Modulation of equine neutrophil adherence and migration by the annexin-1 derived N-terminal peptide, Ac2-26.

Abstract: Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical production at a higher concentration. In the present study, we have further characterised the effects of Ac2-26 on equine neutrophil function. In addition, as anti-inflammatory glucocorticoids are known to up-regulate annexin-1, we have examined the effects of dexamethasone on annexin-1 expression in equine leukocytes. The effects of Ac2-26 alone and on agonist (CXCL8, leukotriene (LT)B(4) and PAF)-induced adherence and migration were examined by measuring adhesion of neutrophils to serum-coated plastic and by use of a ChemoTx migration assay. The role of formyl peptide receptors (FPRs) in mediating the effects of Ac2-26 was examined using the pan-FPR antagonist, BOC-2. Flow cytometry was used to measure the effects of dexamethasone on annexin-1 expression. Pre-incubation with Ac2-26 (10(-5)M) significantly inhibited neutrophil adhesion and migration in response to other agonists but when used alone could also induce these responses. The stimulatory and inhibitory effects of Ac2-26 were reduced by BOC-2, indicating a dependency on FPR activation. Dexamethasone increased the percentage of annexin-1 positive neutrophils and mononuclear cells by 1h post treatment (from 45±5% to 93±1% and 62±14% to 87±9% for neutrophils and monocytes, respectively) but by 4h there was no difference from control cells. No difference was seen between the percentages of annexin-1 positive cells pre- and post-treatment in animals that had undergone a dexamethasone suppression test. The attenuation of agonist-induced adherence and migration by Ac2-26 may play a part in regulating recruitment of equine neutrophils in inflammatory conditions of the horse. However, if high concentrations are produced in vivo following release of annexin-1 from activated cells, direct stimulatory effects may occur which could be either beneficial or detrimental. The therapeutic efficacy of anti-inflammatory steroids in the horse may be mediated in part by increasing annexin-1 expression although this effect appears to be short-lived.