for a further 1 h on the rotating wheel. The beads were then washed twice with lysis buffer and twice with kinase buffer (30 mM Tris-HCl pH 8, and 20 mM MgCl2). The kinase assay was performed by incubating the immunoprecipitation pellet in 25 µl of kinase buffer containing 10 µM ATP, 10 µg MBP, or 10 µg of recombinant p47PHOX and 0.5 µCi [{gamma}32P] ATP for 20 min at room temperature. The reaction was stopped by adding 2x sample buffer. The samples were separated on 13% polyacrylamide gels, and MBP and p47PHOX phosphorylation were detected with a PhosphorImager and autoradiography.

Preparation of phosphorylated p47PHOX
Recombinant glutathione (GSH) S-transferase (GST)-p47PHOX, produced as described by Dang et al. (44) , was coupled to GSH-Sepharose beads. GST-p47PHOX (30 µg) was then phosphorylated on the beads with active recombinant ERK2 from New England Biolabs, Inc (Beverly, MA) in a 40 µl reaction mixture containing 40 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM DTT, and 100 µM ATP (1 µCi/assay) at 30°C for 1 h. The reaction was stopped and recombinant ERK2 was removed by washing the beads 4 times with phosphatase buffer (50 mM Tris-HCl, 0.1 mM EDTA, 10 mM ß-mercaptoethanol, and 0.1% BSA).

Dephosphorylation assay
Dephosphorylation was assayed in phosphatase buffer by incubating 32P-labeled GST-p47PHOX coupled to beads with the cytosolic fraction of IL10-treated PMN or untreated PMN for 1 h at 30°C. The reaction was stopped by adding 2x Laemmli’s sample buffer, and SDS-PAGE was then performed. Dephosphorylation was monitored by autoradiography on the basis of the loss of 32P from phospho-GST-p47PHOX.

Superoxide anion production assay
PMN adherent to plastic were treated with 12.5 ng/ml rhGM-CSF in the presence or absence of various concentrations of rhIL-10, rhIL-4, or rhIL-13 at 37°C for 30 min in sterile Hanks’ buffered salt solution. Total superoxide production was then measured at an end point in the superoxide dismutase (SOD)-inhibitable ferricytochrome c reduction assay (13 , 14) . Superoxide production in whole-blood samples from IL10 (–/–) and IL-10 (+/+) mice was measured in terms of chemiluminescence, using the ABEL cell activation test kit with pholasin from Knight Scientific Limited (Plymouth, UK), as recommended by the manufacturer.

Statistical analysis
All results are mean ± SE. Significant differences were identified with Student’s t test (significance threshold P<0.05).

RESULTS

IL-10 inhibits GM-CSF- but not TNF{alpha}-induced p47PHOX phosphorylation in human PMN
Phosphorylation of p47PHOX is one of the key intracellular events associated with NADPH oxidase activation (45) . We have recently shown that one of the shared mechanisms by which proinflammatory cytokines such as GM-CSF and TNF{alpha} prime the PMN respiratory burst involves partial phosphorylation of p47PHOX on one major peptide (14) . In contrast, IL-10 (IL-10) exerts its anti-inflammatory properties by down-regulating PMN functions such as ROS production (25 26 27 28) but the molecular mechanisms underlying this effect are unknown. We thus examined the effect of IL-10 on GM-CSF- and TNF{alpha}-induced p47PHOX phosphorylation in human PMN. 32P-loaded PMN were incubated with GM-CSF or TNF{alpha} in the presence or absence of IL-10 for 20 min at 37°C. The cells were then lysed, and p47PHOX was immunoprecipitated and analyzed by autoradiography and Western blot. In the absence of added cytokines, weak basal phosphorylation of p47PHOX was detected. IL-10 alone did not significantly modify this basal phosphorylation (6069±747 vs. 6046±1045 cpm in the absence or presence of IL-10 respectively, n=5; Fig. 1 A, left panel). GM-CSF induced marked phosphorylation of p47PHOX, and this effect was significantly inhibited by IL-10 (Fig. 1A , left panel). Indeed, GM-CSF increased p47PHOX phosphorylation by 13 716 cpm ± 1746 and IL-10 decreased p47PHOX phosphorylation to 6150 ± 1214 cpm (P<0.05, n=5). We also examined if this inhibitory effect on GM-CSF-induced p47PHOX phosphorylation was also exerted by IL-4 and IL-13, two other cytokines with anti-inflammatory properties (46) . These two cytokines had no significant inhibitory effect on GM-CSF-induced p47PHOX phosphorylation (Fig. 1A , middle and right panel). We thus focused on IL-10 and showed that its inhibitory effect on GM-CSF-induced p47PHOX phosphorylation was concentration-dependent (Fig. 1B ). Western blots using an Ab directed against p47PHOX showed that the same amount of protein had been loaded in each well.


Figure 1
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  		Figure 1. Effect of IL-10, IL-4, and IL-13 on p47PHOX phosphorylation. A) 32Pi-labeled PMN were incubated with GM-CSF (12.5 ng/ml) in the presence or absence of IL-10 (30 ng/ml), IL-4 (30 ng/ml), or IL-13 (30 ng/ml) at 37°C for 20 min. P47PHOX was then immunoprecipitated with a specific Ab, subjected to SDS-PAGE, blotted on nitrocellulose and detected by autoradiography (P-p47PHOX) or with antip47PHOX Ab (p47PHOX). Data represent 3 experiments. B, left) PMN were incubated with GM-CSF (12 ng/ml) in the presence or absence of various concentrations of IL-10 at 37°C for 20 min. P47PHOX was then detected by autoradiography (P-p47PHOX) or with antip47PHOX Ab (p47PHOX). Right) Graph represents ratio of phosphorylated p47PHOX quantified by phosphorImager analysis to the total amount of p47PHOX, which was quantified by densitometry (mean±SE, n=3; #P<0.05 vs. unstimulated PMN, *P<0.05 vs. GM-CSF-treated PMN in the absence of IL-10). C, left) PMN were incubated with GM-CSF (12 ng/ml) or TNF{alpha} (4.5 ng/ml) in the presence or absence of IL-10 (30 ng/ml) at 37°C for 20 min. P47PHOX was then detected by autoradiography (P-p47PHOX) or with antip47PHOX Ab (p47PHOX). Right) Graph represents ratio of phosphorylated p47PHOX quantified by phosphorImager analysis to total amount of p47PHOX, which was quantified by densitometry (mean±SE, n=5; #P<0.05 vs. unstimulated PMN, *P<0.05 vs. GM-CSF-treated PMN in the absence of IL-10).

GM-CSF and TNF{alpha} are the most potent cytokines in terms of PMN oxidative burst priming. However, IL-10 did not have a significant inhibitory effect on TNF{alpha}-induced p47PHOX phosphorylation, even though it inhibited GM-CSF-induced p47PHOX phosphorylation in the same experiment (Fig. 1C ). This suggests that inhibitory effect of IL-10 on p47PHOX phosphorylation is selective for GM-CSF and may target a specific pathway activated during GM-CSF priming but not during TNF{alpha} priming.

To determine whether IL-10 could only modulate selective neutrophil functions or the global response to GM-CSF, we assessed its effect on GM-CSF-induced CD11b expression at the PMN surface. As shown in Fig. 2 , this effector response was not modified by IL-10, suggesting that IL-10 inhibits selective(s) pathway(s) leading to p47PHOX phosphorylation in PMN. Since GM-CSF induces neither p67PHOX phosphorylation (data not shown) nor Rac 2 activation (47) , we did not analyze the effect of IL-10 on these events known to participate in NADPH oxidase activation.


Figure 2
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  		Figure 2. Effect of IL-10 on CD11b expression at the PMN surface. Isolated PMN were incubated at 37°C with PBS, 60 ng /ml IL-10, 15 ng/ml GM-CSF, or 15 ng/ml GM-CSF plus 60 ng/ml IL-10 for 30 min. Samples (100 µl) were stained at 4°C for 30 min with PE-anti-human CD11b and analyzed by flow cytometry. MFI was recorded as described in Materials and Methods (mean±SE, n=3, *P<0.05 vs. unstimulated PMN).

Molecular mechanisms of IL-10-mediated inhibition of p47PHOX phosphorylation in GM-CSF-treated PMN
We next examined the molecular mechanisms of IL-10-mediated inhibition of p47PHOX phosphorylation in GM-CSF-treated PMN. In human PMN, GM-CSF treatment induces selective activation of the ERK1/ERK2 pathway (48) and the JAK2/STAT3 and/or STAT5 pathway (38 , 49) . As we had recently shown that ERK1/ERK2 are involved in direct p47PHOX phosphorylation on Ser-345 in GM-CSF-treated PMN (50) and that this was the only phosphorylation site induced by GM-CSF on p47PHOX, we tested the effect of IL-10 on this pathway. We examined the activation status of ERK1/ERK2 first by using monoclonal antibodies directed against the activated form of ERK (phosphorylated on residues Y204/T202), and second by in vitro MAP kinase activity assay with myelin basic protein (MBP) as substrate, after ERK1/ERK2 immunoprecipitation. As shown in Fig. 3 A, GM-CSF induced sustained ERK1/ERK2 phosphorylation and IL-10 did not inhibit this effect at any time point studied. Also, IL-10 did not inhibit GM-CSF-induced ERK1/2 phosphorylation at concentrations between 25 and 100 ng/ml (Fig. 3B ). Interestingly, however, IL-10 significantly inhibited GM-CSF-induced ERK1/ERK2 activity, in a concentration-dependent manner, when measured in vitro with the MAP kinase assay after ERK1/ERK2 immunoprecipitation and phosphate incorporation into MBP (Fig. 3C ). In addition, when recombinant p47PHOX was used as substrate, direct phosphorylation of p47PHOX by the ERK1/2 immunoprecipitate was observed in a manner that was inhibitable by cell pretreatment with IL-10 (Fig. 3D , lower panel). We then checked that the MBP and p47PHOX phosphorylating activities measured was due to ERK1/ERK2 activity and not to nonspecific activity precipitated by the anti-ERK1/ERK2 antibodies. For this purpose, immunoprecipitation was performed using an irrelevant isotypic IgG control Ab. In these conditions, neither MBP nor p47PHOX phosphorylating activities were detected in the immunoprecipitate (Fig. 3D , upper and lower panel). Also, when PMN were pretreated with PD98059, an inhibitor of MEK1/2 (the kinase upstream of ERK1/ERK2), the activity recovered in the precipitate was completely inhibited (Fig. 3C ). This demonstrated that the activity measured in the precipitate was indeed ERK1/ERK2 and not a nonspecific activity precipitated by the anti-ERK1/ERK2 antibodies. Furthermore, to confirm that IL-10 also inhibited ERK1/ERK2 activity in intact GM-CSF-treated PMN, we examined the phosphorylation status of p90 ribosomal S6 kinase (p90RSK), which is an endogenous substrate of ERK1/2 in mature hematopoietic cells stimulated by GM-CSF (51) . To analyze the phosphorylation state of p90RSK, we thus used an Ab directed against the ERK1/2-consensus phosphorylation site of p90RSK (antiphospho-p90RSK Thr359/Ser-363). The results showed that GM-CSF induced phosphorylation of p90RSK on Thr359/Ser-363), and that IL-10 significantly inhibited this phosphorylation, in a concentration-dependent fashion (Fig. 4 A, B). Furthermore, the inhibitor of MEK1/2 (PD98059 and UO126) but not the inhibitor of p38MAPK (SB203580) prevented GM-CSF-induced phosphorylation of p90RSK, confirming the involvement of ERK1/ERK2 in this phosphorylation (Fig. 4C ). These results show that IL-10 inhibits ERK1/ERK2 activity in intact GM-CSF-stimulated PMN, and that this inhibition involves neither ERK1/ERK2 dephosphorylation nor inhibition of MEK1/2 activity and related upstream pathways.


Figure 3
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  		Figure 3. IL-10 inhibits GM-CSF-induced ERK1/ERK2 activity without affecting ERK phosphorylation status. A) PMN (25x106/ml) were incubated with buffer alone or with IL-10 (50 ng/ml), GM-CSF (15 ng/ml), or GM-CSF (15 ng/ml) plus IL-10 (50 ng/ml) for various times at 37°C. Western blots were performed with anti-human phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibodies as described in Material and Methods (P-ERK). B, top) PMN (25x106/ml) were stimulated at 37°C for 20 min with 15 ng/ml GM-CSF in the presence or absence of various concentrations of IL-10. Cells were lysed as described in Material and Methods. Western blots were performed with anti-human phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibody as described in Materials and Methods (P-ERK). Membranes were then reprobed with anti-ERK1/2 antibodies (ERK1/2). Bottom) Graph represents ratio of optical density of phosphorylated ERK1/2 to total amount of ERK1/2 (mean±SE, n=4, #P<0.05 vs. unstimulated PMN). C, top) PMN (25x106/ml) were stimulated with 15 ng/ml GM-CSF at 37°C for 20 min, in the presence or absence of various concentrations of IL-10. Where indicated, PMNs were pretreated with PD98059 (50 µM) for 30 min before stimulation. Cells were lysed, ERK1/2 was immunoprecipitated, and kinase assay was performed in the presence of MBP and [{gamma}32P]ATP for 20 min at room temperature as described in Materials and Methods. Reaction was stopped by adding 2x sample buffer. Samples were separated on 13% polyacrylamide gels and MBP phosphorylation was detected by autoradiography (P-MBP). Bottom) Phosphorylated MBP was quantified by PhosphorImager analysis (mean±SE, n=4, #P<0.05 vs. unstimulated PMN, *P<0.05 vs. GM-CSF-treated PMN in the absence of IL-10). D) PMN (25x106/ml) were stimulated with 15 ng/ml GM-CSF at 37°C for 20 min in the presence or absence of various concentrations of IL-10. Cells were lysed and immunoprecipitation was performed using anti-ERK1/2 antibodies (IP anti-ERK1/2) or an irrelevant isotypic IgG control Ab (IP IgG control). Kinase assay was performed with MBP (top) or recombinant p47PHOX (bottom) as substrate. Reaction was stopped by adding 2x sample buffer. Samples were separated on 13% polyacrylamide gels. MBP and p47PHOX phosphorylation were detected by autoradiography (P-MBP and P-p47PHOX, respectively).


Figure 4
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  		Figure 4. IL-10 inhibits GM-CSF-induced phosphorylation of p90RSK at the ERK1/2-consensus phosphorylation site. A) PMN (25x106/ml) were stimulated at 37°C for 20 min with 15 ng/ml GM-CSF in the presence or absence of various concentrations of IL-10. Western blots were performed with an Ab directed against the ERK1/2-consensus phosphorylation site of p90RSK [antiphospho-p90RSK Thr359/Ser-363 as described in Materials and Methods (P-RSK)]. Membranes were then reprobed with antip90RSK Ab (RSK). B) Graph represents ratio of OD of phosphorylated p90RSK to total amount of p90RSK (mean±SE, n=4, #P<0.05 vs. unstimulated PMN, *P<0.05 vs. GM-CSF-treated PMN in the absence of IL-10). C) PMN were treated as in A, except that where indicated they were incubated for 30 min with PD 98059 (50 µM), UO126 (10 µM), or SB203580 (5 µM) before stimulation with GM-CSF.

Early signals triggered by GM-CSF binding to its receptor involves activation of JAK2/STAT3-STAT5 pathway. In keeping with the above results, we checked the effect of IL-10 on the JAK2/STAT3-STAT5 pathway. IL-10 did not inhibit the activation of the JAK/STAT-3 or STAT5 pathway, as shown by the use of antibodies directed against Y705-phosphorylated STAT-3 and Y694-phosphorylated STAT-5 (Fig. 5 A, B). Little or no phosphorylation of STAT-3 and STAT-5 was observed in control and IL-10-treated PMN. GM-CSF induced STAT-3 and STAT-5 phosphorylation, and IL-10 did not inhibit this effect (Fig. 5A, B ).


Figure 5
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  		Figure 5. IL-10 does not inhibit GM-CSF-induced STAT3 or STAT5 phosphorylation. PMN (25x106/ml) were incubated for 20 min at 37°C with buffer alone, or with IL-10 (50 ng/ml), GM-CSF (15 ng/ml), or GM-CSF (15 ng/ml) plus IL-10 (50 ng/ml). Cells were lysed as described in Materials and Methods. Proteins were then separated by SDS-PAGE and transferred to nitrocellulose. A, upper) Western blots were performed with phospho-Stat3 (Tyr705; P-STAT3) polyclonal antibodies as described in Materials and Methods. Membranes were then reprobed with anti-Stat3. lower) Graph represents ratio of OD of phosphorylated Stat3 to total amount of Stat3 (mean±SE, n=4, #P<0.05 vs. unstimulated PMN). B, upper) Western blots were performed with phospho-Stat5 (Tyr694; P-STAT5) polyclonal antibodies as described in Materials and Methods. Membranes were then reprobed with anti-Stat5. lower) Graph represents ratio of OD of phosphorylated Stat5 to the total amount of Stat5 (mean±SE, n=4, #P<0.05 vs. unstimulated PMN).

The above results suggested that IL-10 inhibited p47PHOX phosphorylation by decreasing ERK1/ERK2 activity in GM-CSF-stimulated PMN. However, we could not exclude the possibility that IL-10 may induce a phosphatase activity that directly dephosphorylates Ser-345 on p47PHOX. We thus performed a dephosphorylation assay in vitro, using ERK2-phosphorylated p47PHOX as a substrate for phosphatases. Indeed, in vitro phosphorylation of p47PHOX by ERK2 has been shown to occur also on Ser-345 by comparing the two-dimensional phosphopeptides maps of GM-CSF-induced p47PHOX phosphorylation in PMN and of in vitro ERK2-phosphorylated p47PHOX (data not shown). The experiment was carried out according the following procedure; p47PHOX was phosphorylated in vitro by ERK2, then isolated and incubated with the cytosolic fraction of IL-10-treated or untreated PMN. Basal phosphatase activity that dephosphorylated p47PHOX was observed in the cytosol of untreated cells (Fig. 6 , lane 2) as compared to buffer (Fig. 6 , lane 1). No increase in phosphatase activity was observed after IL-10 treatment (Fig. 6 , lane 3): on the contrary, IL-10 abrogated the basal phosphatase activity observed in untreated cells. This ruled out the possibility that IL-10 induces a phosphatase activity that directly dephosphorylates p47PHOX. The phosphatase activity was examined in the cytosolic fraction because phosphorylation of p47PHOX induced by GM-CSF occurs in the cytosol (13) .


Figure 6
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  		Figure 6. Inhibition of p47PHOX phosphorylation does not involve an IL-10 induced-phosphatase activity. 32P-labeled GST-p47PHOX was prepared as described in Materials and Methods. Dephosphorylation assay was performed in phosphatase buffer by incubating 32P-labeled GST-p47PHOX with the cytosolic fraction of IL-10-treated PMN (lane 3) or cytosolic fraction of untreated PMN (lane 2) for 1 h at 30°C; (lane 1) is control corresponding to 32P-labeled GST-p47PHOX incubated with buffer for 1 h at 30°C. Reaction was stopped by adding 2x Laemli’s sample buffer, then samples were subjected to SDS-PAGE and proteins were transferred to a nitrocellulose sheet. Dephosphorylation was monitored by autoradiography on the basis of loss of 32P from phospho-GST-p47PHOX (P-p47PHOX). Western blot (p47PHOX) was used to check that same amount of GST-p47PHOX was loaded in each well. Experiments were performed twice.

IL-10 negatively regulates ROS production in vitro and in vivo
To determine whether IL-10-mediated inhibition of p47PHOX phosphorylation prevented ROS production in GM-CSF-treated PMN, we used experimental conditions allowing us to measure both events. As GM-CSF does not induce NADPH oxidase activity in suspended PMN (10) , the effect of IL-10 on ROS production was studied in adherent GM-CSF-treated PMN. IL-10 inhibited ROS production in this system in a concentration-dependent manner (Fig. 7 A). In contrast, IL-4 and IL-13, which have no effect on GM-CSF-induced p47PHOX phosphorylation (see above), had no significant effect on GM-CSF-induced superoxide production (Fig. 7B and C ). Furthermore we checked that in the above adherent conditions GM-CSF-induced p47PHOX phosphorylation was inhibited by IL-10. Figure 8 shows that IL-10 inhibits GM-CSF-induced p47PHOX phosphorylation in adherent PMN in a concentration-dependent manner, as in suspended PMN. The effect of IL-10 on NADPH oxidase activity was not measured in suspended PMN in the priming conditions (i.e., priming by GM-CSF and activation by fMLP, because it has been shown that IL-10 directly altered the fMLP response) (52) . Thus, it would have been very difficult to distinguish between the effect of IL-10 on GM-CSF response or fMLP response. Taken together, these results suggest that the inhibitory effect of IL-10 on ERK1/2 activity and p47PHOX phosphorylation may mediate IL-10 down-regulation of ROS production in PMN.


Figure 7
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  		Figure 7. Effect of IL-10, IL-4, and IL-13 on ROS production. PMN adherent to plastic (5x106 cells/well) were treated with GM-CSF (12.5 ng/ml) in the presence or absence of various concentrations of IL-10 (A), IL-4 (B), or IL-13 (C) at 37°C for 30 min in sterile Hanks’ buffered salt solution. Total superoxide production was then measured at an end point in the SOD-inhibitable ferricytochrome c reduction assay as described in Materials and Methods (mean±SE, n>3, *P<0.05). D) ROS production in whole-blood from IL-10 (–/–) mice and IL-10 (+/+) was measured by chemiluminescence using the ABEL cell activation test kit for whole blood with pholasin, as recommended by manufacturer.


Figure 8
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  		Figure 8. GM-CSF-induced P47PHOX phosphorylation is also inhibited by IL-10 in adherent PMN. After being loaded with 32Pi, PMNs were let to adhere on plastic, after which they were treated with GM-CSF (15 ng/ml) in the presence or absence of various concentrations of IL-10 at 37°C for 20 min. P47PHOX was then immunoprecipitated as indicated in Materials and Methods. Data represent 3 experiments.

We also compared ROS production in IL-10 knockout mice (IL-10 –/–) and IL-10 wild type mice (IL-10 +/+) to determine if the absence of IL-10 in vivo could alter ROS production. To maintain PMN in the physiological in vivo cytokine environment of these mice, we decided to use a respiratory burst assay test kit designed for whole blood with pholasin. Pholasin chemiluminescence has been shown to detect mostly superoxide anion released from PMN (52) . Leukocytes from IL-10 (–/–) mice showed higher basal ROS production than leukocytes from IL-10 (+/+) mice, indicating that IL-10 is crucial for negative regulation of ROS production in vivo (Fig. 7D ).

DISCUSSION

Most IL-10 effects on PMN are anti-inflammatory, such as inhibition of LPS-induced proinflammatory cytokine production, prostanoid and platelet-activating factor release (23 24 25) . Inhibition of ROS production has also been observed depending on the stimuli and experimental conditions used (25 26 27 28) ; however the molecular mechanisms underlying this process are unclear. This study sheds light on the molecular mechanisms by which IL-10 inhibits ROS production, a major proinflammatory and bactericidal function of neutrophils. We show for the first time that IL-10 selectively inhibits GM-CSF-induced p47PHOX phosphorylation in a concentration-dependent manner in suspended and adherent neutrophils but does not alter TNF{alpha}- induced p47PHOX phosphorylation. This IL-10-induced inhibition of p47PHOX phosphorylation is due to inhibition of ERK1/2. In parallel, IL-10 reduces ROS production by adherent GM-CSF-treated PMN in keeping with the higher ROS production observed in IL-10 knockout mice compared to their WT counterparts.

Although substantial advances have been made in our understanding of the processes leading to NADPH oxidase activation, the molecular mechanisms involved in NADPH oxidase inactivation are elusive. Inactivation of NADPH oxidase is critical for timely resolution of inflammation, before bystander tissue damage occurs. Rapid inactivation of NADPH oxidase could be due to several processes, including receptor desensitization, uncoupling of transduction pathways, and modulation of the phosphorylation of NADPH oxidase components such as p47PHOX and p67PHOX. An earlier study showed that termination of the respiratory burst during phagocytosis coincided not with dephosphorylation of membrane-associated p47PHOX but with further phosphorylation (54) . A role of phosphatases PP1 and PP2A has also been raised (55) . The results obtained here point to a mechanism that involves inhibition of p47PHOX phosphorylation in GM-CSF-stimulated neutrophils treated with IL-10. However, no phosphatase activity appears to be associated with this inhibition, as we found that IL-10 did not induce a phosphatase activity that could directly dephosphorylate p47PHOX. Since GM-CSF-induced p47PHOX phosphorylation is mediated by ERK1/2 (50) , we tested the effect of IL-10 on this pathway and found that IL-10 inhibited ERK1/2 activity; this was demonstrated in an in vitro MAP kinase assay and by analyzing the phosphorylation status of p90RSK with an Ab directed against the ERK1/2-consensus phosphorylation site of p90RSK (antiphospho-p90RSK Thr359/Ser-363. IL-10-mediated inhibition of ERK1/2 activity has already been observed in LPS- and CD40-stimulated monocytes (56 , 57) . In these earlier studies, however, contrary to our findings, this inhibition was always associated with ERK1/2 dephosphorylation. The results obtained here show that inhibition of ERK1/2 activity is not necessarily associated with lesser ERK1/2 phosphorylation, suggesting that neither MEK1/2 nor MAPK phosphatase is affected by IL-10. Indeed, IL-10 inhibited ERK1/2 activity in an in vitro MAP kinase assay and also inhibited phosphorylation of endogenous p90RSK at the ERK1/2 consensus site, but it did not inhibit ERK1/2 phosphorylation. The inhibition of ERK1/ERK2 activity might occur at a postphosphorylation step. For example, IL-10 might induce one or several inhibitory proteins that interact with phosphorylated ERK1/ERK2, and this might prevent ERK1/ERK2 access to their substrates, such as p47PHOX and p90RSK. This is currently under investigation in our laboratory. The ERK1/2 cascade is regulated through multiple interactions with scaffold proteins, which permit precise regulation of ERK1/2 signaling (58) . Recently, Sur et al. (59) identified a protein called vanishin, which binds ERK and prevents its activation by MEK. This type of regulation might represent an alternative to ERK inactivation by dual-specificity phosphatases (MKP) that remove phosphates from Tyr alone, Thr alone, or both residues together, in the ERK activation loop (60) . It might also permit fine regulation of ERK activity.

In a previous study, we showed that PMN treatment with GM-CSF and TNF{alpha} resulted in partial phosphorylation of p47PHOX on the same site (14) , suggesting that the two cytokines triggered pathways converging on a common serine, which we have now identified as Ser-345. However, inhibition of p47PHOX phosphorylation by IL-10 was observed in GM-CSF-stimulated PMN but not in TNF{alpha}-stimulated PMN. This might be explained by the fact that GM-CSF and TNF{alpha} engage different pathways to phosphorylate p47PHOX on Ser-345 and that only the pathway induced by GM-CSF could be inhibited by IL-10. Data obtained from our laboratory support this hypothesis. Indeed, we and others (61 62 and data not shown) have found that in human neutrophils, TNF{alpha} activates p38 MAPK but not (or weakly) ERK1/2 while GM-CSF activates ERK1/2 but not (or weakly) p38MAPK. In addition, we have shown that ERK1/2 mediates GM-CSF-induced phosphorylation of p47PHOX while p38MAPK controls TNF{alpha}-induced phosphorylation of p47PHOX at the same site (Ser-345) (50) . Finally, we recently obtained evidence that IL-10 cannot inhibit TNF{alpha}-induced p38MAPK activity (data not shown). Thus, IL-10 exerts its inhibitory effect mainly at the concentration of ERK1/ERK2 (not p38MAPK), likely explaining why no inhibition of p47PHOX phosphorylation was observed in TNF{alpha}-stimulated PMN (phosphorylation of p47PHOX being mediated by p38MAPK under TNF{alpha} stimulation).

We recently showed that GM-CSF-induced p47PHOX phosphorylation is mediated by ERK1/2 and that this event is directly related to the priming effect of GM-CSF on superoxide production (50) . IL-10-mediated inhibition of p47PHOX phosphorylation might thus be one mechanism by which this anti-inflammatory cytokine down-regulates GM-CSF-induced superoxide production priming. The ability of IL-10 to inhibit ROS production may depend on the stimuli and experimental conditions used (25 26 27 28) , since conflicting data have been observed (63) . Here we provide evidence that IL-10 down-regulates superoxide production in parallel with inhibition of p47PHOX phosphorylation. Thus, treatment of adherent GM-CSF-stimulated PMN with IL-10 decreased their ability to produce superoxide anion and to induce p47PHOX phosphorylation. In addition, the absence of IL-10 in vivo leads to an increase of basal ROS production in whole blood PMN as shown by the use of IL-10 knockout mice compared to their WT counterparts. This suggests that the anti-inflammatory cytokine IL-10 acts as a negative modulator of ROS generation both in vitro and in vivo.

The intracellular signaling pathways responsible for the observed inhibitory effects of IL-10 remain to be identified. In our experiments the inhibitory effect of IL-10 on p47PHOX phosphorylation and ERK1/2 activity occurred after 20 min of incubation of freshly isolated PMN with IL-10. The intracellular signaling pathways initiating these events must thus occur early. It has recently been shown that, in freshly isolated PMN, IL-10 does not activate the JAK/STAT pathway, although JAK1 and TYK2, as well as STAT1 and STAT3 can be detected in PMN (35 , 38) . It is possible that, in freshly isolated human PMN, IL-10 recruits other, unidentified pathways and that IL-10R signaling occurs independently of JAK/STAT activation.

Together, these results suggest that IL-10 inhibits GM-CSF-induced priming of ROS production by inhibiting p47PHOX phosphorylation through a decrease in ERK1/2 activity. ROS production requires tight regulation for optimal antibacterial activity without detrimental consequences for host tissues. Inhibition of p47PHOX phosphorylation by IL-10 might be a mechanism by which this anti-inflammatory cytokine contributes to the tight regulation of NADPH oxidase activity and thereby diminishes host tissue damage at sites of inflammation.

ACKNOWLEDGMENTS

This work was supported by a grant from Association pour la Recherche sur la Polyarthrite (ARP).

Received for publication December 5, 2005. Accepted for publication February 27, 2006.

REFERENCES

  1. Babior, B. M. (1984) Oxidants from phagocytes: agents of defense and destruction. Blood 64,959-966[Free Full Text]
  2. Babior, B. M. (1999) NADPH oxidase: an update. Blood 93,1464-1476[Free Full Text]
  3. Roos, D., De Boer, M., Kuribayashi, F., Meischi, , Weening, R. S., Segal, A.W., Ahlin, A., Nemet, K., Hossle, J., Bernatowska-Matusskiewicz, P., Middleton-Price, H. H. (1996) Mutations in the X-Linked and Autosomal recessive forms of chronic granulomatous disease. Blood 87,1663-1681[Free Full Text]
  4. Heyworth, P. G., Cross, A. R., Curnutte, J. T. (2003) Chronic granulomatous disease. Curr. Opin. Immunol. 15,578-584[CrossRef][Medline]
  5. Ricevuti, G. (1997) Host tissue damage by phagocytes. Ann. N. Y. Acad. Sci. 832,426-448[Medline]
  6. Vignais, P. V. (2002) The superoxide-generating NADPH oxidase: structural aspects and activation mechanisms. Cell. Mol. Life Sci. 59,1428-1459[CrossRef][Medline]
  7. Groemping, Y., Rittinger, K. (2005) Activation and assembly of the NADPH oxidase: a structural perspective. Biochem. J. 386,401-416[CrossRef][Medline]
  8. Downey, G. P., Fukushima, T., Fialkow, L., Waddell, T. K. (1995) Intracellular signaling in neutrophil priming and activation. Semin. Cell Biol. 6,345-356[CrossRef][Medline]
  9. Hallett, M. B., Lloyds, D. (1995) Neutrophil priming: the cellular signals that say "amber" but not "green". Immunol. Today. 16,264-268[CrossRef][Medline]
  10. Sullivan, G. W., Carper, H. T., Mandell, G. L. (1993) The effect of three human recombinant hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and interleukin-3) on phagocyte oxidative activity. Blood 81,1863-1870[Abstract/Free Full Text]
  11. Berkow, R. L., Wang, D., Larrickm, J. W., Dodson, R. W., Howard, T. H. (1987) Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor. J. Immunol. 139,3783-3791[Abstract]
  12. Elbim, C., Chollet-Martin, S., Bailly, S., Hakim, J., Gougerot-Pocidalo, M. A. (1993) Priming of polymorphonuclear neutrophils by tumor necrosis factor alpha in whole blood: identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides. Blood 82,633-640[Abstract/Free Full Text]
  13. Dang, P. M., Dewas, C., Gaudry, M., Fay, M., Pedruzzi, E., Gougerot-Pocidalo, M. A., El Benna, J. (1999) Priming of human neutrophil respiratory burst by granulocyte/macrophage colony-stimulating factor (GM-CSF) involves partial phosphorylation of p47(phox). J. Biol. Chem. 274,20704-20708[Abstract/Free Full Text]
  14. Dewas, C., Dang, P. M., Gougerot-Pocidalo, M. A., El-Benna, J. (2003) TNF-alpha induces phosphorylation of p47(phox) in human neutrophils: partial phosphorylation of p47phox is a common event of priming of human neutrophils by TNF-alpha and granulocyte-macrophage colony-stimulating factor. J. Immunol. 171,4392-4398[Abstract/Free Full Text]
  15. Zacharowski, P., Breese, E., Wood, E., Del Soldato, P., Warner, T., Mitchell, J. (2005) NSAIDs increase GM-CSF release by human synoviocytes: comparison with nitric oxide-donating derivatives. Eur. J. Pharmacol. 508,7-13[CrossRef][Medline]
  16. El Benna, J., Hayem, G., Dang, P. M., Fay, M., Chollet-Martin, S., Elbim, C., Meyer, O., Gougerot- Pocidalo, M. A. (2002) NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy. Inflammation 26,273-278[CrossRef][Medline]
  17. Fiorentino, D. F., Bond, M. W., Mosmann, T. R. (1989) Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J. Exp. Med. 170,2081-2095[Abstract/Free Full Text]
  18. Moore, K. W., de Waal Malefyt, R., Coffman, R. L., O’Garra, A. (2001) Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19,683-765[CrossRef][Medline]
  19. Cassatella, M. A. (1998) The neutrophil: one of the cellular targets of interleukin-10. Int. J. Clin. Lab. Res. 28,148-161[CrossRef][Medline]
  20. Cassatella, M. A., Meda, L., Bonora, S., Ceska, M., Constantin, G. (1993) Interleukin 10 (IL-10) inhibits the release of proinflammatory cytokines from human polymorphonuclear leukocytes. Evidence for an autocrine role of tumor necrosis factor and IL-1 beta in mediating the production of IL-8 triggered by lipopolysaccharide. J. Exp. Med. 178,2207-2211[Abstract/Free Full Text]
  21. Kasama, T., Strieter, R. M., Lukacs, N.W., Burdick, M. D., Kunkel, S. L. (1994) Regulation of neutrophil-derived chemokine expression by IL-10. J. Immunol. 152,3559-3569[Abstract]
  22. Gasperini, S., Marchi, M., Calzetti, F., Laudanna, C., Vicentini, L., Olsen, H., Murphy, M., Liao, F., Farber, J., Cassatella, M. A. (1999) Gene expression and production of the monokine induced by INF-{gamma} (MIG), IFN-inducible T cell {alpha} Chemoattractant (I-TAC), and IFN-{gamma}-inducible protein (IP-10) chemokines by human neutrophils. J. Immunol. 162,4928-4937[Abstract/Free Full Text]
  23. Cassatella, M. A., Meda, L., Gasperini, S., Calzetti, F., Bonora, S. (1994) Interleukin 10 (IL-10) upregulates IL-1 receptor antagonist production from lipopolysaccharide-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation. J. Exp. Med. 179,1695-1699[Abstract/Free Full Text]
  24. Takeshi Otsuka, N. H., Izuhara, K., Yamaoka, K., Ohshima, K., Tanabe, T., Hara, S., Nemoto, Y., Tanaka, Y., Nakashima, H., Niho, Y. (1997) Regulation by interleukin-10 and interleukin-4 of cyclooxygenase-2 expression in human neutrophils. Blood 89,1621-1628[Abstract/Free Full Text]
  25. Bussolati, B., Mariano, F., Montruchio, G., Piccoli, G., Camusi, (1997) Modulatory effect of interleukin 10 on the production of platelet-activating factor and superoxide anions by human leukocytes. Immunology 90,440-447[CrossRef][Medline]
  26. Capsoni, F., Minonzio, F., Ongari, A. M., Carbonelli, V., Galli, A, Zanussi, C. (1997) Interleukin-10 down-regulates oxidative metabolism and antibody-dependent cellular cytotoxicity of human neutrophils. Scand. J. Immunol. 45,269-275[CrossRef][Medline]
  27. Chaves, M. M., Silvestrini, A. A., Silva-Teixeira, D. N., Nogueira-Machado, J. A. (1996) Effect in vitro of gamma interferon and interleukin-10 on generation of oxidizing species by human granulocytes. Inflamm. Res. 45,313-315[CrossRef][Medline]
  28. Laichalk, L., Danforth, J. M., Standiford, T. J. (1996) Interleukin 10 inhibits neutrophil phagocytic and bactericidal activity. FEMS Immunol. Med. Microbiol. 15,181-187[CrossRef][Medline]
  29. Ho, A. S., Liu, Y., Khan, T. A., Hsu, D. H., Bazan, J. F., Moore, K. W. (1993) A receptor for interleukin 10 is related to interferon receptors. Proc. Natl. Acad. Sci. U. S. A. 90,11267-112671[Abstract/Free Full Text]
  30. Liu, Y., Wei, S. H., Ho, A. S., de Waal Malefyt, R., Moore, K. W. (1994) Expression cloning and characterization of a human IL-10 receptor. J. Immunol. 152,1821-1829[Abstract]
  31. Kotenko, S. V., Krause, C. D., Izotova, L. S., Pollack, B. P., Wu, W., Pestka, S. (1997) Identification and functional characterization of a second chain of the interleukin-10 receptor complex. EMBO J. 16,5894-5903[CrossRef][Medline]
  32. Spencer, S. D., Di Marco, F., Hooley, J., Pitts-Meek, S., Bauer, M., Ryan, A. M., Sordat, B., Gibbs, V. C., Aguet, M. (1998) The orphan receptor CRF2–4 is an essential subunit of the interleukin 10 receptor. J. Exp. Med. 187,571-578[Abstract/Free Full Text]
  33. Grutz, G. (2005) New insights into the molecular mechanism of interleukin-10-mediated immunosuppression. J. Leuko.c Biol. 77,3-15[Abstract/Free Full Text]
  34. Williams, L. M., Ricchetti, G., Sarma, U., Smallie, T., Foxwell, B. M. (2004) Interleukin-10 suppression of myeloid cell activation–a continuing puzzle. Immunology 113,281-292[CrossRef][Medline]
  35. Bovolenta, C., Gasperini, S., McDonald, P. P., Cassatella, M. A. (1998) High affinity receptor for IgG (Fc gamma RI/CD64) gene and STAT protein binding to the IFN-gamma response region (GRR) are regulated differentially in human neutrophils and monocytes by IL-10. J. Immunol. 160,911-919[Abstract/Free Full Text]
  36. Crepaldi, L., Gasperini, S., Lapinet, J. A., Calzetti, F., Pinardi, C., Liu, Y., Zurawski, S., de Waal Malefyt, R., Moore, K.W., Cassatella, M. A. (2001) Up-regulation of IL-10R1 expression is required to render human neutrophils fully responsive to IL-10. J. Immunol. 167,2312-2322[Abstract/Free Full Text]
  37. Elbim, C., Reglier, H., Fay, M., Delarche, C., Andrieu, V., El Benna, J., Gougerot-Pocidalo, M. A. (2001) Intracellular pool of IL10 receptors in specific granules of human neutrophils: differential mobilization by proinflammatory mediators. J. Immunol. 166,5201-5207[Abstract/Free Full Text]
  38. Al-Shami, A., Mahanna, W., Naccache, P. H. (1998) Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Selective activation of Jak2, Stat3, and Stat5b. J. Biol. Chem. 273,1058-1063[Abstract/Free Full Text]
  39. Crepaldi, L., Silveri, L., Calzetti, F., Pinardi, C., Cassatella, M. A. (2002) Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10. Int. Immunol. 14,1145-1153[Abstract/Free Full Text]
  40. Woodman, R. C., Ruedim, J. M., Jesaitism, A. J., Okamuram, N., Quinnm, M. T., Smithm, R. M., Curnuttem, J. T., Babior, B. M. (1991) Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils. J. Clin. Invest. 87,1345-1351[Medline]
  41. Leammli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685[CrossRef][Medline]
  42. Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354[Abstract/Free Full Text]
  43. Nijhuis, E., Lammers, J. W., Koenderman, L., Coffer, P. J. (2002) Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning. J. Leukoc. Biol. 71,115-124[Abstract/Free Full Text]
  44. Dang, P. M., Cross, A. R., Quinn, M. T., Babior, B. M. (2002) Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558 II. Proc. Natl. Acad. Sci. U. S. A. 99,4262-4265[Abstract/Free Full Text]
  45. El Benna, J., Faust, L. P., Babior, B. M. (1994) The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases. J. Biol. Chem. 269,23431-23436[Abstract/Free Full Text]
  46. Opal, S. M., DePalo, V. A. (2000) Anti-inflammatory cytokines. Chest. 117,1162-1172[CrossRef][Medline]
  47. Geijsen, N., Van Delft, S., Raaijmakers, J. A., Lammers, J. W., Collard, J. G., Koenderman, L., Coffer, P. J. (1999) Regulation of p21rac activation in human neutrophils. Blood 94,1121-1130[Abstract/Free Full Text]
  48. Okuda, K., Sanghera, J. S., Pelech, S. L., Kanakura, Y., Hallek, M., Griffin, J. D., Druker, B. J. (1992) Granulocyte-macrophage colony-stimulating factor, interleukin-3, and steel factor induce rapid tyrosine phosphorylation of p42 and p44 MAP kinase. Blood 79,2880-2887[Abstract/Free Full Text]
  49. Brizzi, M. F., Aronica, M.. G., Rosso, A., Bagnara, G. P., Yarden, Y., Pegoraro, L. (1996) Granulocyte-macrophage colony-stimulating factor stimulates JAK2 signaling pathway and rapidly activates p93fes, STAT1 p91, and STAT3 p92 in polymorphonuclear leukocytes. J. Biol. Chem. 271,3562-3567[Abstract/Free Full Text]
  50. Dang, P. M., Stenballe, A., Boussetta, T., Raad, H., Dewas, C., Kroviarski, Y., Hayem, G., Jensen, O. N., Gougerot-Poudalo, M. A., El-Benna, J. (2006) A specific p47PHOX-serine phosphorylated by convergent MAP kinases mediates neutrophil NADPH oxidase priming at inflammatory sites. J. Clin. Invest. In press.
  51. Joseph, D. E., Paul, C. C., Baumann, M. A., Gomez-Cambronero, J. (1996) S6 kinase p90rsk in granulocyte-macrophage colony-stimulating factor-stimulated proliferative and mature hematopoietic cells. J. Biol. Chem. 271,13088-13093[Abstract/Free Full Text]
  52. Thievierge, M., Parent, J. L., Stankova, J., Rola-Pleszczynski, M. (1999) Modulation of formyl peptide receptor expression by IL-10 in human monocytes and neutrophils. J. Immunol. 162,3590-3595[Abstract/Free Full Text]
  53. Muller, T., Davies, E. V., Campbell, A. K. (1989) Pholasin chemiluminescence detects mostly superoxide anion released from activated human neutrophils. J. Biolumin. Chemilumin. 3,105-113[CrossRef][Medline]
  54. DeLeo, F. R., Allen, L. A., Apicella, M., Nauseef, W. M. (1999) NADPH oxidase activation and assembly during phagocytosis. J. Immunol. 163,6732-6740[Abstract/Free Full Text]
  55. Bengis-Garber, C., Gruener, N. (1995) Involvement of protein kinase C and of protein phosphatases 1 and/or 2A in p47 phox phosphorylation in formylmet-Leu-Phe stimulated neutrophils: studies with selective inhibitors RO 31–8220 and calyculin A. Cell. Signal. 7,721-732[CrossRef][Medline]
  56. Geng, Y., Gulbins, E., Altman, A., Lotz, M. (1994) Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway. Proc. Natl. Acad. Sci. U. S. A. 91,8602-8606[Abstract/Free Full Text]
  57. Inoue, Y., Otsuka, T., Niiro, H., Nagano, S., Arinobu, Y., Ogami, E., Akahoshi, M., Miyake, K., Ninomiya, I., Shimizu, S, et al (2004) Novel regulatory mechanisms of CD40-induced prostanoid synthesis by IL-4 and IL-10 in human monocytes. J. Immunol. 172,2147-2154[Abstract/Free Full Text]
  58. Morrison, D. K., Davis, R. J. (2003) Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu. Rev. Cell Dev. Biol. 19,91-118[CrossRef][Medline]
  59. Sur, R., Ramos, J. W. (2005) Vanishin is a novel ubiquitinylated death-effector domain protein that blocks ERK activation. Biochem. J. 387,315-313[CrossRef][Medline]
  60. Farooq, A., Zhou, M. M. (2004) Structure and regulation of MAPK phosphatases. Cell Signal. 16,769-779[CrossRef][Medline]
  61. McLeish, K. R., Knall, C., Ward, R. A., Gerwins, P., Coxon, P. Y., Klein, J. B., Johnson, G. L. (1998) Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-alpha and GM-CSF. J. Leukoc. Biol. 64,537-545[Abstract]
  62. Suzuki, K., Hino, M., Hato, F., Tatsumi, N., Kitagawa, S. (1999) Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. Blood 93,341-349[Abstract/Free Full Text]
  63. Gallova, L., Kubala, L., Ciz, M., Lojek, A. (2004) IL-10 does not affect oxidative burst and expression of selected surface antigen on human blood phagocytes in vitro. Physiol. Res. 53,199-208[Medline]




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