CXCL8 inhibits stimulus-induced equine neutrophil (PMN) migration and adherence

Andrew Brooks1, Karen Rickards2, Fiona Cunningham1. 1Veterinary Basic Sciences, Royal Veterinary College, North Mymms, Hertfordshire, United Kingdom, 2The Donkey Sanctuary, Sidmouth, Devon, United Kingdom.

CXCL8, a chemoattractant for equine PMNs has also been reported to down-regulate the migratory response of human PMN to other stimuli (Lang et al. 2003). This study examined how CXCL8 affects the movement and adhesion of equine PMNs in response to PAF and LTB4 and the possible role of cAMP in mediating the observed effect.

Responses to CXCL8 (10-10-10-8M (chemotaxis) or 10-7M (adhesion and cAMP)), PAF (10-7M) and LTB4 (10-7M)), alone or in combination, were determined using cells from 5 (adherence and migration) or 4 (cAMP) normal horses. Migration was measured using ChemoTx plates (Receptor Technologies Ltd, Leamington Spa, UK), adherence to plastic by determining alkaline phosphatase activity and cAMP concentration by use of an ELISA (R&D Systems, Oxford, UK). Results are expressed as mean±SEM chemotactic index (CI), percentage adherence after subtraction of basal or pmol cAMP/5x107 cells. Data were analysed by 1-way ANOVA and Bonferonni’s correction.

Each of the stimuli caused PMN migration (CI: 2±1, 4±1 and 5±1 (CXCL8 10-10-10-8M), 4±0.2 (PAF) and 4±1 (LTB4), both at 10-7M) and adherence (7±1%, 4±1%, 3±1% for CXCL8, PAF and LTB4 (all at 10-7M)). Co-incubation of PAF with CXCL8 significantly decreased migration (CI: *1±0.4, *1±0.2 and *1±0.1 for CXCL8 10-10-10-8M) and adherence (*-0.1±0.7% PAF (10-7M)+CXCL8 (10-7M)) * = p<0.05 versus PAF (10-7M) alone. Although responses to CXCL8+LTB4 (10-7M) were less than additive, the reductions were much less marked (CI: 5±1, 5±1 and 4±1 (CXCL8 10-10-10-8M); 5±2% adherence (CXCL8 10-7M). This could be explained by a difference in the magnitude of the increase in cAMP obtained using combinations of the two stimuli (pmol cAMP/5x107 cells: 4±0.4 (basal); 13±1 (CXCL8); 21±8 (PAF); 9±2 (LTB4); *31±8 (CXCL8+PAF); 10±2 (CXCL8+LTB4) * = p<0.05 vs. basal; all stimuli used at 10-7M).

These results suggest that, although CXCL8 may initially promote the recruitment of equine PMNs to inflamed tissue by increasing cell adhesion and migration, the chemokine could, by acting together with other chemoattractants, subsequently down-regulate their movement in a cAMP dependent manner.

Lang et al. (2003). Scand. J. Immunol. 57:350-361.