Published as doi: 10.1096/fj.05-5395fje.
(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
SPECIFIC AIMS
Interleukin (IL)-10(IL-10) exerts its antiinflammatory 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)-
, is essential for priming ROS production by PMN. The aim of this study was to determine whether IL-10 inhibits GM-CSF- and TNF-induced p47PHOX phosphorylation and to investigate the molecular mechanisms involved in this effect.
PRINCIPAL FINDINGS
1. IL-10 inhibits GM-CSF- but not TNF-induced p47PHOXphosphorylation in human PMN
32P-loaded PMNs in suspension were incubated with GM-CSF or TNF 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 ± 1746 cpm 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 antiinflammatory properties. 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
). GM-CSF and TNF are the most potent cytokines in terms of PMN oxidative burst priming. However, IL-10 did not have a significant inhibitory effect on TNF-induced p47PHOX phosphorylation, even though it inhibited GM-CSF-induced p47PHOX phosphorylation in the same experiment (Fig. 1C
). This suggests that the 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 priming.
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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). P47PHOX was detected by autoradiography (P-p47PHOX) or with antip47PHOX Ab (p47PHOX). B, PMN were incubated with GM-CSF (12 ng/ml) in the presence or absence of various concentrations of IL-10. P47PHOX was then detected by autoradiography (P-p47PHOX) or with antip47PHOX Ab (p47PHOX). C) PMN were incubated with GM-CSF (12 ng/ml) or TNF (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).
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2. IL-10 inhibits GM-CSF-induced extracellular signal-regulated kinase 1/2 activity without affecting extracellular signal-regulated kinase phosphorylation status
We next examined the molecular mechanisms of IL-10-mediated inhibition of p47PHOX phosphorylation in GM-CSF-treated PMN. As we had recently shown that extracellular signal-regulated kinase 1/2 (ERK1/2) are involved in direct p47PHOX phosphorylation on Ser-345 in GM-CSF-treated PMN (Dang et al., in press), 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. 2
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. 2B
). 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. 2C
). 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. 2D
, lower panel). The activity measured was due to ERK1/ERK2 activity and not to nonspecific activity precipitated by the anti-ERK1/ERK2 antibodies, as neither MBP nor p47PHOX phosphorylating activities were detected in the immunoprecipitate when irrelevant isotypic control antibody (Ab) were used instead of ERK1/2 antibodies (Fig. 2D
, upper and lower panel). Also, when PMNs 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. 2C
). 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, an endogenous substrate of ERK1/2 in mature hematopoietic cells stimulated by GM-CSF) by using 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. Furthermore, inhibitor of MEK1/2 (PD98059 and UO126) but not inhibitor of p38MAPK (SB203580), prevented GM-CSF-induced phosphorylation of p90RSK, confirming the involvement of ERK1/ERK2 in this phosphorylation. 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 or related upstream pathways.
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Figure 2. 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 antibodies (P-ERK). B)PMN (25x106/ml) were stimulated where indicated with 15 ng/ml GM-CSF in the presence or absence of various concentrations of IL-10. Western blots were performed with anti-human phospho-p44/42 MAPK antibody C) PMN (25x106/ml) were stimulated with 15 ng/ml GM-CSF 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. ERK1/2 was immunoprecipitated, and kinase assay was performed in the presence of MBP and [ 32P]ATP. MBP phosphorylation was detected by autoradiography (P-MBP). 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. 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. MBP and p47PHOX phosphorylation were detected by autoradiography (P-MBP and P-p47PHOX respectively).
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3. 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, 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. In contrast, IL-4 and IL-13, which have no effect on GM-CSF-induced p47PHOX phosphorylation, had no significant effect on GM-CSF-induced superoxide production. Furthermore, GM-CSF-induced p47PHOX phosphorylation was inhibited in adherent PMN in a concentration-dependent manner, as in suspended PMN. 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. Finally, we showed that the absence of IL-10 in vivo altered ROS production by leukocytes tested in whole blood: leukocytes of IL-10 knockout mice showed higher basal ROS production than leukocytes of wild-type (WT) mice, indicating that IL-10 is also crucial for negative regulation of ROS production in vivo.
CONCLUSIONS AND SIGNIFICANCE
IL-10 (IL-10) exerts its antiinflammatory properties by down-regulating neutrophil functions such as ROS production. This study sheds light on the molecular mechanisms by which IL-10 inhibits ROS production, a major proinflammatory and bactericidal function of neutrophils. Significant findings include 1) IL-10 selectively inhibits GM-CSF-induced p47PHOX phosphorylation in a concentration-dependent manner in suspended and adherent neutrophils but does not alter TNF-induced p47PHOX phosphorylation; 2) this IL-10-induced inhibition of p47PHOX phosphorylation is due to inhibition of ERK1/2 activity, as shown by immunocomplex kinase assay and by analyzing the phosphorylation status of the endogenous substrate of ERK1/2, p90RSK, in intact PMN; 3) 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 with their WT counterparts.
In a previous study, we showed that PMN treatment with GM-CSF and TNF resulted in partial phosphorylation of p47PHOX on the same site, 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-stimulated PMN. This might be explained by the fact that GM-CSF and TNF engage different pathways to phosphorylate p47PHOX on Ser-345 and that only the pathway induced by GM-CSF could be inhibited by IL-10. (Dang P.M. et al., in press and unpublished observations).
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. 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.
Based on our findings, we propose a working model in which IL-10 inhibits GM-CSF-induced priming of ROS production by inhibiting p47PHOX phosphorylation through a decrease in ERK1/2 activity (Fig. 3
). 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.
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Figure 3. Schematic model of molecular mechanisms by which IL-10 inhibits priming of ROS production induced by GM-CSF in PMN. GM-CSF induces partial phosphorylation of p47PHOX via activation of ERK1/2, and this results in priming of ROS production. When present, IL-10 inhibits ERK1/2 activity at a postphosphorylation step, and this inhibition leads to a decrease in GM-CSF-induced p47PHOX phosphorylation; as a result, GM-CSF-induced priming of ROS production is inhibited.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5395fje
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
