Expression of annexin-1 in equine leucocytes and the effects of the N-terminal annexin-1 peptide, Ac2-26, on equine neutrophil superoxide production.
Abstract: N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil superoxide production was measured by the reduction of cytochrome (cyt) C following stimulation with Ac2-26 and the formyl peptide receptor (FPR) agonists, FMLP, WKYMVm and WKYMVM. Responses were examined in the presence of the pan-FPR antagonist, BOC-2, and the role of p42/44 MAPK in agonist-induced effects was determined using PD98059. The effect of Ac2-26 on superoxide production in response to serum-treated zymosan (STZ) was also investigated, and the roles of FPR and p42/44 MAPK ascertained. Annexin-1 was detected in both equine neutrophils and mononuclear cells using a polyclonal rabbit anti-human annexin-1 antibody. Ac2-26 (5x10(-5)M) induced superoxide production in cytochalasin B-primed (48+/-8 versus 21+/-9 (unstimulated cells) nmol cyt C/10(6) neutrophils) and un-primed cells (37+/-10 versus 11+/-5 nmol cyt C/10(6) neutrophils). FMLP and WKYMVm, but not WKYMVM, also caused superoxide production in primed neutrophils, suggesting the response was mediated by FPR receptor binding. This was supported by the marked inhibitory effect of BOC-2 on the responses to Ac2-26 and FMLP although, interestingly, the effects of WKYMVm were not significantly reduced (50+/-5 (WKYMVm) versus 45+/-5 (WKYMVm+BOC-2) nmol reduced cyt C/10(6) neutrophils). Inhibition of p42/44 MAPK activation with PD98059 significantly attenuated superoxide production in response to Ac2-26, FMLP and WKYMVm and Western blotting showed that Ac2-26 induced p42/44 MAPK activation. At a concentration which did not cause superoxide production, Ac2-26 (10(-5)M) significantly reduced the response to STZ (84+/-17% inhibition). This inhibitory effect was attenuated by both BOC-2 and PD98059. These results suggest that if activation of equine leucocytes in vivo leads to the release and subsequent cleavage of annexin-1, the N-terminal peptides formed could bind to neutrophil FPR and decrease free radical production in response to particulate stimuli. This could help to reduce local tissue damage but, as Ac2-26 can also stimulate superoxide production at higher concentrations in an FPR-dependent manner, the amount of free radical production may depend on the concentration of peptide present.
Copyright 2009 Elsevier B.V. All rights reserved.
Publication Date: 2009-12-16 PubMed ID: 20079938DOI: 10.1016/j.vetimm.2009.12.002Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article is about a study exploring the presence of annexin-1 in equine leucocytes and the impact of its N-terminal peptide on the production of superoxide in equine neutrophils. The researchers have taken a look at how the N-terminal peptide regulates free radical production, essentially affecting inflammation and cellular damage.
Objective of the Study
- The main objective of the research was to investigate the presence and function of annexin-1 in equine leucocytes, focusing specifically on how the N-terminal peptide (Ac2-26) affects equine neutrophil superoxide production.
Methods of the Study
- The researchers used immuno-blotting to determine annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK.
- The production of superoxide in equine neutrophils was assessed through the reduction of cytochrome C post-stimulation with Ac2-26 and several FPR agonists. The responses were examined in the presence of the pan-FPR antagonist, BOC-2, and the role of p42/44 MAPK in these effects was determined using PD98059.
- An examination of superoxide production in response to serum-treated zymosan (STZ) was also carried out, and the roles of FPR and p42/44 MAPK were ascertained.
Results of the Study
- Annexin-1 was found in both equine neutrophils and mononuclear cells through using a polyclonal rabbit anti-human annexin-1 antibody.
- Ac2-26 was observed to induce superoxide production in both cytochalasin B-primed and un-primed cells.
- FMLP and WKYMVm were able to prompt superoxide production in primed neutrophils, which suggested the response was mediated by FPR receptor binding. The inhibitory influence of BOC-2 on the responses to Ac2-26 and FMLP reinforce this assumption.
- Using PD98059 to inhibit the activation of p42/44 MAPK significantly lessened superoxide production in response to Ac2-26, FMLP, and WKYMVm. Plus, Western blotting revealed that Ac2-26 led to the activation of p42/44 MAPK.
- At a concentration that did not induce superoxide production, Ac2-26 was seen to significantly decrease the response to STZ. This reduction was lessened by both BOC-2 and PD98059.
Conclusion of Study
- The findings suggest that when there is activation of equine leucocytes in vivo leading to the release and subsequent cleavage of annexin-1, the N-terminal peptides formed could bind to neutrophil FPR and lessen free radical production in response to particulate triggers.
- This would be beneficial in minimizing local tissue damage. However, it’s also important to consider that Ac2-26 also prompts superoxide production at higher concentrations in an FPR-dependent manner, meaning that the level of free radical production may rely on the peptide concentration present.
Cite This Article
APA
Pickles KJ, Brooks AC, Rickards KJ, Cunningham FM.
(2009).
Expression of annexin-1 in equine leucocytes and the effects of the N-terminal annexin-1 peptide, Ac2-26, on equine neutrophil superoxide production.
Vet Immunol Immunopathol, 135(3-4), 226-233.
https://doi.org/10.1016/j.vetimm.2009.12.002 Publication
Researcher Affiliations
- Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Campus, Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, UK.
MeSH Terms
- Amino Acid Sequence
- Animals
- Annexin A1 / blood
- Annexin A1 / chemistry
- Annexin A1 / immunology
- Annexin A1 / pharmacology
- Flavonoids / pharmacology
- Horses / blood
- Horses / immunology
- Humans
- In Vitro Techniques
- Leukocytes / immunology
- Leukocytes / metabolism
- MAP Kinase Signaling System / drug effects
- Molecular Sequence Data
- N-Formylmethionine Leucyl-Phenylalanine / pharmacology
- Neutrophils / drug effects
- Neutrophils / immunology
- Oligopeptides / pharmacology
- Peptides / chemistry
- Peptides / immunology
- Peptides / pharmacology
- Rabbits
- Superoxides / blood
Citations
This article has been cited 3 times.- Bayless RL, Sheats MK, Jones SL. Withaferin A Inhibits Neutrophil Adhesion, Migration, and Respiratory Burst and Promotes Timely Neutrophil Apoptosis. Front Vet Sci 2022;9:900453.
- Lice I, Sanches JM, Correia-Silva RD, Corrêa MP, Icimoto MY, Silva AAR, Sánchez-Vinces S, Porcari AM, Moreira V, Gil CD. Effects of Formyl Peptide Receptor Agonists Ac(9-12) and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models. Cells 2022 Jan 11;11(2).
- Correia-Silva RD, Corrêa MP, de Castro ME, Almeida JS, D'Ávila SCGP, Oliani SM, Greco KV, Gil CD. Regulatory role of annexin A1 in NLRP3 inflammasome activation in atopic dermatitis: insights from keratinocytes in human and murine studies. J Mol Med (Berl) 2025 Apr;103(4):435-451.
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