Original Paper Received: November 27, 2013 Accepted after revision: April 18, 2014 Published online: August 27, 2014
Neuroimmunomodulation DOI: 10.1159/000363061
Neuro-Immuno-Endocrine Modulation in Marathon Runners André L.L. Bachi a, b Francisco J.O. Rios c, d Pedro Henrique Carr Vaisberg e Marcia Martins e Matheus Cavalcante de Sá e Angélica B. Victorino f Roberta Foster g Ana Paula R. Sierra h Maria Augusta P. Dal´Molin Kiss h Mauro Vaisberg e
a Department of Microbiology and Immunology, Federal University of São Paulo, b Institute of Physical Activity and Sport Science, Cruzeiro do Sul University, and c Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; d Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, UK; Departments of e Otorhinolaryngology, f Neurology and Neurosurgery and g Gynaecology, Federal University of São Paulo, and h School of Physical Education and Sport, University of São Paulo, São Paulo, Brazil
Abstract Objective: Sports practice alters the homeostasis of athletes. To achieve homeostatic equilibrium, the integrated action of the neuroendocrine and immune systems is necessary. Here we studied the relation between cytokines, hormones and mood states in marathon runners. Methods: A total of 20 male recreational marathon runners (mean age = 35.7 ± 9 years) and 20 male sedentary individuals (mean age = 35.5 ± 7 years) were recruited. We compared the serum levels of growth hormone (GH), cortisol and interleukins 8 and 10 and the amounts of these two cytokines spontaneously produced by peripheral blood mononuclear cells. Blood samples of the sedentary group were collected at rest. Blood from the marathon runners was collected at rest (baseline: 24 h before the race), immediately after a marathon and 72 h after a marathon. Mood state analysis in both groups was performed using the 24-item Brunel Mood Scale (BRUMS). Results: Our results showed that, at rest, levels of interleukins 8 and 10 in the supernatant of culture cells, the serum
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concentration of GH, and tension and vigour (evaluated using the BRUMS), were significantly higher in athletes compared to sedentary people. Immediately after the race all serum parameters analysed were statistically higher than baseline values. At 72 h after the marathon, serum levels of hormones and interleukins returned to values at rest, but the concentrations of interleukins in the supernatant of culture cells showed a significant reduction compared to values at rest. Conclusion: The higher serum levels of GH in athletes at rest and the higher production of cytokines in culture without previous stimulus suggest that marathon runners present mechanisms that may be associated with preparing the body to perform prolonged strenuous exercise, such as a marathon. © 2014 S. Karger AG, Basel
Introduction
Sports practice requires neuro-immuno-endocrine adaptations to maintain homeostasis, both during the practice of exercise and at rest. These adaptations will influence the activity of the cardiovascular, respiratory and musculoskeletal systems [1]. Beyond neuro-immunoAndre Luis Lacerda Bachi Rua Botucatu 862 Vila Clementino CEP 04023-062 São Paulo, SP (Brazil) E-Mail abachi @ unifesp.br
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Key Words Cytokines · Exercise · Stress hormone · Behaviour · Brunel Mood Scale
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Neuroimmunomodulation DOI: 10.1159/000363061
Table 1. Data of physical characteristics and mood state analysis for sedentary individuals at rest and marathon runners on 2 different occasions: at rest (baseline) and 72 h after a marathon
Variables
Sedentary group Marathon runners (n = 20) (n = 20) baseline 72 h after marathon
Age, years BMI Tension Vigour Anger Depression Fatigue Confusion
35.5 ± 7 26.6 ± 3.8 2.6 ± 0.8 9.2 ± 3.3 0.7 ± 0.4 0.7 ± 0.5 2.1 ± 0.7 0.6 ± 0.3
35.7 ± 9 24.6 ± 2.7 4.7 ± 0.51 11.9 ± 31 0.2 ± 0.11 0.5 ± 0.4 0.6 ± 0.31 0.4 ± 0.2
1 ± 0.62 8 ± 3.32 0.9 ± 0.52 0.3 ± 0.2 2.6 ± 0.82 0.4 ± 0.2
Mood state analysis: BRUMS. 1 Statistically significant difference in comparison to sedentary individuals. 2 Statistically significant difference in comparison to baseline value.
Methods Study Subjects In this study we evaluated 20 male recreational marathon runners and 20 male sedentary individuals (control group) living in the city of São Paulo. The two groups were paired for age and body mass index (BMI; data shown in table 1). All individuals were aware of the possible risks involved in the study and gave their informed consent to participate. Both the study protocol and consent form were approved by the UNIFESP-EPM Ethics Committee. Exclusion criteria included the following: (1) the use of medications, alcohol or drugs, (2) smoking and (3) individuals that presented obesity and pathologies such as systemic arterial hypertension and liver, renal, metabolic, inflammatory and neoplastic diseases. Collection of the Samples Blood samples were collected from sedentary people at rest. Blood from the marathon runners was collected on three different occasions: (1) at rest (baseline), (2) immediately after the marathon and (3) 72 h after the marathon. Blood draws for all individuals were performed early morning (between 6 and 7 a.m.) after 12 h of fasting, except when collecting after the marathon. After blood clotting, the tubes were centrifuged at 2,500 rpm for 10 min to obtain 500 μl of serum, which was stored at –80 ° C prior to determining cytokine and hormone concentrations. The marathon runners ran an average of 80–100 km/week or trained for an average of 90–120 min/day to prepare for the race. The last training session was performed 24 h before the blood draw. We recommended that all marathon runners ingested 400–500 ml of water before the marathon. Water was provided for the athletes by the marathon organizers during the race.
Bachi et al.
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endocrine adaptations, alterations in behaviour are of extreme importance in the capacity to accomplish an exercise, because anxiety or other altered mood states will prejudice performance. For this reason, the relationship between immune and endocrine systems and the neuropsychological set is of great importance for athletes [2]. The marathon is a challenge to athletes, with a degree of difficulty that involves not only physical but also psychological needs, and it demands careful preparation. Training will prepare the athlete for the exhaustive demands of the race, allowing them to tolerate the strenuous physical and psychological effort. A myriad of cytokines, hormones and neurotransmitters are involved, supporting the challenge posed by extremely strenuous effort, and interactions between them are essential [3]. It is well known that hormones and neurotransmitters interact with the immune system [4], and the importance of cytokines for maintaining homeostasis has been demonstrated in different organic systems. During exercise, among many other functions, these molecules also act in brain signalling, being one of the mechanisms by which sports practice may influence brain functions [5, 6]. The binding of growth hormone (GH) to human peripheral blood leucocytes was first described in the 1980s [7] and can lead to the induction of cytokines production [8]. This is of great interest given the importance of GH as a stress hormone that, together with other hormones such as prolactin and cortisol, is essential in maintaining homeostasis during exercise. During prolonged and intensive exercise an elevation of many cytokines is described, among them interleukins (IL)-8 and IL-10 [9–11]. While IL-8 is a pro-inflammatory cytokine, IL-10 presents anti-inflammatory actions [12]. Several studies have demonstrated that both cytokines have many effects on the brain and behaviour [13– 15]. However, several aspects involving hormones, cytokines and mood states in marathon runners remain unclear, and understanding the interactions between these molecules would improve knowledge about the performance and behaviour of athletes. In an attempt to understand these aspects, we proposed to investigate how physical and psychological changes induced in marathon runners by training and by the race can affect mood states, hormones and cytokines. For this, we evaluated mood states (using the Brunel Mood Scale, BRUMS) and levels of cytokines (IL-8 and IL-10) and hormones (GH and cortisol) in marathon runners and sedentary people.
Cell Culture of Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll-Paque 1.077 density gradient (Ficoll-PaqueTM PLUS, GE Healthcare, Sweden) after centrifugation. Cells (1 × 106) were seeded in cell culture medium (RPMI 1640 containing 2% FBS) in 96-well plates and the cells were maintained, without stimulation, in an incubator with 5% CO2 under controlled humidity and temperature (37 ° C) for 24 h. Cell culture supernatants were collected and stored at –80 ° C for later analysis of cytokine concentrations.
Determination of Cytokine Concentrations in Serum and Supernatant of PBMCs The concentration of the cytokines IL-8 and IL-10 in the serum and in the supernatant of PBMCs previously stored at –80 ° C was measured using ELISA (R&D Systems Inc., Minneapolis, Mass., USA), according to the manufacturer’s instructions.
Determination of GH and Cortisol Concentrations in Serum The concentration of GH (Growth Hormone, hGH, Test System; Monobind Inc., Lake forest, Calif., USA) and cortisol (Cortisol kit; Diagnostic Biochem Canada, DBC, Dorchester, Ont., Canada) in serum stored at –80 ° C was measured using ELISA, according to the manufacturer’s instructions.
Statistical Analysis Data on age, BMI and mood states are shown as means and standard deviations (SD). Serum cytokine and hormone data are shown as medians with the respective quartiles. Student’s t test was used to analyse differences in age and BMI. The Mann-Whitney test was used to determine whether the differences between the results for sedentary individuals and those for marathon runners at rest were significant. The Friedman test and Müller-Dunn posttest were used to determine whether the differences between the baseline results for the marathon runners and the results immediately after and 72 h after the marathon were significant. The significance level was set at 5% (p < 0.05).
nificantly higher, anger and fatigue were lower in athletes compared to the sedentary group. The status of depression and confusion did not differ between the two groups. At 72 h after the marathon, the mood state of individuals of both groups evaluated using the BRUMS was similar. PBMCs Produce Higher Levels of Cytokines before a Marathon As shown in figure 1, 24 h before the marathon race the levels of IL-8 (p < 0.001; fig. 1a) and IL-10 (p < 0.002; fig. 1b) produced by PBMCs (1 × 106 cells per well) of marathon runners were significantly higher than those in the sedentary group. However, the serum levels of IL-8 (fig. 1c) and IL-10 (fig. 1d) observed in marathon runners did not differ in relation to the sedentary group when at rest. Immediately after the race, we observed that the levels of IL-8 (fig. 1a) and IL-10 (fig. 1b) in PBMC culture were unchanged, but that the serum concentrations of IL-8 (p < 0.001; fig. 1c) and IL-10 (p < 0.001; fig. 1d) were higher than baseline levels. At 72 h after the end of the marathon, the concentrations of IL-8 (p < 0.001; fig. 1a) and IL-10 (p < 0.05; fig. 1b) in PBMC culture were significantly lower than baseline values, but the serum levels of IL-8 (fig. 1c) and IL-10 (fig. 1d) had returned to baseline values. Levels of GH, but Not Cortisol, Increase before the Marathon Race As shown in figure 2, although the serum levels of GH (p < 0.01; fig. 2a) were significantly higher in marathon runners than in the sedentary group, the cortisol levels (fig. 2b) did not differ between groups when at rest. Immediately after the end of the marathon, the serum concentrations of GH (p < 0.001; fig. 2a) and cortisol (p < 0.001; fig. 2b) were significantly higher in relation to baseline levels. At 72 h after the marathon race, the levels of GH (fig. 2a) and cortisol (fig. 2b) had returned to baseline values.
Discussion
Marathon Runners Present Changes in Mood State before Strenuous Exercise As shown in table 1, marathon runners presented different mood states from controls when at rest. While the status of tension (p < 0.01) and vigour (p < 0.02) was sig-
We were able to demonstrate that, at rest, marathon runners showed higher serum levels of GH, an elevated production of cytokines IL-8 and IL-10 in PBMC culture (without any stimulation) and, using the BRUMS, increased tension and vigour and lower anger and fatigue compared to sedentary individuals. Immediately after the marathon race, the levels of all serum parameters analysed had increased, but the con-
Neuro-Immuno-Endocrine Behaviour in Marathon Runners
Neuroimmunomodulation DOI: 10.1159/000363061
Results
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Determination of Mood State The 24-item BRUMS [16–18] was used to analyse the mood state of sedentary individuals at rest and that of marathon runners on two different occasions: at rest (24 h before) and 72 h after the marathon race. The BRUMS considers 6 factors: tension, vigour, anger, depression, fatigue and confusion, as presented in table 1. Each of these 6 factors is composed of four items as follows: (1) tension – panicky, anxious, worried, nervous; (2) vigour – lively, energetic, active, alert; (3) anger – annoyed, bitter, angry, bad tempered; (4) depression – depressed, downhearted, unhappy, miserable; (5) fatigue – worn out, exhausted, sleepy, tired, and (6) confusion – confused, muddled, mixed-up, uncertain.
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Fig. 1. Levels of IL-8 (a) and IL-10 (b) produced by PBMCs (1 × 106 cells per well) after 24 h of culture and serum concentrations of IL-8 (c) and IL-10 (d) in a group of sedentary subjects at rest and in a group of marathon run-
ners measured on 3 different occasions: at rest (baseline), immediately after a marathon and 72 h after a marathon. Data are presented as means (± SD). * p < 0.05: significance level.
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Neuroimmunomodulation DOI: 10.1159/000363061
Bachi et al.
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Fig. 2. Serum concentrations of GH (a) and cortisol (b) in a group of sedentary subjects at rest and in a group of marathon runners measured on 3 different occasions: at rest (baseline), immediately after a marathon and 72 h after a marathon. Data are presented as means (± SD). * p < 0.01, ** p < 0.001: significance levels.
It is thus reasonable to hypothesize that the raised GH found before the race, probably due to the psychological stress arising from anticipating the strenuous physical and psychological stress elicited by the marathon, can act as a protective mechanism against brain injury during the race. Prolonged and high-intensity exercise is associated with elevations of many cytokines, including IL-8 and IL10 [9, 10, 34]. The increased production of cytokines may be associated with different cell types from the immune and other systems [35–37]. In the current study, we observed an increase in IL-8 and IL-10 levels immediately after the race that is consistent with the literature [9, 38]. However, we also found an increase in IL-8 and IL-10 production in PBMC cultures of cells collected from the marathon runners before the race – an unexpected finding. IL-10 is the prototypical cytokine with anti-inflammatory action [12] and also has many effects on the brain and behaviour [14]. It has previously been demonstrated that GH can stimulate the peripheral production of IL-10 [8], and therefore we can conclude that a virtuous cycle is established by exercise to protect the brain against injury. Voorhees et al. [15] showed the importance of IL-10 in behaviour, demonstrating that prolonged restraint stress reduces IL-10 and causes persistent depressive-like behaviour, which is reversed by recombinant IL-10, highlighting the importance of this cytokine in mood state in an animal model. Studies using IL-10 knockout mice showed that an absence of IL-10 expression results in constitutively depressive behaviour and that treatment with recombinant IL-10 improves this alteration in mood state [39]. IL-8, a pro-inflammatory cytokine mainly produced by monocytes, endothelial cells [13] and skeletal muscle [35, 36], classically acts as a chemokine that attracts primarily neutrophils and angiogenic factors [35, 40]. Moreover, Marsland et al. [41] showed increased production of IL-8 in situations of perceived stress during chronic stress and Xiong et al. [42] demonstrated important influences of IL-8 on neuronal function, as well as on mood state [13]. Differences in cytokine profiles observed in serum and culture supernatant of PBMCs is not an unexpected finding, considering that in several studies cited in the literature the production and secretion of cytokines in cultures of PBMCs (with or without stimulation) may show different values from those obtained in serum [43, 44]. Analysis of serum concentrations of cytokines may also have differences in relation to cytokine concentrations mea-
Neuro-Immuno-Endocrine Behaviour in Marathon Runners
Neuroimmunomodulation DOI: 10.1159/000363061
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centrations of IL-8 and IL-10 in the supernatant of PBMC cultures did not differ in relation to values at rest. At 72 h after the marathon, while the levels of IL-8 and IL-10 observed in PBMC culture were significantly lower than basal levels, serum concentrations of cytokines and hormones had returned to basal values, as had the mood state. Our findings demonstrate that important adjustments in neuro-immuno-endocrine systems and mood states are turned on both by the marathon itself and by the training period. Increased levels of stress hormones, like cortisol and GH, observed during and after a short period of exercise may be associated with mechanisms involved in the maintenance of homeostasis [19]. Stress, exercise and a variety of neurogenic stimuli are involved in the increase in GH secretion [20]. In general, exercise training decreases the serum levels of cortisol [21] but not of GH [22]. According to Steinacker et al. [23], after an intensive period of exercise training rowers showed elevated baseline levels of GH in the pre-event period. It is well known that intensive periods of training are routine before a marathon, and this could explain our finding of an altered pattern of GH secretion. The GH and somatotropic axis have multiple functions, among them affecting metabolic control [24] and brain functions, including cognition [5] and neuroprotection [6]. Running a marathon requires an unusual level of psychological conditioning and, as demonstrated by Nagane et al. [25], depending on psychological requirements, GH secretion may be altered. Myiabo et al. [26] showed that GH response is a more accurate indicator than cortisol response to psychological stress. So, the expectation of the challenge of a marathon, theoretically involving great physical and psychological effort, could alter GH secretion, even during periods of physical rest. It is important to highlight recent findings of studies of marathon runners. In contrast to the classical descriptions of the protection of organic structures such as the brain, heart and kidney during prolonged strenuous exercise, several authors have shown that after a marathon race, organs such as the heart [27] and kidney [28] are negatively affected. Therefore, the elevation of GH before the race can represent a protective mechanism of the central nervous system (CNS) [25] that can occur through a direct action of GH and by activation of the GH-IGF1 axis, according to many descriptions obtained from the literature [29–33].
sured in other sites such as the CNS [45]. Therefore, the cytokine evaluation in serum does not necessarily reflect cytokine levels in the CNS, which are primarily produced by astrocytes and glial cells [46, 47]. The activation state observed in the culture of PBMCs (not stimulated) before the exercise correlated with elevated levels of GH and can reflect the state of activation of cells in the periphery. For obvious reasons we cannot demonstrate the state of activation of cells from the CNS in marathon runners or the cytokine levels in the CNS of these individuals. Moreover, until now there is no animal model of a marathon that could be used to evaluate these aspects. However, the induction of cytokine production by GH in the periphery may be a clue to understanding the changes in mood states, considering the fact that GH, both derived from peripheral blood and produced in the CNS [29, 33], acts in astrocytes and glial cells, leading to
the production of IL-10 and IL-8, which, as previously mentioned, act on mood states. Therefore, it is possible to suggest a relationship between changes in GH and cytokines and mood states in these athletes. Analysis of our results in the light of data from the literature suggests that marathon runners increase their physical and psychological capacities during the preparation in order to cope with prolonged strenuous exercise. In addition, these changes are related to hormonal and immunological modifications. Further studies are required to obtain a more complete understanding of the complex organic and psychological functions of marathon runners. Acknowledgement This study was supported by the Fundação de Amparo à Pesquisa de São Paulo (FAPESP), São Paulo, Brazil (2010/50025-1).
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Neuroimmunomodulation DOI: 10.1159/000363061
Neuro-Immuno-Endocrine Modulation in Marathon Runners André L.L. Bachi a, b Francisco J.O. Rios c, d Pedro Henrique Carr Vaisberg e Marcia Martins e Matheus Cavalcante de Sá e Angélica B. Victorino f Roberta Foster g Ana Paula R. Sierra h Maria Augusta P. Dal´Molin Kiss h Mauro Vaisberg e
a Department of Microbiology and Immunology, Federal University of São Paulo, b Institute of Physical Activity and Sport Science, Cruzeiro do Sul University, and c Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; d Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, UK; Departments of e Otorhinolaryngology, f Neurology and Neurosurgery and g Gynaecology, Federal University of São Paulo, and h School of Physical Education and Sport, University of São Paulo, São Paulo, Brazil
Abstract Objective: Sports practice alters the homeostasis of athletes. To achieve homeostatic equilibrium, the integrated action of the neuroendocrine and immune systems is necessary. Here we studied the relation between cytokines, hormones and mood states in marathon runners. Methods: A total of 20 male recreational marathon runners (mean age = 35.7 ± 9 years) and 20 male sedentary individuals (mean age = 35.5 ± 7 years) were recruited. We compared the serum levels of growth hormone (GH), cortisol and interleukins 8 and 10 and the amounts of these two cytokines spontaneously produced by peripheral blood mononuclear cells. Blood samples of the sedentary group were collected at rest. Blood from the marathon runners was collected at rest (baseline: 24 h before the race), immediately after a marathon and 72 h after a marathon. Mood state analysis in both groups was performed using the 24-item Brunel Mood Scale (BRUMS). Results: Our results showed that, at rest, levels of interleukins 8 and 10 in the supernatant of culture cells, the serum
© 2014 S. Karger AG, Basel 1021–7401/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/nim
concentration of GH, and tension and vigour (evaluated using the BRUMS), were significantly higher in athletes compared to sedentary people. Immediately after the race all serum parameters analysed were statistically higher than baseline values. At 72 h after the marathon, serum levels of hormones and interleukins returned to values at rest, but the concentrations of interleukins in the supernatant of culture cells showed a significant reduction compared to values at rest. Conclusion: The higher serum levels of GH in athletes at rest and the higher production of cytokines in culture without previous stimulus suggest that marathon runners present mechanisms that may be associated with preparing the body to perform prolonged strenuous exercise, such as a marathon. © 2014 S. Karger AG, Basel
Introduction
Sports practice requires neuro-immuno-endocrine adaptations to maintain homeostasis, both during the practice of exercise and at rest. These adaptations will influence the activity of the cardiovascular, respiratory and musculoskeletal systems [1]. Beyond neuro-immunoAndre Luis Lacerda Bachi Rua Botucatu 862 Vila Clementino CEP 04023-062 São Paulo, SP (Brazil) E-Mail abachi @ unifesp.br
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Key Words Cytokines · Exercise · Stress hormone · Behaviour · Brunel Mood Scale
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Neuroimmunomodulation DOI: 10.1159/000363061
Table 1. Data of physical characteristics and mood state analysis for sedentary individuals at rest and marathon runners on 2 different occasions: at rest (baseline) and 72 h after a marathon
Variables
Sedentary group Marathon runners (n = 20) (n = 20) baseline 72 h after marathon
Age, years BMI Tension Vigour Anger Depression Fatigue Confusion
35.5 ± 7 26.6 ± 3.8 2.6 ± 0.8 9.2 ± 3.3 0.7 ± 0.4 0.7 ± 0.5 2.1 ± 0.7 0.6 ± 0.3
35.7 ± 9 24.6 ± 2.7 4.7 ± 0.51 11.9 ± 31 0.2 ± 0.11 0.5 ± 0.4 0.6 ± 0.31 0.4 ± 0.2
1 ± 0.62 8 ± 3.32 0.9 ± 0.52 0.3 ± 0.2 2.6 ± 0.82 0.4 ± 0.2
Mood state analysis: BRUMS. 1 Statistically significant difference in comparison to sedentary individuals. 2 Statistically significant difference in comparison to baseline value.
Methods Study Subjects In this study we evaluated 20 male recreational marathon runners and 20 male sedentary individuals (control group) living in the city of São Paulo. The two groups were paired for age and body mass index (BMI; data shown in table 1). All individuals were aware of the possible risks involved in the study and gave their informed consent to participate. Both the study protocol and consent form were approved by the UNIFESP-EPM Ethics Committee. Exclusion criteria included the following: (1) the use of medications, alcohol or drugs, (2) smoking and (3) individuals that presented obesity and pathologies such as systemic arterial hypertension and liver, renal, metabolic, inflammatory and neoplastic diseases. Collection of the Samples Blood samples were collected from sedentary people at rest. Blood from the marathon runners was collected on three different occasions: (1) at rest (baseline), (2) immediately after the marathon and (3) 72 h after the marathon. Blood draws for all individuals were performed early morning (between 6 and 7 a.m.) after 12 h of fasting, except when collecting after the marathon. After blood clotting, the tubes were centrifuged at 2,500 rpm for 10 min to obtain 500 μl of serum, which was stored at –80 ° C prior to determining cytokine and hormone concentrations. The marathon runners ran an average of 80–100 km/week or trained for an average of 90–120 min/day to prepare for the race. The last training session was performed 24 h before the blood draw. We recommended that all marathon runners ingested 400–500 ml of water before the marathon. Water was provided for the athletes by the marathon organizers during the race.
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endocrine adaptations, alterations in behaviour are of extreme importance in the capacity to accomplish an exercise, because anxiety or other altered mood states will prejudice performance. For this reason, the relationship between immune and endocrine systems and the neuropsychological set is of great importance for athletes [2]. The marathon is a challenge to athletes, with a degree of difficulty that involves not only physical but also psychological needs, and it demands careful preparation. Training will prepare the athlete for the exhaustive demands of the race, allowing them to tolerate the strenuous physical and psychological effort. A myriad of cytokines, hormones and neurotransmitters are involved, supporting the challenge posed by extremely strenuous effort, and interactions between them are essential [3]. It is well known that hormones and neurotransmitters interact with the immune system [4], and the importance of cytokines for maintaining homeostasis has been demonstrated in different organic systems. During exercise, among many other functions, these molecules also act in brain signalling, being one of the mechanisms by which sports practice may influence brain functions [5, 6]. The binding of growth hormone (GH) to human peripheral blood leucocytes was first described in the 1980s [7] and can lead to the induction of cytokines production [8]. This is of great interest given the importance of GH as a stress hormone that, together with other hormones such as prolactin and cortisol, is essential in maintaining homeostasis during exercise. During prolonged and intensive exercise an elevation of many cytokines is described, among them interleukins (IL)-8 and IL-10 [9–11]. While IL-8 is a pro-inflammatory cytokine, IL-10 presents anti-inflammatory actions [12]. Several studies have demonstrated that both cytokines have many effects on the brain and behaviour [13– 15]. However, several aspects involving hormones, cytokines and mood states in marathon runners remain unclear, and understanding the interactions between these molecules would improve knowledge about the performance and behaviour of athletes. In an attempt to understand these aspects, we proposed to investigate how physical and psychological changes induced in marathon runners by training and by the race can affect mood states, hormones and cytokines. For this, we evaluated mood states (using the Brunel Mood Scale, BRUMS) and levels of cytokines (IL-8 and IL-10) and hormones (GH and cortisol) in marathon runners and sedentary people.
Cell Culture of Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll-Paque 1.077 density gradient (Ficoll-PaqueTM PLUS, GE Healthcare, Sweden) after centrifugation. Cells (1 × 106) were seeded in cell culture medium (RPMI 1640 containing 2% FBS) in 96-well plates and the cells were maintained, without stimulation, in an incubator with 5% CO2 under controlled humidity and temperature (37 ° C) for 24 h. Cell culture supernatants were collected and stored at –80 ° C for later analysis of cytokine concentrations.
Determination of Cytokine Concentrations in Serum and Supernatant of PBMCs The concentration of the cytokines IL-8 and IL-10 in the serum and in the supernatant of PBMCs previously stored at –80 ° C was measured using ELISA (R&D Systems Inc., Minneapolis, Mass., USA), according to the manufacturer’s instructions.
Determination of GH and Cortisol Concentrations in Serum The concentration of GH (Growth Hormone, hGH, Test System; Monobind Inc., Lake forest, Calif., USA) and cortisol (Cortisol kit; Diagnostic Biochem Canada, DBC, Dorchester, Ont., Canada) in serum stored at –80 ° C was measured using ELISA, according to the manufacturer’s instructions.
Statistical Analysis Data on age, BMI and mood states are shown as means and standard deviations (SD). Serum cytokine and hormone data are shown as medians with the respective quartiles. Student’s t test was used to analyse differences in age and BMI. The Mann-Whitney test was used to determine whether the differences between the results for sedentary individuals and those for marathon runners at rest were significant. The Friedman test and Müller-Dunn posttest were used to determine whether the differences between the baseline results for the marathon runners and the results immediately after and 72 h after the marathon were significant. The significance level was set at 5% (p < 0.05).
nificantly higher, anger and fatigue were lower in athletes compared to the sedentary group. The status of depression and confusion did not differ between the two groups. At 72 h after the marathon, the mood state of individuals of both groups evaluated using the BRUMS was similar. PBMCs Produce Higher Levels of Cytokines before a Marathon As shown in figure 1, 24 h before the marathon race the levels of IL-8 (p < 0.001; fig. 1a) and IL-10 (p < 0.002; fig. 1b) produced by PBMCs (1 × 106 cells per well) of marathon runners were significantly higher than those in the sedentary group. However, the serum levels of IL-8 (fig. 1c) and IL-10 (fig. 1d) observed in marathon runners did not differ in relation to the sedentary group when at rest. Immediately after the race, we observed that the levels of IL-8 (fig. 1a) and IL-10 (fig. 1b) in PBMC culture were unchanged, but that the serum concentrations of IL-8 (p < 0.001; fig. 1c) and IL-10 (p < 0.001; fig. 1d) were higher than baseline levels. At 72 h after the end of the marathon, the concentrations of IL-8 (p < 0.001; fig. 1a) and IL-10 (p < 0.05; fig. 1b) in PBMC culture were significantly lower than baseline values, but the serum levels of IL-8 (fig. 1c) and IL-10 (fig. 1d) had returned to baseline values. Levels of GH, but Not Cortisol, Increase before the Marathon Race As shown in figure 2, although the serum levels of GH (p < 0.01; fig. 2a) were significantly higher in marathon runners than in the sedentary group, the cortisol levels (fig. 2b) did not differ between groups when at rest. Immediately after the end of the marathon, the serum concentrations of GH (p < 0.001; fig. 2a) and cortisol (p < 0.001; fig. 2b) were significantly higher in relation to baseline levels. At 72 h after the marathon race, the levels of GH (fig. 2a) and cortisol (fig. 2b) had returned to baseline values.
Discussion
Marathon Runners Present Changes in Mood State before Strenuous Exercise As shown in table 1, marathon runners presented different mood states from controls when at rest. While the status of tension (p < 0.01) and vigour (p < 0.02) was sig-
We were able to demonstrate that, at rest, marathon runners showed higher serum levels of GH, an elevated production of cytokines IL-8 and IL-10 in PBMC culture (without any stimulation) and, using the BRUMS, increased tension and vigour and lower anger and fatigue compared to sedentary individuals. Immediately after the marathon race, the levels of all serum parameters analysed had increased, but the con-
Neuro-Immuno-Endocrine Behaviour in Marathon Runners
Neuroimmunomodulation DOI: 10.1159/000363061
Results
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Determination of Mood State The 24-item BRUMS [16–18] was used to analyse the mood state of sedentary individuals at rest and that of marathon runners on two different occasions: at rest (24 h before) and 72 h after the marathon race. The BRUMS considers 6 factors: tension, vigour, anger, depression, fatigue and confusion, as presented in table 1. Each of these 6 factors is composed of four items as follows: (1) tension – panicky, anxious, worried, nervous; (2) vigour – lively, energetic, active, alert; (3) anger – annoyed, bitter, angry, bad tempered; (4) depression – depressed, downhearted, unhappy, miserable; (5) fatigue – worn out, exhausted, sleepy, tired, and (6) confusion – confused, muddled, mixed-up, uncertain.
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Fig. 1. Levels of IL-8 (a) and IL-10 (b) produced by PBMCs (1 × 106 cells per well) after 24 h of culture and serum concentrations of IL-8 (c) and IL-10 (d) in a group of sedentary subjects at rest and in a group of marathon run-
ners measured on 3 different occasions: at rest (baseline), immediately after a marathon and 72 h after a marathon. Data are presented as means (± SD). * p < 0.05: significance level.
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Neuroimmunomodulation DOI: 10.1159/000363061
Bachi et al.
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Fig. 2. Serum concentrations of GH (a) and cortisol (b) in a group of sedentary subjects at rest and in a group of marathon runners measured on 3 different occasions: at rest (baseline), immediately after a marathon and 72 h after a marathon. Data are presented as means (± SD). * p < 0.01, ** p < 0.001: significance levels.
It is thus reasonable to hypothesize that the raised GH found before the race, probably due to the psychological stress arising from anticipating the strenuous physical and psychological stress elicited by the marathon, can act as a protective mechanism against brain injury during the race. Prolonged and high-intensity exercise is associated with elevations of many cytokines, including IL-8 and IL10 [9, 10, 34]. The increased production of cytokines may be associated with different cell types from the immune and other systems [35–37]. In the current study, we observed an increase in IL-8 and IL-10 levels immediately after the race that is consistent with the literature [9, 38]. However, we also found an increase in IL-8 and IL-10 production in PBMC cultures of cells collected from the marathon runners before the race – an unexpected finding. IL-10 is the prototypical cytokine with anti-inflammatory action [12] and also has many effects on the brain and behaviour [14]. It has previously been demonstrated that GH can stimulate the peripheral production of IL-10 [8], and therefore we can conclude that a virtuous cycle is established by exercise to protect the brain against injury. Voorhees et al. [15] showed the importance of IL-10 in behaviour, demonstrating that prolonged restraint stress reduces IL-10 and causes persistent depressive-like behaviour, which is reversed by recombinant IL-10, highlighting the importance of this cytokine in mood state in an animal model. Studies using IL-10 knockout mice showed that an absence of IL-10 expression results in constitutively depressive behaviour and that treatment with recombinant IL-10 improves this alteration in mood state [39]. IL-8, a pro-inflammatory cytokine mainly produced by monocytes, endothelial cells [13] and skeletal muscle [35, 36], classically acts as a chemokine that attracts primarily neutrophils and angiogenic factors [35, 40]. Moreover, Marsland et al. [41] showed increased production of IL-8 in situations of perceived stress during chronic stress and Xiong et al. [42] demonstrated important influences of IL-8 on neuronal function, as well as on mood state [13]. Differences in cytokine profiles observed in serum and culture supernatant of PBMCs is not an unexpected finding, considering that in several studies cited in the literature the production and secretion of cytokines in cultures of PBMCs (with or without stimulation) may show different values from those obtained in serum [43, 44]. Analysis of serum concentrations of cytokines may also have differences in relation to cytokine concentrations mea-
Neuro-Immuno-Endocrine Behaviour in Marathon Runners
Neuroimmunomodulation DOI: 10.1159/000363061
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centrations of IL-8 and IL-10 in the supernatant of PBMC cultures did not differ in relation to values at rest. At 72 h after the marathon, while the levels of IL-8 and IL-10 observed in PBMC culture were significantly lower than basal levels, serum concentrations of cytokines and hormones had returned to basal values, as had the mood state. Our findings demonstrate that important adjustments in neuro-immuno-endocrine systems and mood states are turned on both by the marathon itself and by the training period. Increased levels of stress hormones, like cortisol and GH, observed during and after a short period of exercise may be associated with mechanisms involved in the maintenance of homeostasis [19]. Stress, exercise and a variety of neurogenic stimuli are involved in the increase in GH secretion [20]. In general, exercise training decreases the serum levels of cortisol [21] but not of GH [22]. According to Steinacker et al. [23], after an intensive period of exercise training rowers showed elevated baseline levels of GH in the pre-event period. It is well known that intensive periods of training are routine before a marathon, and this could explain our finding of an altered pattern of GH secretion. The GH and somatotropic axis have multiple functions, among them affecting metabolic control [24] and brain functions, including cognition [5] and neuroprotection [6]. Running a marathon requires an unusual level of psychological conditioning and, as demonstrated by Nagane et al. [25], depending on psychological requirements, GH secretion may be altered. Myiabo et al. [26] showed that GH response is a more accurate indicator than cortisol response to psychological stress. So, the expectation of the challenge of a marathon, theoretically involving great physical and psychological effort, could alter GH secretion, even during periods of physical rest. It is important to highlight recent findings of studies of marathon runners. In contrast to the classical descriptions of the protection of organic structures such as the brain, heart and kidney during prolonged strenuous exercise, several authors have shown that after a marathon race, organs such as the heart [27] and kidney [28] are negatively affected. Therefore, the elevation of GH before the race can represent a protective mechanism of the central nervous system (CNS) [25] that can occur through a direct action of GH and by activation of the GH-IGF1 axis, according to many descriptions obtained from the literature [29–33].
sured in other sites such as the CNS [45]. Therefore, the cytokine evaluation in serum does not necessarily reflect cytokine levels in the CNS, which are primarily produced by astrocytes and glial cells [46, 47]. The activation state observed in the culture of PBMCs (not stimulated) before the exercise correlated with elevated levels of GH and can reflect the state of activation of cells in the periphery. For obvious reasons we cannot demonstrate the state of activation of cells from the CNS in marathon runners or the cytokine levels in the CNS of these individuals. Moreover, until now there is no animal model of a marathon that could be used to evaluate these aspects. However, the induction of cytokine production by GH in the periphery may be a clue to understanding the changes in mood states, considering the fact that GH, both derived from peripheral blood and produced in the CNS [29, 33], acts in astrocytes and glial cells, leading to
the production of IL-10 and IL-8, which, as previously mentioned, act on mood states. Therefore, it is possible to suggest a relationship between changes in GH and cytokines and mood states in these athletes. Analysis of our results in the light of data from the literature suggests that marathon runners increase their physical and psychological capacities during the preparation in order to cope with prolonged strenuous exercise. In addition, these changes are related to hormonal and immunological modifications. Further studies are required to obtain a more complete understanding of the complex organic and psychological functions of marathon runners. Acknowledgement This study was supported by the Fundação de Amparo à Pesquisa de São Paulo (FAPESP), São Paulo, Brazil (2010/50025-1).
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