There have been many reports of altered immune function after acute exercise, both increase and decrease, depending on the immune cell investigated (Rowbottom and Green, 2000). The influence of chronic exercise has also been studied in both animal and human models. Several studies using training protocols of varying durations and intensities in human and different animal species support the findings of increased resting levels of endogenous cytotoxicity after voluntary exercise (Pedersen and Hoffman-Goetz, 2000).

Strenuous exercise increases production of several pro- and anti-inflammatory cytokines, endogenous cytokine inhibitors and chemokines (Pedersen et al., 2001). Unaccustomed and strenuous exercise can also cause neutrophil activation and muscle damage that involves protein degradation and ultra structural changes (Komulainen et al., 1998; Peake et al., 2005; Sorichter et al., 1999). High intensity exercise results in neutrophil translocation to the active skeletal muscle, subsequently resulting in oxidative stress. High blood levels of neutrophil myeloperoxidase (MPO) and elastase levels can be elevated immediately post-exercise (Quindry et al., 2003). In addition, phagocyte infiltration in to muscle is found after strenuous exercise (Sorichter et al., 1999; Aoi et al., 2004; Niess et al., 1999; Tidball et al., 1995). Several studies demonstrated that exercise induces production of cytokines, especially interleukin-6 (IL-6) and interleukin-1 receptor antagonist (IL-1ra) (Ostrowski et al., 2000; Ronsen et al., 2002; Rosendal et al., 2005). Ostrowski et al., 2000. found that marathon running resulted in a 100-fold increase in plasma IL-6 concentration whereas a less strenuous treadmill running resulted in only a 25-fold increases.

Plomgaard et al., 2005 reported that skeletal muscle is recently recognized as an endocrine organ with the capacity to produce signaling peptides in response to muscle contractions. It has also been demonstrated that resting healthy human muscles express cytokines in a fiber type specific manner, such as tumor necrosis factor (TNF)-alpha and interleukin-18 (IL-18) is solely expressed by type II fibers, whereas the expression of IL-6 in more prominent in type I compared to type II fibers (Plomgaard et al., 2005).

Several experimental and clinical evidences show that IL-17 family members are involved in specific inflammatory processes leading to the mobilization of granulocytes. Most published evidence supports a role for IL-17A and possible IL-17F, as a promoter of granulopoeisis, neutrophil accumulation, and neutrophil activation in the lung, joint space, central nervous system, and intestinal tissue (Kolls and Linden, 2004). In human, it has been demonstrated that IL-17, a cytokine derived from activated CD4 cells, increases the release of the major human chemoattractant, the C-X-X chemokine, interleukin-8 (IL- 8), in human bronchial epithelial and venous endothelial cells in vitro (Laan et al., 1999). On the other hand, it has been showed that IL-17 increases neutrophil elastase and myeloperoxidase concentration in rat airways in vivo (Hoshino et al., 2000). However, the positive in vivo finding on IL-17 contrasts with the negative finding in vitro (Hoshino et al., 2000; Prause et al., 2004).

Although IL-17 is a T cell-derived, proinflammatory cytokine that is suggested to be involved in the development of various inflammatory diseases (Nakae et al., 2003; Sigal, 2004), IL-6 secretion (Borish and Steinke, 2003) and neutrophil activation in tissue (Hoshino et al., 2000), there is not any knowledge about the effects of moderate or strenuous exercise on its production. Likewise, the role of IL-17 and its association with neutrophil activation in inflammatory process in skeletal muscle has not been elucidated in exercise models. Additionally, acute effects of moderate and strenuous running exercise on immune functions have not been evaluated in trained athletes. In this study, we aimed to compare the acute effects of single bout of moderate and strenuous exercise on the production of IL-17, IL-6, IL-1ra and inflammatory response of skeletal muscle in regularly trained rats.

Twenty-five Wistar-Albino adult male rats were used in the study. Principles of laboratory animal care (NIH publication) were followed, and Inonu University Ethic Committee of Experimental Animals (Malatya, Turkey) approved this study protocol. The control (n = 10), 30 min (moderate) exercise (n = 8) and 60 min (strenuous) exercise (n = 7) groups of rats were housed in three groups in colony cages, at ambient temperature of 23 °C with a 12-h/12-h light/dark cycle. The animals had free access to water and pellet chow containing 1.8-2.2 % of calcium, 1.1 % of phosphorus and 2650 kcal/kg energy.

Moderate and strenuous exercises were performed by using an animal treadmill system that allows to four animals to run at the same time. In this study, we used a training protocol for treadmill running described by Rico et al., 1999. As previously described in above cited article, our training protocol was consisted of treadmill running 5 days per week for 13 weeks. The steep grade treadmill incline was used to stimulate high- intensity muscle activity in rats. The rats in moderate and strenuous exercise groups started training 8 weeks old and acquainted to the treadmill exercise with a minimal progression during a week. In this period, running time was gradually increased from 15 min to 30 and 60 min per session for moderate and strenuous exercise groups, respectively. Treadmill speed increased to 45 cm·sec^-1 with increments of 2-3° to reach a final grade inclination of 18°. The chronological progression in the treadmill speed, grade inclination, and running time are shown in the Table 1 and Table 2. Control rats were kept in the cages at the same environmental conditions and inspected daily to control their health.

To evaluate the acute effects of single bout moderate and strenuous exercise, an additional single bout running was performed 48 hours after the training protocols were over. Ten minutes after a single bout of treadmill training (13 weeks), blood samples were withdrawn from the abdominal aorta under intraperitoneal ketamine and xylasine anesthesia. Serum IL-6, IL-17 and IL-1ra levels were measured by ELISA (Organon Technica Microwell, Italy) using commercial ELISA kits (rat IL-1ra and IL-6 Biosource International, Inc., USA; rat IL-17 Market Inc., USA). The minimum detectable limits of IL-1ra, IL-6 and IL-17 were to be 12, 8, 5 pg·ml^-1, respectively.

In order to evaluate inflammation in skeletal muscles, MPO activity of gastrocnemius muscle was also determined by biochemical technique using UV-Visible Spectrophotometer (Shimadzu UV-1601, Japan) (Bradley et al., 1982). Tissue samples, approximately 200 mg, were homogenized with homogenizer (Tempest Virtishear, Model 278069; The Virtis Company, Gardiner, NY, USA) in 3 ml of 50 mmol·l^-1 potassium phosphate buffer, pH 6. One milliliter of homogenate was centrifuged at 10,000 g for 10 min at 4°C, and pellet was suspended in 1 ml of (50 mmol·l^-1) potassium phosphate buffer, containing 0.5% hexadecyl-trimethyl ammonium bromide (Sigma) to negate peroxidase activity of hemoglobin and myoglobin and to solubilize membrane-bound MPO. Oxidized o-dianisidine forms a stable chromophore absorbing at a 460 nm wavelength. One unit of MPO activity is defined as that required, to degrade 1 µmol of H₂O₂ per minute at 25°C. Protein concentration was determined with Lowry method (Lowry et al., 1951). Values were calculated as mU of MPO per milligram of protein.

SPPS for Windows 13.0 version was used to analyze of data. Normality of data was analyzed by Shapiro Wilks normality test. Because of the not normal distribution of all variables (p < 0.05), Wilcoxon tests were used to compare body weights of rats between pre- and post-exercise periods. Kruskal-Wallis and Bonferroni Mann-Whitney U tests were used to compare variables among the groups. Spearman’s correlation was also used to correlate variables, and p < 0.05 was regarded to be statistically significant.

Initial body weights of rats in control, moderate and strenuous exercise groups and their alterations at the end of training protocol were shown in the Table 3. Increase in body weight at the end of training was found to be significant only in moderate exercise group when compared to initial values (p < 0.01).

Mean and standard errors of serum IL-1ra levels were found 86.50 ± 19.87; 107.50 ± 14.33; 121.91 ± 22.34 pg/ml in control, moderate and strenuous exercise groups, respectively. They were 56.37 ± 13.58; 232.93 ± 104.63; 165.03 ± 87.18 pg·ml^-1 for IL-6 and, 52.41 ± 17.98; 90.86 ± 35.13; 308.74 ± 74.60 pg·ml^-1 for Il-17. Additionally, MPO activities of gastrocnemius muscle were also 26.13 ± 1.43; 26.56 ± 0.95; 32.37 ± 5.24 U·g^-1 wet tissue in the above mentioned groups, respectively.

Comparison of serum IL-1ra, IL-6, IL-17 levels and muscle MPO activity of rats in control, moderate and strenuous exercise groups were also shown in the Figure 1. In both moderate and strenuous exercise groups, there were no significant changes in serum IL-6, IL-1ra levels or MPO activity when compared to those of sedentary controls. However, IL-17 level was significantly increased in the strenuous exercise group (p < 0.01) whereas not changed in the moderate exercise group when compared to the controls (p > 0.05). There was not any significant change in any parameters between two exercise groups. Additionally, in moderate exercise group, there was a negative correlation between IL-6 and IL-17 levels (r = -0.857 and p = 0.014).

Our results show that a single bout of strenuous exercise increases serum IL-17 level in rats whereas a single bout of moderate exercise does not change its production in rats trained for two types of exercise model by treadmill running for 13 weeks. In our study, we also found that single bouts of both moderate and strenuous exercise elevated serum IL-6 levels in trained groups of rat, but these elevations were not statistically different from the basal levels of control group. Additionally, IL-1ra level slightly increased in both exercise groups and this result was not correlated to increase in serum IL-6 level in these exercise groups. In both moderate and strenuous exercise groups, there were no significant changes in MPO activities in gatrocnemius muscle and in blood lactate levels when compared to those of controls.

Skeletal muscle has been recognized as an endocrine organ, and muscle cell cultures express several cytokines with potential hormonal effects (Akerstrom et al., 2005). Contracting muscle fibers produce and release IL-6, and plasma levels of this cytokine are markedly elevated in response to physical exercise (Keller et al., 2005). Increased plasma IL-6 is observed after intense, prolonged exercise (Haddad et al., 2005; Rosendal et al., 2005).

It is well known that IL-6 stimulates the production of IL-1ra, which binds to and blocks the IL-1 receptor, thus exerting strong anti-inflammatory effects. With exercise, peak IL-1ra is found 1-2 h after the peak of IL-6. From this, it is assumed that the level of IL-1ra reflects the production of IL-6 (Ronsen et al., 2002). In our study, insignificant increases in IL-6 levels in moderate and strenuous exercise groups may be due to limited number of animals in control and exercise groups or the training protocols we used. However, our findings indicate that IL-6 production may not correlate with the duration of chronic running exercise because the elevation of IL-6 in the strenuous exercise group was less than the moderate exercise group (Figure 1). We found that serum IL-1ra level slightly increased in both exercise groups and this result was not correlated to non-significant increase in serum IL-6 level in these exercise groups. The elevation of IL-6 level may be related to duration of exercise. It was demonstrated that there was a negative correlation between serum IL-6 concentration and running time and positive correlation between plasma IL-6 concentration and running intensity (Ostrowski et al., 2000).

Based on our present data and the literature, one could speculate that a single bout of strenuous exercise increases IL-17 production. This cytokine activates macrophages and fibroblasts, secretion of cytokines and stromal cells, including their expression of intercellular adhesion molecule-1 (ICAM-1), and secretion of cytokines (IL-6, IL-8, IL-11), granulocyte-colony stimulating factor [G-CSF]), prostaglandin E2, and nitric oxide (Borish et al. 2003; Sigal, 2004). MPO activity is another marker of neutrophil activation. It is a catalyzing enzyme that metabolizes superoxide radicals produced during neutrophil activation (Dularay et al., 1990). It has been speculated that IL-17 can activate neutrophils in association with their recruitment into the airways in vivo (Hoshino et al., 2000). However, similar activation of MPO by IL-17 for the skeletal muscle is not known. In our study, in contrast to plasma IL-6 level, tendencies of increases in serum IL-1ra and IL-17 are parallel to the duration of treadmill exercise. Increase in MPO levels of gastrocnemius muscle of rats especially in strenuous exercise group may also be related to the duration of running exercise. In the whole blood or plasma, biochemical markers strictly correlated with MPO activity may help to determine tissue damage in skeletal muscle of human. From our results, serum or plasma IL-17 level may be a useful biochemical marker to determine acute exercise-induced inflammation in skeletal muscles of trained animals or human subjects. However, we did not show any significant correlation between serum IL-17 level and muscle MPO activity because of limited number of rats in our experimental groups.

To maintain resistance training goals intensity and volume are important variables to be manipulated. Moreover, the present study showed that the rest interval time between sets and exercises is also a critical variable to meet different training goals; therefore it directly affects the number of repetitions during the progression of the sets, the total number of repetitions per exercise and the total number of repetitions of the session. These results indicate that these values can be reduced by the use of shorter intervals (two versus five-minute), and these reductions are less evident during the progression of the sets in the initial exercises. However, the influence of the rest intervals between sets and exercises in a training session is still a controversial issue in the literature, since it is highly related with the order in which a certain exercise is placed in the session. This way, studies that further investigate the influence of the relationship between different rest intervals and exercises orders for the same training session are recommended, especially in daily practical sessions, where frequently more than one exercise for the same or distinct muscle group are used.