Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release

Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65–16,624 pg/ml), IFNγ (4–80 U/ml), IL-4 (0–5069 pg/ml), IL-10 (49–1862 pg/ml), and IL-17 (4–1244 U/ml)]. Each animal's age influenced leukocyte composition, cell morphology and cytokine release (TNFα, IL-4, IL-10) ex vivo. Geldings showed smaller monocytes and higher spontaneous production of IL-10 when compared to the mares included. The stimulation to spontaneous release ratios of TNFα, IL-4 and IL-17 differed in Warmblood and Thoroughbred types. Sampling time influenced leukocyte composition and cell morphology. In summary, many animal factors – age being the dominant one – should be considered for studies involving the analysis of equine leukocytes. In addition, high inter-individual variances argue for individual baseline measurements.