Exercise-induced increases in inflammatory cytokines in muscle and blood of horses

Authors

  • N. R. LIBURT,

    1. Equine Science Center, Department of Animal Sciences, Rutgers State University of New Jersey; and Maxwell H. Gluck Equine Research Center, Department of Veterinary Sciences, University of Kentucky, USA.
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  • A. A. ADAMS,

    1. Equine Science Center, Department of Animal Sciences, Rutgers State University of New Jersey; and Maxwell H. Gluck Equine Research Center, Department of Veterinary Sciences, University of Kentucky, USA.
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  • A. BETANCOURT,

    1. Equine Science Center, Department of Animal Sciences, Rutgers State University of New Jersey; and Maxwell H. Gluck Equine Research Center, Department of Veterinary Sciences, University of Kentucky, USA.
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  • D. W. HOROHOV,

    1. Equine Science Center, Department of Animal Sciences, Rutgers State University of New Jersey; and Maxwell H. Gluck Equine Research Center, Department of Veterinary Sciences, University of Kentucky, USA.
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  • K. H. McKEEVER

    Corresponding authorSearch for more papers by this author

Summary

Reasons for performing study: Studies have demonstrated increases in mRNA expression for inflammatory cytokines following exercise in horses and have suggested those markers of inflammation may play a role in delayed onset muscle soreness. However, measurement of mRNA expression in white blood cells is an indirect method. No studies to date have documented the cytokine response to exercise directly in muscle in horses.

Hypothesis: This study tested the hypothesis that exercise increases cytokine markers of inflammation in blood and muscle.

Methods: Blood and muscle biopsies were obtained from 4 healthy, unfit Standardbred mares (∼500 kg). The randomised crossover experiment was performed with the investigators performing the analysis blind to the treatment. Each horse underwent either incremental exercise test (GXT) or standing parallel control with the trials performed one month apart. During the GXT horses ran on a treadmill (1 m/s increases each min until fatigue, 6% grade). Blood and muscle biopsies were obtained 30 min before exercise, immediately after exercise and at 0.5, 1, 2, 6 and 24 h post GXT or at matched time points during the parallel control trials. Samples were analysed using real time-PCR for measurement of mRNA expression of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 (IL-1). Data were analysed using t tests with the null hypothesis rejected when P<0.10.

Results: There were no changes (P>0.10) in IL-1, IL-6, IFN-gamma or TNF-alpha during control. Exercise induced significant increases in IFN-gamma, IL1 and TNF-alpha in blood and significant increases in IFN-gamma, IL-6 and TNF-alpha in muscle. There were no significant changes in mRNA expression of IL-1 in muscle or IL-6 in blood following the GXT. These cytokine markers of inflammation all returned to preGXT levels by 24 h post GXT.

Conclusion: High intensity exercise results in a transient increase in the expression of inflammatory cytokines in muscle and blood.

Introduction

A growing body of research has demonstrated increases in mRNA expression for a variety of cytokines following exercise in man, horses and other species (Haahr et al. 1991; Pedersen et al. 1998; Moldoveanu et al. 2001; Malm 2002; Keller et al. 2004; Streltsova et al. 2006; Liburt et al. 2009). The cytokines are part of a complex set of signalling pathways that interplay with the immune and endocrine system as part of the multifaceted response to a variety of physiological challenges including exercise (Moyna et al. 1996; LaManca et al. 1999; Pedersen and Hoffman-Goetz 2000). The growing list of actions of these low molecular weight proteins are complex with many involved in the modulation of immune function and inflammation (LaManca et al. 1999; Pedersen and Hoffman-Goetz 2000; Malm 2002). To that end, a number of papers have examined the effects of exercise on immune function and cytokines in horses (Horohov et al. 1996, 1999; Ainsworth et al. 2003; Streltsova et al. 2006; Donovan et al. 2007; Adams et al. 2009; Liburt et al. 2009). For example, Horohov et al. (1996) found that the interleukin-2 (IL-2) exercise-induced augmentation of lymphokine activated killer (LAK) cell activity was associated with altered IL-2 response following exercise. In another study, it was reported that there were differences between the cytokine and immune response to exercise in young vs. old horses (Horohov et al. 1999). Other investigations have focused on clinical situations reporting a relationship between exercise, endotoxaemia and cytokines in horses (Barton et al. 2003). Data are controversial in more recent studies that have examined the cytokine response in blood collected from horses, with some papers reporting that cytokine levels increase following physical activity, while other papers have found that mRNA expression for cytokine in white blood cells remained unchanged. For example, Ainsworth and co-workers (2003) found that there was no change in mRNA expression of interleukin-12 (IL-12), IFN-gamma and interleukin-4 (IL-4) equine by mononuclear cells 24 h after strenuous physical activity. Similarly, Colahan et al. (2002) reported that prolonged acute exercise did not alter mRNA expression for IL-2, IL-4, IL-6 or IL-10. However, they did document an effect on mRNA for TNF-alpha and IL-1 at 23 and 30 days post exercise. However, this lack of a response is in contrast to 2 recent studies (Streltsova et al. 2006; Liburt et al. 2009) that reported increases in TNF-alpha and IFN-gamma in the first hours following an intense acute exercise challenge. Timing of the sampling may be the key difference between the studies reported in the literature (Streltsova et al. 2006; Liburt et al. 2009) as the later studies obtained blood samples immediately after exercise with intervals spaced out to 24 h post exertion. The responses of all the studies mentioned above also reflect the use of blood as a marker for inflammation, which is an indirect marker as it reflects a whole body response and not the response of key metabolically active tissues such as muscle.

Functionally, the cytokines play an important role in initiating the inflammatory response to cell damage, an important response that is more than just a set of symptoms or cardinal signs such as rubor, tumor, calor and dolor (Pedersen and Hoffman-Goetz 2000; Malm 2002; Suzuki et al. 2002). When it comes to the inflammatory response to cell damage, some cytokines have been classified as ‘pro-inflammatory’, some as ‘anti-inflammatory’ and some have multiple roles, even modulating the effects of other cytokines (Ostrowski et al. 1999; Pedersen and Hoffman-Goetz 2000; Malm 2002; Suzuki et al. 2002). At its worst, inflammation can lead to severe secondary problems and damage; however, short of this, it can be part of a sequelae of protective symptoms that, when it comes to exercise, can result in a loss of performance capacity (Pedersen and Hoffman-Goetz 2000; Malm 2002; Peake et al. 2005a,b). Exertion-related inflammation can result if the intensity, resistance or duration of an acute bout of exercise is excessive enough to cause muscle cell damage (Northoff et al. 1998; Ostrowski et al. 1998b; Pedersen and Hoffman-Goetz 2000; Malm 2002). If severe enough, this damage results in a larger inflammatory response with a release of cell enzymes, a cascade of pro-inflammation cytokines and an endocrine response that many studies of nonequine species have characterised as markers of delayed onset muscle soreness (DOMS) (Northoff et al. 1998; Ostrowski et al. 1998b; Pedersen and Hoffman-Goetz 2000; Malm 2002; Peake et al. 2005a,b). DOMS is complex, multifaceted and the cellular changes required to affect performance varies from individual to individual (Peake et al. 2005a,b). However, not all inflammation is bad and many cytokines modulate the inflammatory response and in that capacity facilitate a host of other processes that important for the repair of tissue (Malm 2002; Youdim et al. 2002; Vassilakopoulos et al. 2003; Peake et al. 2005a,b; Nieman et al. 2007). This ‘good’ inflammation is an appropriate part of the adaptive response to repeated bouts of exercise. This inflammation initiates the signalling for protein synthesis which ultimately leads to muscle hypertrophy to increase strength and performance. Therefore, recent studies have tried to find markers of subtle damage that can be used to prevent or minimise the ergolytic effects of DOMS (Nieman et al. 2007). Treatments that can modulate the cytokine response to exercise may be efficacious in preventing loss of performance while maintaining the signaling needed to produce adaptation and hypertrophy (Youdim et al. 2002; Vassilakopoulos et al. 2003; Peake et al. 2005a,b; Nieman et al. 2007). To that end, measurements of plasma cytokine concentrations are difficult due to a variety of factors including low concentrations and rapid clearances from the blood. Furthermore, plasma concentrations are tissue nonspecific and do not necessarily reflect changes in muscle. Similarly, measurement of mRNA expression in white blood cells is also an indirect method. While there is limited information on the cytokine response in muscle in man (Malm 2002; Nieman et al. 2003, 2007) and rodents (Rosa Neto et al. 2009; Batista et al. 2010); there are no published studies to date that have documented the cytokine response to exercise directly in muscle in horses. This study tested the hypothesis that exercise increases cytokine markers of inflammation in peripheral blood and muscle.

Materials and methods

Animals and experimental design

The Rutgers University Institutional Animal Care Review Board approved all methods and procedures used in this experiment. Blood and muscle biopsies were obtained from 4 healthy, unfit Standardbred mares (age ∼500 kg) in a randomised crossover experiment that was conducted with the investigators who performed the analysis blind to the treatment. During the trial each horse underwent either an incremental exercise test (GXT) or standing parallel control on round one and the opposite treatment during the second round of the experiment. The trials were performed one month apart to allow complete recovery from all the tests. All the horses were housed as a group on 3 acre dry lots. They were fed ∼6 kg/day alfalfa and grass hay and 3 kg/day of a commercially available grain twice per day, morning and evening. Water was provided ad libitum.

Incremental exercise tests

During the incremental exercise tests, the mares ran on a high-speed horse treadmill (Sato I)1 at a fixed 6% grade (Kearns and McKeever 2002; McKeever et al. 2006). The GXTs started at an initial speed of 4 m/s for 1 min. Speed was then increased to 6 m/s, followed by incremental increases of 1 m/s every 60 s (omitting 5 m/s) until the horses reached fatigue. Fatigue was defined as the point where the horse could not keep up with the treadmill despite humane encouragement (Kearns and McKeever 2002; McKeever et al. 2006). At the point of fatigue, the treadmill was stopped and post exercise blood and muscle samples collected. Blood and muscle biopsies were obtained 30 min before exercise, immediately after exercise and at 0.5, 1, 2, 6 and 24 h post GXT or at matched time points during the parallel standing control trials. The blood samples were collected via venipuncture into tubes containing RNA preservatives for measurement using white blood cells (PAXgene™ Blood RNA Tubes).2 Muscle biopsy samples were obtained from the middle gluteal muscle using sterile techniques, local lidocaine anaesthesia and a Bergstrom biopsy needle. A minimum of 1 g of muscle tissue was placed in a cryovial containing preservatives (RNALater Solution and preservative, Cat # AM7021)3 at 4°C overnight to allow for thorough penetration of the solution into the sample and then frozen at -20°C until processing.

Muscle sample preparation

To prepare the tissue for homogenisation, samples were cut into 1–2 mm pieces and then transferred into a 5 ml tube that was filled with 500 µl of RNA-STAT60 Solution (# NC9489785)4. Using a hand-held homogeniser (PowerGen 125)5, several pieces were homogenised into the RNA-Stat60 solution until it appeared cloudy. To avoid contamination between samples, each sample was homogenised using a different plastic generator and generators were washed and sterilised before being used again. The homogenate was transferred into a clean 1.5 ml microcentrifuge tube and the overall volume of the RNA-STAT60 Solution was brought up to 1 ml. Total RNA was subsequently isolated from the tubes using the manufacturer's directions. The RNA was quantified using a spectrophotometer (BioPhotometer)6. In all cases, OD260/280 ratios were greater than 1.9 and RNA yields were greater than 50 µg/ml. One microgram of RNA was reverse transcribed into cDNA in an 80 µl reaction containing 20 units of AMV reverse transcriptase, 0.5 µg of oligo dT primers, 40 units of RNAsin and 5 mmol/l MgCl27.

Real time-polymerase chain reaction (RT-PCR)

Samples were analysed using real time-PCR for measurement of mRNA expression of IFN-gamma, IL-1, IL-6 and TNF-alpha. Total RNA was isolated from the PAXgene tubes using the PAXgene RNA Blood Kit (which can technically be classified as a spin column kit) and RNA from tissue was isolated using phenol-chloroform extraction (BioPhotometer6). The RT reactions resulted in 80 N of cDNA, and each reaction consists of 16 N of AMV reverse transcription buffer (5x), 16 N of MgCl2, 4 N of dNTP, 1 µl RNasin, 1 µl Oligo dT primer, and 0.5 µl of AMV Reverse Transcriptase in addition to the 1 N of RNA. Cytokine-specific cDNA was amplified and quantitated by ‘real-time’ PCR (ABI 7500HT Fast Real-Time PCR System)8, using the Taq thermostable DNA polymerase and primers based on the sequences for equine cytokines and β-glucoronidase (β-gus).Specific primers and FAM-labelled probes for interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-1 (IL-1) and (-gus, provided as kits (ABI, Assay-by-Design)8 were added to 10 µl reactions in 384 well plates, which consisted of 5 µl of TaqMan Gene Expression Master Mix (ABI)8, 0.5 of primer-probe and 4.5 of cDNA. The following PCR conditions were employed; 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s, as recommended by the manufacturer.

Differences in RNA isolation and cDNA construction between samples were corrected using (β-gus as an internal control for each sample (Breathnach et al. 2006). Relative differences in cytokine mRNA expression resulting from exercise were determined by relative quantification. Relative quantification provides accurate comparison between the initial levels of target cDNA in a sample without requiring that the exact copy number be determined (Livak and Schmittgen 2001). The pre-exercise samples were selected as the calibrator and the change in cytokine gene expression post expression relative to the calibrator (because this is a relative measure the convention in the literature is that there are no units for cytokine expression in the graphs) was then determined for each sample.

Statistical analysis

Data were analysed using analysis of variance for repeated measures and t tests when appropriate (Sigmastat version 3.1)9. Data were subjected to and passed tests for normality. An a priori decision was made to reject the null hypothesis when P<0.10 so as to avoid a type II error. The use of a P value <0.10 is acceptable with low sample numbers (Little and Hills 1978) Said resource includes probability tables with the 0.10, 0.05 and 0.01 levels with a reference to Fischer's original calculations and rationale for the justification based on the trade-off made in a priori decisions related to protecting against a type I error vs. making a type II error when sample numbers are low. The reader should be cautioned, however, taking into account the caveat regarding reduced probability due to the use of the 90% rather than 95% level for setting the alpha level.

Results

There were no changes (P>0.10) in IFN-gamma, IL-1, IL-6, or TNF-alpha mRNA expression in the blood or muscle samples collected during the parallel control trial (Figs 1a–d). By contrast, gene expression for cytokines post exercise exhibited 4 different patterns (Figs 2a–d). IFN-gamma mRNA expression in peripheral blood increased (Fig 2a, P<0.10) rapidly following exercise whereas there was a delayed and transient increase (Fig 2a, P<0.10) in its expression in muscle at 1 h post GXT. There was no significant change in mRNA expression for IL-1 in muscle (Fig 2b); however, there was a significant increase in blood that peaked at the sample taken at 2 h post exercise and returned to baseline (Fig 2b, P>0.10) by 24 h of recovery. On the other hand, exercise induced a significant increase in mRNA expression for IL-6 in muscle (Fig 2c) but no change (Fig 2c, P>0.10) in blood. Finally, there was an immediate but short term increase in mRNA expression for TNF-alpha in muscle (Fig 2d, P<0.10) with a delayed increase (Fig 2d, P<0.10) in blood that peaked at 6 h post GXT.

Figure 1.

Mean ± s.e. values for the expression of mRNA for IFN-gamma, IL-1, IL-6 and TNF-alpha in muscle (left) and blood (right) from samples collected during a standing parallel control trial at time points matching those used during the exercise trial depicted in Figure 2.

Figure 2.

Mean ± s.e. values for the expression of mRNA for IFN-gamma, IL-1, IL-6 and TNF-alpha in muscle and blood from samples collected at before (-0.5 h), immediately after (0 h) and at 0.5, 1, 2, 6 and 24 h after a high intensity incremental exercise test. An asterisk (*) denotes a mean that is different (P<0.01) from the pre-exercise (-0.5 h) sample.

Discussion

The major finding of the present study was that there was an upregulation of mRNA for key cytokines in muscle, as well as blood, following intense exercise but no change during the parallel standing control trial. The latter documents that the observed increases were due to exercise, not due to multiple sampling. Studies of humans have documented exercise-induced increases in plasma concentrations of TNF-alpha, IL-1Beta, IL-6, IL-1ra, IL-10 and IFN-gamma during and after exercise (Ostrowski et al. 1998a,b, 1999; Moldoveanu et al. 2001; Pedersen and Hoffman-Goetz 2000; Kimura et al. 2001; Helge et al. 2003; Hiscock et al. 2003; Nieman et al. 2007). Those increases appear to be affected by the intensity, mode (eccentric vs. concentric), and duration of the exercise challenge (Ostrowski et al. 1998a,b, 1999; Pedersen and Hoffman-Goetz 2000). In other studies, Pedersen and Hoffman-Goetz (2000) have documented that a variety of exercise challenges cause increases in urinary concentrations of TNF-alpha, IL-1-beta, IL-6, IL-2 receptors and IFN-gamma (Pedersen and Hoffman-Goetz 2000). Plasma and urinary concentrations are not tissue specific and thus, no inference can be made to the effects of exercise on key tissues such as muscle. To that end, recent investigations of exercising humans have focused on the cytokine response in muscle with a focus on there role in muscle soreness and effects on performance (Peake et al. 2005a,b; Nieman et al. 2007). The present study is the first that documents a similar response in horses. The measurement using muscle biopsies is a more direct measurement in the tissue of interest especially when compared to measurements of plasma concentrations of cytokines or measurement of mRNA upregulation in white blood cells. This is an important consideration since the application of information gained from muscle biopsies is especially useful when examining delayed onset muscle soreness and a variety of countermeasures that can modulate the response to strenuous exercise (Youdim et al. 2002; Vassilakopoulos et al. 2003; Nieman et al. 2003, 2004, 2007; Peake et al. 2005a,b). One should note, however, that the present study as designed only addresses the basic question focused on the effect of exercise on increases in mRNA expression for cytokines in muscle and white blood cells. The study was not designed to examine acute injury or muscle soreness. However, the rationale for the study included the potential application of said measurements in future studies where markers of DOMS or more severe damage could be measured and used to assess a variety of interventions. Thus, the present paper only presents a ‘proof of concept’ experiment addressing the very specific hypothesis that intense exercise would evoke a cytokine mRNA response in muscle as well as in white blood cells.

The second major insight gained from the data generated in the present study centres on the timing of the release of the cytokines in blood vs. muscle. At a minimum the data allows one to make some suggestions as to the origin for their release or stimulus for the release of the cytokines, but only to the extent that it is either intra- or extra muscular in origin. Still, the pattern of release gives some new insight into how exercise affects cytokine production. For example, if the increase is seen in muscle first and then in white blood cells it suggests the stimulus originated in the muscle. On the contrary if the upregulation occurs in blood first and then muscle, this suggests that exercise exerted its stimulating effects on other tissues. Whilst it is true that we do not know the source of the mRNA in the samples, due to current limitations in technology, it is fair to say that we can discriminate between local (i.e. muscle) and systemic (i.e. peripheral blood cell) sources. It is also true that post transcriptional mechanisms likely play a role in regulating cytokine production (Rosa Neto et al. 2009; Batista et al. 2010); however, our results indicate that active transcription of the gene is occurring, indicating a response to exercise has occurred. Additional insight can be gained in future studies if there are simultaneous measurements of cytokine protein levels in plasma. However, since we measured mRNA expression in both PBMC and muscle biopsies this is less of an issue. To that end, the cytokines measured in the present study exhibited responses to exercise that followed 4 different expression patterns. Each of the cytokines and expression patterns will be discussed in brief below.

Interferon-gamma

Interferon-gamma is a major pro-inflammatory cytokine synthesised by T-helper 1 (Th1) and natural killer (NK) cells (Elenkov 2004). It acts on stimulated macrophages, T cytotoxic cells, NK cells, B lymphocytes and is involved in nitric oxide synthesis (Elenkov 2004), antibody production and antiviral activity (Ijzermans and Marquet 1989). Results have been mixed regarding the effects of exercise on IFN-gamma. Most studies of man and species other than horses have shown that excessively intense or long exercise causes an inhibition of IFN-gamma production with some papers suggesting that this leads to immunosuppression (Haahr et al. 1991; Weinstock et al. 1997; Northoff et al. 1998; Kimura et al. 2001; Suzuki et al. 2002). However, it has also been reported that gene expression for IFN-gamma (LaManca et al. 1999) as well as the concentration of IFN-gamma in plasma (Baum et al. 1997; Kimura et al. 2001), are increased during the recovery from exertion (Moyna et al. 1996). Baum et al. (1997) observed increases in IFN-gamma concentration mRNA expression 24 h after moderate exercise but marked decreases in IFN-gamma concentration levels 30 min after exhaustive exercise in man and, therefore, suggested that the IFN-gamma response is affected by the type of exercise performed. Interestingly, studies of horses have demonstrated that intense short term exercise causes an increase in IFN-gamma mRNA expression in the immediate period following exercise (Streltsova et al. 2006; Liburt et al. 2009). The pattern of the upregulation of mRNA for IFN-gamma in the present study is consistent with its role in immune function with an initial rise in the blood followed by a secondary increase in muscle. Since the exercise response in the present study was not of a nature that would cause severe muscle damage one could speculate that the transient increase in IFN-gamma may indicate a role in the adaptive response in muscle following exertion. The increased IFN-gamma expression also parallels the increased LAK cell activity seen post exercise, suggesting that the 2 responses may also be linked (Horohov et al. 1996).

Interleukin-1

Interleukin-1 is a pro-inflammatory cytokine produced by macrophages, monocytes and dendritic cells. This cytokine has 2 distinct subtypes, one (IL-1α) that stays primarily in the cytosol of cells and the second (IL-1β) that becomes active upon cleavage by a protease produced in monocytes (Moldoveanu et al. 2001). An increase in IL-1 is associated with a variety of number of generalised secondary systemic wide responses including fever, lethargy, modulation of inflammation and stimulation of cell proliferation (Moldoveanu et al. 2001). IL-1β upregulates a wide variety of genes and it can boost its own expression as well as that of IL-2 and IL-6 (Moldoveanu et al. 2001). IL-1β can also induce an increase in the synthesis and release of enzymes involved in the synthesis of leucotreines, prostaglandins and nitric oxide (NO) (Moldoveanu et al. 2001), substances involved in the mediation of the sequelae of symptoms associated with an inflammatory response. To that end, IL-1β, when injected into a joint, can cause inflammatory and degenerative changes on joint surfaces as well as bone reabsorption (Moldoveanu et al. 2001). These degenerative effects can be even more pronounced when it acts synergistically with TNF-α to promote catabolism of lean tissue (Moldoveanu et al. 2001).

Data on exercise-induced alterations in IL-1β are mixed with some nonhorse papers documenting that IL-1β is among the first cytokine to increase in response to exercise and some reporting no change due to exertion. The former showed increases in plasma IL-1β concentrations and an upregulation of mRNA expression for IL-1β in blood mononuclear cells in man following running and cycling (Ostrowski et al. 1998a, 1999; Moldoveanu et al. 2001). An increase in IL-1β mRNA was also documented in muscle samples following a marathon (Ostrowski et al. 1998b). Other studies have reported no increase following exercise with low concentrations near the level of detection (Petersen and Hoffman-Goetz 2000). Information on the response to exercise in the horse is very limited; however, one study has documented an upregulation in the expression of mRNA for IL-1 at 23 and 30 days post exercise (Colahan et al. 2002). In the present study, there was a transient upregulation of mRNA expression for IL-1β in blood but not in muscle. This is consistent with its role in inflammation.

Interleukin-6

Systemically, Interleukin-6 has been documented to function in the multifaceted cascade of events involved in the onset and modulation of inflammation (Pedersen and Hoffman-Goetz 2000). In that role there are conflicting studies that suggest IL-6 has both anti-inflammatory and pro-inflammatory roles (Pedersen et al. 2001). However, when it comes to the biological role during exercise, the preponderance of information on the effect of IL-6 demonstrates that it is an important signalling protein in the integrated control of glucose homeostasis in muscle (Steensberg et al. 2000, 2001a,b, 2002, 2003). This has led researchers to classify IL-6 as a ‘myokine’ or a muscle cytokine. In this role IL-6 has been shown to increase when the demand of an exercise challenge alters i.m. glucose concentrations (Pedersen et al. 2001, 2003; Helge et al. 2003; Steensberg et al. 2003). One major observation from human-based studies is that upregulation of IL-6 occurs in muscle but not in the blood when intensity or duration of exercise causes a metabolic challenge without muscle damage (Nieman et al. 1998; Ostrowski et al. 2000; Pedersen et al. 2001, 2003; Helge et al. 2003; Steensberg et al. 2003). The lack of an IL-6 response in the blood of exercising horses in the present study is consistent with studies in man, and similar to the response seen in 2 other investigations involving horses that performed intense short term exercise (Streltsova et al. 2006; Liburt et al. 2009). The important observation in the present study was a significant increase in IL-6 in muscle during exercise but not during parallel control. This increase in the expression of mRNA for muscle IL-6 is consistent with observations made in man and other species (Pedersen et al. 2001, 2003; Steensberg et al. 2003). Other studies of horses subjected to a GXT that measured blood IL-6 as well as muscle enzymes have reported no evidence of muscle damage (Streltsova et al. 2006; Liburt et al. 2009). One could potentially suggest that the response in muscle without a similar response in blood in the present study may suggest that IL-6 plays more of a role in acute challenges to muscle metabolism rather than in a response to muscle damage. This would be consistent with the aforementioned studies from man and other species.

Tumour necrosis factor alpha

Tumour necrosis factor alpha is a pro-inflammatory cytokine that has been used as a general marker of inflammation (Pedersen and Hoffman-Goetz 2000; Suzuki et al. 2002). TNF-alpha, an acute inflammatory cytokine, plays a role in the response to muscle damage (Kimura et al. 2001), muscle proteolysis (Nawabi et al. 1990) and impaired skeletal muscle glucose uptake (Steensberg et al. 2002. Its role in the latter may be associated with the fact that it is expressed in human skeletal muscle and has been shown in mechanistic studies to stimulate IL-6 production (Pedersen et al. 2003). There are mixed results from human studies as far as the TNF-alpha response in blood during and following exercise (Pedersen and Hoffman-Goetz 2000). Some papers document an exercise-induced increase in mRNA expression (Ostrowski et al. 1999; Kimura et al. 2001) and others report no change (Ostrowski et al. 1998b). Still, other experiments have demonstrated decreases (Ferrier et al. 2004; Keller et al. 2004) in TNF-alpha concentrations following exercise. Type of exercise, intensity, and duration are all factors that may be responsible for the divergent nature of the reported effect of exercise in nonhorse studies.

Studies of horses have reported upregulation of mRNA for TNF-alpha in horses during clinical situations where inflammation is present (e.g. arthritis, laminitis. obesity) (Horohov et al. 1996, 1999; Ainsworth et al. 2003; Streltsova et al. 2006; Donovan et al. 2007; Adams et al. 2009). It has also been shown to increase following experimentally-induced inflammation where horses have been challenged with joint lesions or where they have been challenged with pathogens during vaccine development (Horohov et al. 1996, 1999; Ainsworth et al. 2003; Streltsova et al. 2006). Intense exercise has been also been shown to cause an increase TNF-alpha in man (Haahr et al. 1991; Kimura et al. 2001) and horses (Streltsova et al. 2006; Liburt et al. 2009). In the present study, TNF-alpha was increased in the muscle immediately after exercise but returned rapidly to baseline. Interestingly, the increase of TNF-alpha in the blood did not occur until 6 h post GXT. This would suggest a few different scenarios that may explain the pattern for its upregulation. First, acute exercise caused intracellular changes that induced the upregulation in muscle. The secondary increase seen in the blood could be due to spill over of TNF-alpha; or due to secondary signalling stimuli, or due to inflammatory processes in other tissues altered by acute exercise or the recovery process. These explanations are purely speculative; however, the response appears to be 2-fold suggesting 2 separate mechanisms or locations for the stimulus for the upregulation.

In summary, the present study is the first we are aware of in horses to document the cytokine response simultaneously in blood and muscle in a controlled experiment that utilised a parallel control trial to eliminate the effects of repeated sampling. Interpretation of the data suggests that, while a GXT can cause an increase in cytokines, the response is more in line with the low level response needed for adaptation of muscle rather than a massive response associated with the inflammation associated with muscle damage. While the present study did not measure local or whole body concentrations of the cytokine proteins it did measure mRNA upregulation in the tissue of interest, muscle. The application of the information gained in the present investigation is that there is a tool to conduct applied and more mechanistic studies. For example, to screen the effects of various nutraceuticals or drugs with anti-inflammatory properties that may affect the cytokine response to a variety of exercise challenges.

Acknowledgements

The authors would like to thank the graduate and undergraduate students from Rutgers University and the Rutgers Equine Science Center for their contributions to this project. Support was provided by the United States Department of Defense, US Army Natick Soldier Center, the New Jersey State Equine Initiative and the New Jersey Agricultural Experimentation Station.

Conflicts of interest

The authors declare no potential conflicts.

Manufacturers' addresses

1 Equine Dynamics, Inc., Lexington, Kentucky, USA.

2 Quiagen, Inc., Santa Clarita, California, USA.

3 Applied Biosystems/Ambion, Austin, Texas, USA.

4 Tel-Test, Friendswood, Texas, USA.

5 Fisher Scientific, Bridgewater, New Jersey, USA.

6 Eppendorf, Hamburg, Germany.

7 Promega, Madison, Wisconsin.

8 Applied Biosystems, Foster City, California, USA.

9 SPSS, Inc., Chicago, Illinois, USA.

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