Effect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses J. P. LEJEUNE*, C. SANDERSEN † , D. VOTION ‡ , I. CAUDRON, L. VANDER HEYDEN, T. FRANCK ‡ , J. CEUSTERS ‡ , A. MOUITHYS-MICKALAD ‡ , A. NIESTEN ‡ , G. DE LA REBIÈRE DE POUYADE †‡ and D. SERTEYN †‡ Equine European Centre of Mont-le-Soie, University of Liege, Mont-le-Soie, Vielsalm, Belgium; † Equine Clinic, Faculty of Veterinary Medicine, University of Liege, Bât. B41 Boulevard de Colonster Liège - Sart-Tilman, Belgium; and ‡ Centre of Oxygen: Research and Development (C.O.R.D.), University of Liege, Bât. B6, Allée de la Chimie, Liège, Belgium. Keywords: horse; equine physiology; intensive exercise; elastase; myeloperoxidase; inflammation Summary Reasons for performing study: Intensive exercise induces a systemic inflammatory response characterised by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by its proteinase activities. Objective: To compare plasmatic NE concentrations before and after different forms of intensive exercise. Materials and methods: EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or 2 star eventing competition (n = 51), or a 120 or 160 km endurance race (n = 32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcox on test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. Results: At T0, mean NE levels were 14.43  3.63 ng/ml for EvH and 11.7  2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57  24.06 ng/ml) EvH and (95.74  22.70 ng/ml) EndH (P<0.05). NE was significantly (P<0.0001) correlated to MPO in EvH (r = 0.293) and EndH (r = 0.594) and to CK (r = 0.297) in EndH (P<0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. Conclusions: Significant increase of NE (EndH) was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and NE indicates that not all horses show the same intensity of systemic inflammatory response. Introduction Ultra-endurance exercise, such as an Ironman triathlon, induces muscle damage and a systemic inflammatory response (Neubauer et al. 2008). Prolonged strenuous physical exercise also attenuates many components of immunity in man (Pedersen and Hoffman- Goetz 2000; Malm 2004; Gleeson 2007). It was shown that intense physical exercise can induce the degranulation of neutrophils that leads to an increase in plasma concentration of neutrophil marker proteins including myeloperoxidase (MPO) and elastase (NE) (Kokot et al. 1988; Camus et al. 1998; Gleeson et al. 1998; Walsh et al. 2000; Morozov et al. 2001, 2006). The extent of degranulation tends to increase with increasing intensity of exercise (Peake et al. 2004). The knowledge of exercise-induced inflammatory reaction in horses is sparse even if horses are particularly sensitive and exposed to excessive inflammatory responses (De la Rebière de Pouyade et al. 2009a). Neutrophil contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes (Franck et al. 2009). Consequently, the activation of neutrophils is one of the key events of the inflammatory process. MPO is considered as a valuable marker of this activation (Franck et al. 2005). Some studies have demonstrated the effect of strenuous exercise on the innate immune system in horses (Robson et al. 2003; Donovan et al. 2007). These studies demonstrated an impairment of the neutrophil function that could persist for several days after intense exercise. Further, increased levels of circulating MPO have been shown to occur in the post exercise period in eventing and endurance horses (Art et al. 2006). In this last study, Art and collaborators found a positive correlation between MPO and neutrophil counts and between MPO and creatine kinase (CK) levels in endurance horses. Elastase is the second neutrophil enzyme of the inflammatory reaction studied in man. NE released by activated neutrophils is recognised as an inflammatory and prognostic marker in various conditions (Gross et al. 1993; Zorn et al. 2003; Braga et al. 2006; Langhorst et al. 2008) and is implicated in inflammatory tissue damage (Jochum et al. 1994; Ginsburg 1999). This enzyme also plays a role in ischaemia-reperfusion injuries (Bzeizi et al. 1996; Okajima et al. 2004; Aoki et al. 2005; De la Rebière de Pouyade et al. 2009b). Elastase was regularly pointed out in lung pathology in man and horses for its elastinolytic properties (Dubin et al. 1994; Dagleish et al. 1999). Despite its wellknown proteinolytic *Corresponding author email: jph.lejeune@ulg.ac.be [Paper received for publication 10.01.10; Accepted 21.06.10] © 2010 EVJ Ltd 12 EQUINE VETERINARY JOURNAL Equine vet. J. (2010) 42 (Suppl. 38) 12-16 doi: 10.1111/j.2042-3306.2010.00242.x