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Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling.

Pupjalis D, Goetsch J, Kottas DJ, Gerke V, Rescher U - EMBO Mol Med (2011)

Bottom Line: Supernatants from apoptotic neutrophils or the annexin A1 peptidomimetic Ac2-26 significantly reduced IL-6 signalling and the release of TNF-α from endotoxin-challenged monocytes.Ac2-26 activated STAT3 in a JAK-dependent manner, resulting in upregulated SOCS3 levels, and depletion of SOCS3 reversed the Ac2-26-mediated inhibition of IL-6 signalling.This identifies annexin A1 as part of the anti-inflammatory pattern of apoptotic cells and links the activation of FPRs to established signalling pathways triggering anti-inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Centre, Institute of Medical Biochemistry, University of Muenster, Muenster, Germany.

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Ac2-26 downregulates IL-6 signalling via upregulation of SOCS3Ac2-26 treatment dampens IL-6-induced STAT3 activation. Cell lysates of monocytes, which were stimulated for the indicated periods of time with either IL-6, Ac2-26 or a combination of both (90 min Ac2-26 followed by 15 min IL-6 or 90 min IL-6 followed by 60 min Ac2-26 as a control for the sensitivity of the cells towards activation), were analysed for the amount of pY-STAT3 by immunoblotting. Membranes were reprobed with a tubulin antibody to ensure equal loading.SOCS3 is required for mediating the inhibitory effects of Ac2-26 on IL-6 signalling. Monocytes depleted of cellular SOCS3 by phosphorothioate ODN complementary to SOCS3 mRNA (antisense) or treated with sense ODN employed as a control were treated with either IL-6 or Ac2-26/IL-6 as above. The cellular amounts of pY-STAT3 and SOCS3 were analysed by immunoblotting. Tubulin was used as an internal control for equal loading. Representative blots of at least three independent experiments are shown.
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fig07: Ac2-26 downregulates IL-6 signalling via upregulation of SOCS3Ac2-26 treatment dampens IL-6-induced STAT3 activation. Cell lysates of monocytes, which were stimulated for the indicated periods of time with either IL-6, Ac2-26 or a combination of both (90 min Ac2-26 followed by 15 min IL-6 or 90 min IL-6 followed by 60 min Ac2-26 as a control for the sensitivity of the cells towards activation), were analysed for the amount of pY-STAT3 by immunoblotting. Membranes were reprobed with a tubulin antibody to ensure equal loading.SOCS3 is required for mediating the inhibitory effects of Ac2-26 on IL-6 signalling. Monocytes depleted of cellular SOCS3 by phosphorothioate ODN complementary to SOCS3 mRNA (antisense) or treated with sense ODN employed as a control were treated with either IL-6 or Ac2-26/IL-6 as above. The cellular amounts of pY-STAT3 and SOCS3 were analysed by immunoblotting. Tubulin was used as an internal control for equal loading. Representative blots of at least three independent experiments are shown.

Mentions: To investigate whether Ac2-26-induced SOCS3 expression negatively regulates gp130 cytokines signalling, we first analysed the effect of Ac2-26 on IL-6 induced STAT3-activation. It is well known that IL-6 together with the signal transducer gp130 activates STAT3. As expected, IL-6 stimulation rapidly and robustly increased pY-STAT3 levels, reaching a maximum already 15 min after stimulation. This is in clear contrast to Ac2-26-elicited STAT3 phosphorylation, which occurred much later (Fig 7A, lanes 2 and 3). To test whether Ac2-26 elicited STAT3 activation is perturbed by pre-treatment with IL-6, monocytes were first stimulated with IL-6. After 90 min, when IL-6 induced STAT3 phosphorylation was already back to basal levels (not shown), Ac2-26 was given as a second stimulus and Ac2-26-elicited STAT3 phosphorylation was analysed. As presented in Fig 7A (lane 4), the STAT3 phosphorylation signal obtained in these cells was almost equivalent to the signal seen in cells stimulated with Ac2-26 only. This shows that STAT3 activation in response to Ac2-26 is not inhibited by pre-treatment with IL-6. Next, we tested for cross-inhibition of IL-6 signalling by Ac2-26 pre-treatment. Monocytes were pre-cultivated for 90 min in the presence of Ac2-26. At this time point, annexin-mediated STAT3 activation was back to basal levels (see Fig 4A). As shown in Fig 7A (lane 5), a subsequent 15 min stimulation with IL-6 resulted in decreased STAT3 phosphorylation as compared to treatment with IL-6 alone (Fig 7A, lane 2). Ac2-26 inhibited the IL-6-mediated STAT3 phosphorylation for up to 30 min of IL-6 application (not shown). To test whether the Ac2-26-induced upregulation of SOCS3 expression documented above (Fig 6) is the cause of impaired IL-6 signalling in the Ac2-26-treated monocytes, we performed stimulation experiments in monocytes, which had been treated with antisense phosphorothioate oligodeoxynucleotides (ODN; Sigma-Genosys) complementary to SOCS3 mRNA. A sense phosphorothioate ODN served as control. As expected, the response pattern of sense ODN treated monocytes was identical to that of control cells. In marked contrast, Ac2-26 was not able to interfere with IL-6-induced STAT3 activation in antisense ODN-treated monocytes (Fig 7B). These findings demonstrate that Ac2-26 negatively regulates IL-6 signalling via upregulation of SOCS3.


Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling.

Pupjalis D, Goetsch J, Kottas DJ, Gerke V, Rescher U - EMBO Mol Med (2011)

Ac2-26 downregulates IL-6 signalling via upregulation of SOCS3Ac2-26 treatment dampens IL-6-induced STAT3 activation. Cell lysates of monocytes, which were stimulated for the indicated periods of time with either IL-6, Ac2-26 or a combination of both (90 min Ac2-26 followed by 15 min IL-6 or 90 min IL-6 followed by 60 min Ac2-26 as a control for the sensitivity of the cells towards activation), were analysed for the amount of pY-STAT3 by immunoblotting. Membranes were reprobed with a tubulin antibody to ensure equal loading.SOCS3 is required for mediating the inhibitory effects of Ac2-26 on IL-6 signalling. Monocytes depleted of cellular SOCS3 by phosphorothioate ODN complementary to SOCS3 mRNA (antisense) or treated with sense ODN employed as a control were treated with either IL-6 or Ac2-26/IL-6 as above. The cellular amounts of pY-STAT3 and SOCS3 were analysed by immunoblotting. Tubulin was used as an internal control for equal loading. Representative blots of at least three independent experiments are shown.
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Related In: Results  -  Collection

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fig07: Ac2-26 downregulates IL-6 signalling via upregulation of SOCS3Ac2-26 treatment dampens IL-6-induced STAT3 activation. Cell lysates of monocytes, which were stimulated for the indicated periods of time with either IL-6, Ac2-26 or a combination of both (90 min Ac2-26 followed by 15 min IL-6 or 90 min IL-6 followed by 60 min Ac2-26 as a control for the sensitivity of the cells towards activation), were analysed for the amount of pY-STAT3 by immunoblotting. Membranes were reprobed with a tubulin antibody to ensure equal loading.SOCS3 is required for mediating the inhibitory effects of Ac2-26 on IL-6 signalling. Monocytes depleted of cellular SOCS3 by phosphorothioate ODN complementary to SOCS3 mRNA (antisense) or treated with sense ODN employed as a control were treated with either IL-6 or Ac2-26/IL-6 as above. The cellular amounts of pY-STAT3 and SOCS3 were analysed by immunoblotting. Tubulin was used as an internal control for equal loading. Representative blots of at least three independent experiments are shown.
Mentions: To investigate whether Ac2-26-induced SOCS3 expression negatively regulates gp130 cytokines signalling, we first analysed the effect of Ac2-26 on IL-6 induced STAT3-activation. It is well known that IL-6 together with the signal transducer gp130 activates STAT3. As expected, IL-6 stimulation rapidly and robustly increased pY-STAT3 levels, reaching a maximum already 15 min after stimulation. This is in clear contrast to Ac2-26-elicited STAT3 phosphorylation, which occurred much later (Fig 7A, lanes 2 and 3). To test whether Ac2-26 elicited STAT3 activation is perturbed by pre-treatment with IL-6, monocytes were first stimulated with IL-6. After 90 min, when IL-6 induced STAT3 phosphorylation was already back to basal levels (not shown), Ac2-26 was given as a second stimulus and Ac2-26-elicited STAT3 phosphorylation was analysed. As presented in Fig 7A (lane 4), the STAT3 phosphorylation signal obtained in these cells was almost equivalent to the signal seen in cells stimulated with Ac2-26 only. This shows that STAT3 activation in response to Ac2-26 is not inhibited by pre-treatment with IL-6. Next, we tested for cross-inhibition of IL-6 signalling by Ac2-26 pre-treatment. Monocytes were pre-cultivated for 90 min in the presence of Ac2-26. At this time point, annexin-mediated STAT3 activation was back to basal levels (see Fig 4A). As shown in Fig 7A (lane 5), a subsequent 15 min stimulation with IL-6 resulted in decreased STAT3 phosphorylation as compared to treatment with IL-6 alone (Fig 7A, lane 2). Ac2-26 inhibited the IL-6-mediated STAT3 phosphorylation for up to 30 min of IL-6 application (not shown). To test whether the Ac2-26-induced upregulation of SOCS3 expression documented above (Fig 6) is the cause of impaired IL-6 signalling in the Ac2-26-treated monocytes, we performed stimulation experiments in monocytes, which had been treated with antisense phosphorothioate oligodeoxynucleotides (ODN; Sigma-Genosys) complementary to SOCS3 mRNA. A sense phosphorothioate ODN served as control. As expected, the response pattern of sense ODN treated monocytes was identical to that of control cells. In marked contrast, Ac2-26 was not able to interfere with IL-6-induced STAT3 activation in antisense ODN-treated monocytes (Fig 7B). These findings demonstrate that Ac2-26 negatively regulates IL-6 signalling via upregulation of SOCS3.

Bottom Line: Supernatants from apoptotic neutrophils or the annexin A1 peptidomimetic Ac2-26 significantly reduced IL-6 signalling and the release of TNF-α from endotoxin-challenged monocytes.Ac2-26 activated STAT3 in a JAK-dependent manner, resulting in upregulated SOCS3 levels, and depletion of SOCS3 reversed the Ac2-26-mediated inhibition of IL-6 signalling.This identifies annexin A1 as part of the anti-inflammatory pattern of apoptotic cells and links the activation of FPRs to established signalling pathways triggering anti-inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Centre, Institute of Medical Biochemistry, University of Muenster, Muenster, Germany.

Show MeSH