Med. Weter. 2014, 70 (9) 568
Praca oryginalna Original paper
Long-distance endurance rides are the kind of strenu-ous effort that triggers stress and many metabolic, hor-monal, and immunological changes in the horse’s body, which lead to systemic effects, including the exercise-induced acute-phase response (APR). A typical APR is defined as the first non-specific response to any distur-bances in homeostasis, and has been widely described as a systemic response during inflammation caused by both infectious and non-infectious factors (7, 11, 12, 18, 24). At the site of injury of any origin, vascular and cellular responses are activated within minutes, and pro-inflammatory cytokines are released by macrophages. These reactions, in turn, promote the production of more pro-inflammatory cytokines: interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and especially interleukin-6 (IL-6) and mediators, which diffuse to the extracellular fluid and circulate in the blood. These cytokines activate receptors on various target cells and conduct the immune response by promoting further changes, including the
synthesis of acute-phase proteins (APPs) in the liver (7, 18, 24). The production of APPs is one of the most important components of the acute-phase response, and their concentrations in serum closely reflect the onset of the APR (7, 12, 18), although several important species-specific variations clearly occur (5, 18). The pattern of protein synthesis in the liver is drastically altered within a few hours after the inflammatory stimulus. The shift towards APP production is promoted by at least five re-dundant cytokines (11), but TNF-α, IL-1, and especially IL-6 play a key role in this process by activating hepa-tocytic receptors (7, 11, 24). The reaction is suppressed by IL-1, IL-4, APPs that modulate cytokine production, and locally by Kupffer cells, which secrete either IL-6 (when stimulated by pro-inflammatory cytokines) or IL-10, which results in the suppression of local IL-6 production (7). If the inflammatory response is limited spontaneously or by treatment, the level of acute-phase proteins returns to normal within days or weeks.
Changes in blood cytokine concentrations
in horses after long-distance endurance rides
ANNA CYWINSKA, AGNIESZKA TURŁO, LUCJAN WITKOWSKI*, EWA SZARSKA**, ANNA WINNICKA
Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Poland
*Laboratory of Veterinary Epidemiology and Economic Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Poland
**Military Institute of Hygiene and Epidemiology, Warsaw, Poland
Received 08.04.2014 Accepted 08.07.2014
Cywinska A., Turło A., Witkowski L., Szarska E., Winnicka A.
Changes in blood cytokine concentrations in horses after long-distance endurance rides
Summary
Long-distance endurance rides involve strenuous effort, which induces numerous changes in the horse’s body, including the exercise-induced acute-phase response (APR). Such a reaction has also been reported in humans and dogs after exertion, but it varies depending on the species and the type of exercise. In horses, the exercise-induced APR is manifested as an increase in serum amyloid A (SAA) after the effort, but the mechanism and regulation of this process has not been clearly understood. The aim of this study was therefore to determine the changes in the concentration of cytokines that are believed to regulate this type of reaction.
Twelve horses competing in 120 km and 160 km endurance rides were included in the study. The routine haematological and biochemical blood test, as well as the measurements of SAA, IL-1, IL-4, IL-6, IL-10, and TNFα, were carried out before and after competitions. Typical haematological changes and increases in SAA levels were accompanied by increases in IL-6 and IL-10 concentrations, which were also positively correlated before the effort.
Taking into account the sampling time, it is postulated that the exercise-induced APR is promoted by type 1 cytokines. It has also been concluded that the exercise-induced APR in horses in regular training is accompanied by strong a anti-inflammatory response, which prevents clinical disorders after long-distance rides. Then, the overall “anti-inflammatory state” defined previously in race horses is also likely to occur in endurance horses.
Med. Weter. 2014, 70 (9) 569 Strenuous exertion has been shown to induce unique
patterns of the acute-phase response in humans (6, 16, 19, 22), dogs (25, 26), and horses (2-4, 14, 15). These reactions are believed to be related to skeletal muscle damage and differ from a typical APR in inflammation. In humans, the exercise-induced APR was characterized by changes in pro-inflammatory cytokines, C-reactive protein (CRP), haptoglobin, and hepcidin levels (6, 16). In dogs competing in endurance and moderate-duration racing, the level of CRP increased after the race, but, in contrast to humans, the concentration of IL-6 remained unchanged (25, 26). In horses, this reaction has been observed after long, but not after moderate, distances in endurance rides, and is characterized by a marked in-crease in the concentration of a major equine APP, serum amyloid A (SAA), but not other acute-phase proteins (3). The blood concentrations of pro-inflammatory cytokines have not been examined in sports horses, but increases in mRNA for interferon-γ (IFN-γ), IL-1, and TNF-α in blood mononuclear cells and increases in mRNA for IL-1 and IL-6 in muscle cells have been reported after treadmill exercise (13). The expression of mRNA for pro-inflammatory cytokines has also been changed at the beginning of race training, as reported by Horohov et al. (10) in 2-year-old thoroughbred horses. No data on changes in cytokine concentrations or mRNA expression in endurance hoses are available.
Thus, the aim of this study was to determine the cy-tokine profile in the exercise-induced APR in endurance horses.
Material and methods
Horses and competition. Twelve endurance-trained Ara-bian horses (3 mares, 3 stallions, and 6 geldings, 8-15 years old) participating in long-distance endurance rides in Poland, CEI (Concours Endurance International) 2* (120 km) and CEI (Concours Endurance International) 3* (160 km, champion-ships competition), were included in this study. All horses successfully completed the distance, and all veterinary health checks were performed during and following the ride and routine doping control. The horses were privately owned and trained, and were prepared for the start in endurance events by their owners. The owners, Veterinary Commission, and the local Ethical Committee agreed to the procedures. The compe-titions were held in June (160 km ride), July, and September (two 120 km rides) under similar, clear weather conditions, temperature 22-24°C, and in similar terrain (Mazovia). All horses were dewormed and vaccinated at similar times, did not receive medications or suffer from an infection in the preceding 3 weeks (according to the owners).
Blood samples. Peripheral blood samples were obtained by jugular venipuncture before (5.00 a.m.) and 2 hours after the competition. The samples were aspirated into 20 ml sy-ringes and immediately transferred into sterile EDTA tubes for haematological tests and into tubes with no anticoagulant for serum analyses. The EDTA-3K tubes were kept in a re-frigerator (+4°C) and analyzed within 6 hours after collection. Routine hematological parameters: haematocrit (HCT), hae-moglobin concentration (HGB), red blood cell count (RBC), platelet count (PLT), and white blood cell count (WBC), were determined with an automated haematology analyzer (Abacus,
France). Differential counts were determined manually from smears by counting 100 cells, and the neutrophil to lympho-cyte ratio (N:L) was calculated. The tubes with no antico-agulant were centrifuged at 4380 rpm for 5 minutes, serum was aspirated, immediately frozen, and stored at –20°C until analyzed. Serum samples were used for the measurement of creatine phosphokinase (CPK) activity, total protein concen-tration (TP), and serum amyloid A concenconcen-trations. Creatine phosphokinase activity was assayed by the kinetic method (POINTE SCIENTIFIC, USA), and total protein levels were determined with a Biuret Reagent (POINTE SCIENTIFIC, USA).
Serum amyloid A and cytokine (IL-1, IL-4, IL-6, IL-10 and TNF-α) concentrations were measured by a double-sandwich ELISA. PHASE Serum Amyloid A (TRIDELTA Ltd., Ireland) and ELISA Kits for Interleukin 1, 4, 6, 10 and Tumor Necrosis Factor (USCN Life Science Inc.) were used according to the manufacturers’ protocols.
Serum SAA concentrations were corrected for TP concen-trations to avoid the influence of haemoconcentration, which resulted in an increase in total protein level and may falsely increase the concentrations of acute-phase proteins. To com-pare the SAA concentrations before and after exertion, the values obtained after exertion were recalculated taking into account the changes in total protein concentrations according to the following formula:
SAA concentrationcorr = SAA concentration × TP1/TP2, where: SAA concentrationcorr – corrected serum SAA, SAA concentration – serum SAA concentration measured by ELISA kit, TP1 – total protein level before exertion, TP2 – total protein level after exertion.
Statistical analysis. Statistical procedures, means and standard errors of the mean were computed with Statistica 7.0 for Windows. Results are expressed as means ± standard errors of the mean (SEM). The Kolmogorov-Smirnov test in-dicated that the data were not normally distributed. Statistical comparisons by the Mann-Whitney U test were performed to compare the values obtained before and after the rides, and the results from horses participating in 120 km and 160 km rides. Spearman’s correlation was used to test the associations among changes in cytokine and SAA concentrations. P ≤ 0.05 was considered significant.
Results and discussion
The average times for completing the events were 8.09 ± 0.01 h and 11.11 ± 0.10 h – for 120 km and 160 km, respectively. The average speeds at these distances were 15.36 ± 0.54 km/h and 15.25 ± 0.47 km/h, respectively. There were no significant differences between pre- and post-exercise values of the parameters in the horses that completed 120 km and 160 km rides, so they were analyzed together.
All haematological and biochemical (Tab. 1) param-eters determined before the competition varied within normal ranges for equine species (9) in all horses. After the exertion, typical leukogram changes were observed, including a significant increase in WBC, a decrease in lymphocyte numbers (p ≤ 0.05), as well as significant increases in neutrophil numbers, the neutrophil to lym-phocyte ratio (N:L), CPK activity (p ≤ 0.01), and SAA concentration (p ≤ 0.001).
Med. Weter. 2014, 70 (9) 570
In all horses, the concentrations of IL-6 and IL-10 (Fig. 1) increased after the ride, and the post-exercise mean levels of these cytokines were significantly higher (p ≤ 0.05 and p ≤ 0.01, respectively). A positive correla-tion (r = 0.67, p ≤ 0.05) between pre-exercise values of these parameters was observed. The concentration of IL-1 increased in 7 horses after the ride, but decreased in 5. No significant differences between the mean pre- and post-exercise levels of IL-1 occurred. The concen-trations of TNF were similar before and after the ride, and the mean level of this parameter remained almost unchanged. The concentrations of IL-4 were undetect-able (below 31 pg/ml as indicated by the manufacturer) in all horses before the rides, and in most horses afterwards – no significant differences were identified. None of the cytokine levels was in a significant correlation with an increase in SAA concentrations.
In all horses examined, typical (9, 23) changes in leukogram parameters and CPK activity were noted after the ride, indicating exercise-induced stress as described elsewhere (3). Marked increases in SAA concentrations also confirmed the exercise-induced APR in all animals. The cytokine profile showed that IL-6 in involved this process, but the roles of IL-1 and TNF-α were not clearly elucidated.
In a typical APR in inflammation, two types of pro-inflammatory cytokines, acting through different hepa-tocyte receptors, and so that two types of APPs are dis-tinguished (18). IL-1 type cytokines (including IL-1 and TNF-α) elicit a primary autostimulatory signal, which stimulates the secretion of IL-6 in various cells, acting as a secondary signal. This pathway has been proposed to induce the synthesis of the first-line, rapid-reacting APPs (type), including CRP and SAA. The second pathway, IL-6 type dependent, promote the synthesis of the second-line APPs, such as haptoglobin. Additionally, IL-6 synergistically induces the production of type 1 APPs, but this type of cytokines are also believed to exert a negative feed-back on the production of IL-1 type cytokines. In contrast, type 2 APPs are neither induced nor synergistically stimulated by IL-1 (18).
In our study, IL-6 concentrations in blood markedly increased in all horses, and, as a result, increases in SAA levels occurred. We have previously reported that haptoglobin concentrations were not changed after long distance rides (3) and together with the fact that IL-1 concentrations increased in 7 out of 12 horses, so mean pre-and post-exercise values did not differ significantly), type 2 reaction cannot be favored. It should also be taken into account that blood samples examined in our study were collected after exertion that lasted at least 8 hours, when increases in APP concentrations had already oc-curred as a result of changes in the cytokine profile. Thus, it seems likely that the type 1 APR was stimulated, and blood samples were taken when secondary stimulation by IL-6 promoted SAA synthesis and then suppressed IL-1 and TNF-α production.
This hypothesis seems in line with the findings dealing with shorter exertions (10, 13). Two patterns of cyto-kine profiles in blood and muscles have been reported after treadmill exercise in unconditioned horses (13). In blood, transient increases in type 1 cytokine mRNA (TNF-α and IL-1) were observed immediately after exertion and 2 hours later, respectively. In muscles, increases occurred in mRNA for TNF-α (immediately post-exercise) and IL-6 (0.5 hour later), but not for IL-1. The cytokine pattern in blood mach the type 1 reaction. The different muscle pattern was consistent with findings presented in human and equine studies (8, 20, 21), and it has been postulated that the upregulation of IL-6 is a reaction to an acute challenge to muscle metabolism, rather than an APR reflecting muscle damage (13). Other authors (10) observed the upregulation of mRNA for IL-1 and IL-6 in blood following exercise bouts in young thoroughbred horses in training. The increased expression of IL-1β was dependent on exercise intensity and related to the damage of muscle fibers, as indicated Tab. 1. Haematological and biochemical parameters
Parameter Before competition After competition RBC [× 1012/l] 9.39 ± 0.35 9.62 ± 0.52 HCT [%] 37.95 ± 0.8 40.16 ± 3.85 HGB [g/dl] 13.51 ± 0.35 14.1 ± 0.62 WBC [× 109/l] 8.12 ± 0.72 14.54 ± 1.82* Neutrophils [× 109/l] 5.73 ± 0.61 12.14 ± 1.32** Lymphocytes [× 109/l] 3.12 ± 0.49 1.95 ± 0.19* Eosinophils [× 109/l] 0.1 ± 0.07 0.02 ± 0.01 N:L 1.82 ± 0.39 6.23 ± 1.54** CPK [U/l] 151.42 ± 24.59 3455.06 ± 1423.52** SAA [ng/ml] 693.17 ± 222.05 13367.06 ± 1573.76*** Explanations: RBC – red blood cell counts, HCT – haematocrit, HGB – haemoglobin concentration, WBC – white blood cell counts, N:L – neutrophil to lymphocyte ratio, CPK – creatine phosphokinase, SAA – serum amyloid A. Significant differen-ces between the values before and after competition: *p < 0.05; **p < 0.01; *** p < 0.001
Fig. 1. Cytokine concentrations
Explanations: IL-1 – interleukin 1, IL-4 – interleukin 4, IL-6 – interleukin 6, IL-10 – interleukin 10, TNF-α – tumor necrosis factor-α 0 50 100 150 200
IL1 IL4 IL6 IL10 TNFα
pg/mol
Before exercise After exercise
Med. Weter. 2014, 70 (9) 571 by the concentration of malondialdehyde. The authors
also emphasize the overall “anti-inflammatory state” in trained horses, confirmed by a decreased baseline of the expression of type 1 cytokines over the training period (IL-1β and TNF-α). Such a condition has also been documented by other authors (17, 27). Cappelli et al. (1) have shown that genes involved in inflammatory reaction and known to be modulated during effort (TLR4, IL-1β, IL-1RII, IL-18, IL-6 and CEBPβ) are expressed to a greater extent in race horses than in sedentary animals. However, a time-course comparison in athletic horses revealed that genes exhibiting the highest levels of expression at rest did not show significant changes after various endurance races. It has been suggested that the genes expressed at higher levels were more able to respond to homeostatic changes induced by the race, and further production of mRNA transcripts was unnecessary. Thus, it can be interpreted as an adaptation reaction. These authors, however, exam-ined athletic horses before and after various rides (of unknown duration), so the onset of changes is not well defined.
The expression of IL-6 mRNA in a study by Horohov et al. (10) was inversely correlated with proinflammatory cytokine mRNA, and the authors interpreted it as reflect-ing the mechanism that generated an anti-inflammatory environment (10). The results obtained in our study can-not be directly compared with those for race horses or unconditioned horses exercised on a treadmill, because of certain differences in the type and duration of exercise and so that sampling time. However, they are largely in line with the hypothesis presented by Horohov et al. (10). In endurance horses, the exercise-induced APR has been identified after long-distance competitions, but no clini-cal consequences occur. Thus, it seems to confirm the general anti-inflammatory conditions in horses prepared for strenuous competitions. This is additionally con-firmed by increases in IL-10 concentrations, positively correlated with IL-6 levels before exertion.
In conclusion, changes in the cytokine profile after long-distance competitions in endurance horses mach the APR promoted by type 1 rather than type 2 cytokines, as confirmed by the increase in SAA concentration. We also favor the hypothesis indicating the general anti-inflammatory state, as indicated by the increased IL-10 concentration after exertion and the lack of clinical con-sequences regardless of the increased SAA level. There is little evidence for such a state in race horses, and in endurance horses a similar condition has been reported for the first time in our study. Further investigations are therefore needed to understand the onset and cytokine regulation of the exercise-induced APR resulting from endurance effort.
References
1. Cappelli K., Barrey E., Capomaccio S., Felicetti M., Silvestrelli M., Supplizi
A. V.: Exercise influence on gene-expression profile in endurance horse PBMC.
Ital. J. Anim. Sci. 2009, 8 (Suppl. 2), 730.
2. Cywinska A., Gorecka R., Szarska E., Witkowski L., Dziekan P., Schollen-
berger A.: Serum amyloid A as a potential indicator of the status of endurance
horses. Equine Vet. J. 2010, 42, (Suppl 38), 23-27.
3. Cywińska A., Szarska E., Gorecka R., Witkowski L., Hecold M., Berezowski A.,
Schollenberger A., Winnicka A.: Acute phase protein concentrations after
limited distance and long distance endurance rides in horses. Res. Vet. Sci. 2012, 93, 1402-1406.
4. Cywinska A., Witkowski L., Szarska E., Schollenberger A., Winnicka A.: Serum amyloid A (SAA) concentration after training sessions in Arabian race and endurance horses. BMC Vet. Res. 2013, 9, 91. doi: 10.1186/1746-6148-9-91. 5. Eckersall P. D.: Recent advances and future prospects for the use of acute
phase proteins as markers of disease in animals. Vet. Med. Rev. 2000, 151, 577-584.
6. Fallon K. E.: The acute phase response and exercise: the ultramarathon as prototype exercise. Clin. J. Sport Med. 2001, 11, 38-43.
7. Gruys E., Toussaint M. J. M., Niewold T. A., Koopmans S. J.: Acute phase reaction and acute phase proteins. Journal of Zhejiang University SCIENCE 2005, 11, 1045-1056.
8. Helge J. W., Stallknecht B., Pedersen B. K., Galbo H., Kiens B., Richter E. A.: The effect of graded exercise on IL-6 release and glucose uptake in human skeletal muscle. J. Physiol. 2003, 546, 299-305.
9. Hinchcliff H., Kaneps A., Geor R.: Equine Sports Medicine and Surgery. Saunders 2004, 937-1013.
10. Horohov D. W., Sinatra S. T., Chopra R. K., Jankowitz S., Betancourt A.,
Bloomer R. J.: The effect of exercise and nutritional supplementation on
proinflammatory cytokine expression in young racehorses during training. J. Equine Vet. Sci. 2012, 32, 805-815.
11. Khan F. A., Khan M. F.: Inflammation and acute phase response. Int. J. App. Biol. Pharm. Technol. 2010, 1, 313-321.
12. Kilicarslan A., Uysal A., Roach E. C.: Acute Phase Reactants. Acta Med. 2013, 2, 2-7.
13. Liburt N. R., Adams A. A., Betancourt A., Horohov D. W., McKeever K. H.: Exercise-induced increases in inflammatory cytokines in muscle and blood of horses. Equine Vet. J. 2010, 42, (Suppl 38), 280-288.
14. Niedźwiedź A., Kubiak K., Nicpoń J.: Plasma total antioxidant status in horses after 8-hours of road transportation. Acta Vet. Scand. 2013, 55, doi: 10.1186/1751-0147-55-58.
15. Niedźwiedź A., Zawadzki M., Filipowski H., Nicpoń J.: Influence of 8-hour road transportation on selected physiological parameters in horses. Bull. Vet. Inst. Pulawy 2012, 56, 193-197.
16. Peeling P., Dawson B., Goodman C., Landers G., Trinder D.: Athletic induced iron deficiency: new insights into the role of inflammation, cytokines and hormones. Eur. J. App. Physiol. 2008, 103, 381-391.
17. Pedersen B. K.: The anti-inflammatory effect of exercise: its role in diabetes and cardiovascular disease control. Essays Biochem. 2006, 42, 105-117. 18. Petersen H. H., Nielsen J. P., Heegaard P. M. H.: Application of acute phase
protein measurements in veterinary clinical chemistry. Vet. Res. 2004, 35, 163-187.
19. Scharhag J., Meyer T., Gabriel H. H. W., Schlick B., Faude O., Kindermann W.: Does prolonged cycling of moderate intensity affect immune cell function? Br. J. Sports Med. 2005, 39, 171-177.
20. Steensberg A., Toft A. D., Schjerling P., Halkjaer-Kristensen J., Pedersen B. K.: Plasma interleukin-6 during strenuous exercise: role of epinephrine. Am. J. Physiol. Cell Physiol. 2001, 281, C1001-1004.
21. Streltsova J. M., McKeever K. H., Liburt N. R., Gordon M. E., Filho H. M.,
Horohov D. W., Rosen R., Franke W.: Effect of orange peel and black tea
extracts on markers of performance and cytokine markers of inflammation in horses. Equine Comp. Exerc. Physiol. 2006, 3, 121-130.
22. Suzuki K., Peake J., Nosaka K., Okutsu M., Abbiss C. R., Surriano R.,
Bishop D., Quod M. J., Lee H., Martin D. T., Laursen P. B.: Changes in markers
of muscle damage, inflammation and HSP70 after an Ironman triathlon race. Eur. J. App. Physiol. 2006, 98, 525-534.
23. Szarska E.: Investigations of blood parameters for evaluating of health status and training effects in race and sport horses. Zeszyty Naukowe AR, Wroclaw 2003, 471, 1-115.
24. Tizard I. R.: Veterinary immunology, an introduction. 8th ed., Saunders Elsevier
2009.
25. Wakshlag J. J., Kraus M. S., Gelzer A. R., Downey R. L., Vacchani P.: The influence of high-intensity moderate duration exercise on cardiac troponin I and C-reactive protein in sled dogs. J. Vet. Int. Med. 2010, 24, 1388-1392. 26. Wakshlag J. J., Stokol T., Geske S. M., Greger C. E., Angle C. T., Gillette R. L.:
Evaluation of exercise-induced changes in concentrations of C-reactive pro-tein and serum biochemical values in sled dogs competing in a long-distance endurance race. Am. J. Vet. Res. 2010, 71, 1207-1213.
27. Woods J. A., Vieira V. J., Keylock K. T.: Exercise, inflammation, and innate immunity. Immunol. Allergy Clin. North Am. 2009, 29, 381-393.
Corresponding author: dr hab. Anna Cywinska, Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Poland; e-mail: anna_cywinska@sggw.pl
