title: 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.
authors.affiliation: Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Campus, Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, UK.
authors.forename: Kirstie J
authors.initials: KJ
authors.lastname: Pickles
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Brooks
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Rickards
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Cunningham
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017305:Annexin A1 / Q000097:blood* / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006736:Horses / Q000097:blood* / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000276:immunology / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D011817:Rabbits; D013481:Superoxides / Q000097:blood
chemical_list: D017305:Annexin A1; D005419:Flavonoids; D009842:Oligopeptides; D010455:Peptides; C096153:annexin A1 peptide (2-26); D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; C033373:butyloxycarbonyl-phenylalanyl-leucyl-phenylalanyl-leucyl-phenylalanine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one