Futsal is a dynamic sport characterized by aerobic and anaerobic metabolic demands. A competitive futsal season includes weekly microcycles of training, tapering, competition, and recovery. During a futsal match, players may suffer lesions resulting from muscle fatigue and contact and collision among players. Additionally, the demands involved in playing 2–3 matches per week elevate the stress imposed on players, thereby increasing the risk of injury and diminishing their performance because of fatigue, muscle damage, and inflammation (7).
Strenuous physical exercise can result in muscle injury, promoting release of the skeletal muscle enzymes creatine kinase (CK), and lactate dehydrogenase (LDH). Both CK and LDH are found in cellular cytosol, so the appearance of these enzymes in serum indicates cellular lesion (5). Inflammatory response to cellular lesion with soreness is initiated by fluid, plasma protein, and leukocyte infiltration into the injured muscle. Cytokines (e.g., tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, IL-6) and chemokines (IL-8) are mediators that participate in the onset of the inflammatory response (6,17,19). However, when inflammation and strenuous exercise are prolonged, the initial inflammatory response results in the production of antiinflammatory cytokines such as IL-1ra, IL-4, and IL-10, which may lead to immunosuppression and heightened susceptibility to invasive microorganisms, negatively affecting the athlete's performance. Exercise-induced muscle damage is also associated with phagocyte (neutrophils and macrophages) infiltration into muscle, production of reactive oxygen species (ROS), and systemic elevation in the production of cytokines and other inflammatory mediators such as leukotrienes and prostaglandins (17).
Futsal athletes seem to be prone to illness, particularly during playing seasons. The aim of this study was to investigate how playing a futsal match affects the functional characteristics of the innate immune response, particularly the production of inflammatory mediators, leukocyte functions and the possible relationship with host defense capacity. Specific knowledge about subclinical systemic inflammation and neutrophil function in athletes after a match may help coaches, doctors, and nutritionists devise strategies for controlling the inflammatory process, avoiding infection, and tissue injury. Regardless of the origin, however, excessive or deficient neutrophil function in players can be controlled. Thus, if one considers that incremental levels of proinflammatory cytokine and acute-phase proteins are an important characteristic of acute-phase response, it is reasonable to suppose that the administration of antioxidants and antiinflammatories, for example, n-3 fatty acids, may be a useful adjunct to athlete health management. A knowledge of the immune system alterations that occur during training and matches may enable the maintenance and improvement of the performance of futsal athletes. Although futsal has gained worldwide status as a sport, few studies have focused on the health of futsal players and on strategies to prevent lesions. Chronic or acute inflammation modifies the metabolism of fatty acids, carbohydrates and iron, calcium concentrations, and the number and functions of circulating leukocytes (19). Therefore, studies exploring different aspects of lesion and inflammation are important to improve the performance of athletes.
This work involved an investigation of the effects of a futsal match on the players' serum CK and LDH activities. After examining possible muscle lesions by measuring muscle enzyme activity, we determined the systemic inflammatory status of the athletes based on their serum levels of C-reactive protein (CRP), proinflammatory (IL-1β, TNF-α, IL-8, and IL-6), and antiinflammatory (IL-10 and IL-1ra) cytokines. Changes in the production of cytokines may lead to inappropriate activation of leukocytes and even increased susceptibility to invasive microorganisms, impairing the athlete's health. Therefore, the study was completed by measuring neutrophil functions (phagocytic capacity, release of ROS and cytokines) and the proportion of cells with signs of necrosis and apoptosis.
Experimental Approach to the Problem
In this study, we demonstrated that the players' participation in a futsal match induced muscle damage and inflammation, as indicated by their augmented serum CK and LDH activities and IL-6 and CRP levels after a match. After intense prolonged exercise is done, metabolically exhausted muscle fibers exhibit diminished membrane resistance and augmented serum CK and LDH activities in plasma (5). Once muscle damage occurs, the cells in the inflammatory focus release a series of mediators that will simulate tissue repair. As a result, CRP, TNF-α, IL-1β, IL-6, and IL-8 levels also increase in plasma. In the conditions of this study, no change was found in the serum levels of TNF-α, IL-1β, IL-8, IL-10, and IL-1ra measured before and immediately after the match. The fact that no changes were observed in the plasma levels of these cytokines is likely because of the low sensitivity of the methods used for cytokine determination.
With the approval of the Ethics Committee of Cruzeiro do Sul University (protocol number 178/2008), 16 volunteers participated in the study. All the athletes signed an informed consent form agreeing to submit to the procedures involved in the study. The group presented the following characteristics (mean ± SD): age 26.4 ± 3.2 years, body mass 70.2 ± 6.9 kg, height 172.8 ± 5.7 cm, body fat 12.0 ± 2.3%, V[Combining Dot Above]O2peak 59.7 ± 5.1 ml·kg−1·min−1, and sports experience of 4.4 ± 0.9 years. All the participants played during approximately the same amount of time (5 minutes playing and 5 minutes recovering, playing a total of 10 minutes in each period) until they completed 2 equal periods of 20 minutes. The subjects with a history of infection, viruses, chronic lesions, diabetes, rheumatoid arthritis, hormonal dysfunction, lupus, or other inflammatory and hematology diseases (such as hemoglobinopathies) and who were on medication were excluded from the study.
Twenty milliliters of venous blood were collected before and immediately after a competitive futsal match. The blood samples were drawn from 1 of the 3 main veins of the antecubital fossa (the cephalic, basilic, and median cubital). In each case, the vein was chosen based on the identification of the optimal site by both visual and tactile exploration. The blood samples were drawn into 2 BD Vacutainer tubes, the first containing heparin, which was used for plasma collection and cell separation, whereas the second one was a dry gel tube for serum collection. All the samples were collected in the field (athletic clothing) 1 hour before and immediately after the match. After collecting the samples, the serum was stored at room temperature (25–30° C), whereas the plasma was stored on ice. The laboratory tests were performed within 1 hour of venipuncture, which was the time required to transport the samples from the field to the laboratory. In the laboratory, the blood was centrifuged (400g, 10 minutes), and serum and plasma were separated from the cell components. Neutrophils were isolated immediately and cellular function was tested. The enzymatic activity of CK and LDH was measured not later than 48 hours after collecting the plasma. The storage temperature of 200 μl of plasma was between 2 and 8° C, away from light because of the samples' low stability. The volume of samples used for CK, LDH, and polymerase chain reaction (PCR) determination was 10 μl for each biochemical marker and 100 μl for cytokine determination. However, whenever necessary, the samples were diluted to fall within the linearity range of the methods. All the dilutions were performed with suitable dilution reagents, according to the instructions of the kit manufacturer. The concentration of diluted samples was determined by multiplying by the dilution factor.
Serum was collected and stored at −80° C before cytokine and PCR determination by enzyme-linked immunosorbent assay (ELISA) and immunoturbidimetric assay, respectively. The samples were stored for not >3 months.
Fetal bovine serum, Hepes, Histopaque, lipopolysaccharide (LPS—Escherichia coli 026:B6), penicillin, RPMI 1640 medium supplemented with l-glutamine, sodium bicarbonate, propidium iodide (PI), hydroethidine, lucigenin, phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine, and streptomycin were supplied by Sigma Chemical Co. (St. Louis, MO, USA).
Determination of Creatine Kinase and Lactate Dehydrogenase Activities
Serum CK and LDH activities were measured according to the methods established by Oliver and Zammit and Newsholme (16,20), respectively. The kits were supplied by Bioclin Diagnostics (São Paulo, Brazil), and the measurements were carried out according to the manufacturer's instructions. The control serum was used to check the accuracy and precision of the doses, with a maximum error of 5%. The sensitivity of the methods was 5.0 U·L−1 for both CK and LDH. The reference values, 200–480 U·L−1 for the LDH method and 0–25 U·L−1 for the CK method, were obtained by determining LDH in healthy male and female populations according to the manufacturer's kit. The instruments were calibrated for all the determinations, and a control serum was used to check the data obtained.
Determination of Plasma Cytokines
Plasma levels of IL-1β, TNF-α, IL-8, IL-6, IL-10, and IL-1ra were determined by the ELISA, according to the manufacturer's instructions (DuoSet Kit—Quantikine, R&D System, Minneapolis, MN, USA). The IL-6 and IL-10 methods were linear for protein concentrations in the range of 25–2,000 pg·ml−1. The TNF-α method was considered linear for protein concentrations in the range of 6.0–1,000 pg·ml−1 of IL-β 5.0–2,500 pg·ml−1. The IL-1ra and IL-8 were linear at concentrations in the range of 3.0–250 pg·ml−1. A standard curve was built for each set of samples and cytokines assayed, yielding a correlation coefficient in the range of 0.98–0.99. For these determinations, the intraassay coefficient of variance was 3–5%, whereas the interassay coefficient of variance was 8–10%.
Determination of Serum C-Reactive Protein Levels
The CRP levels were determined by a highly sensitive immunoturbidimetric method (Bioclin Diagnostics, São Paulo, Brazil) according to the manufacturer's instructions. The linearity in the determination of CRP was 5.0–200 μg·ml−1, with a correlation coefficient of 0.99. The equipment was calibrated for all the determinations, and the data obtained were compared with a control serum.
The experiments were performed within 1 hour of venipuncture. Human neutrophil (>98%) preparations were isolated from peripheral blood of human donors under endotoxin-free conditions using Histopaque (Sigma Chemical Co.) according to the manufacturer's instructions. Briefly, blood was diluted vol/vol with 10 mM phosphate buffered saline (PBS) Dulbecco at pH 7.4 and carefully layered on 10 ml of a commercial gradient of Ficoll-Hypaque (Histopaque, d = 1.077). The tube was centrifuged at 400g at room temperature for 20 minutes. The supernatant, rich in mononuclear cells, was discarded, and 10 ml of 5% dextran was added to the pellet. The tube was homogenized and kept on ice for 45 minutes to allow for erythrocyte sedimentation. The resulting supernatant rich in granulocytes was recovered, washed with PBS Dulbecco, and the pellet hypotonically treated with 10 ml of distilled water to promote lysis of contaminated red blood cells. After 1 minute, isotonicity was restored by adding 5 ml of 2.7% NaCl and 15 ml of PBS Dulbecco. The cells were centrifuged at 400g for 5 minutes at room temperature and suspended in RPMI 1640 medium. The purity of the cell preparation was >98%.
Cell Culture and Cytokine Determination
After purification, neutrophils (2.5 × 106 cells per milliliter) were suspended in RPMI1640 medium supplemented with 0.3 g·L−1 glutamine, 2.32 g·L−1 Hepes, 2 g·L−1 sodium bicarbonate, 100 μg·ml−1 streptomycin, 100 IU·ml−1 penicillin, and 10% fetal bovine serum. The cells were then counted in a Neubauer chamber and immediately cultured at 37° C and 5% CO2, with and without LPS (5 μg·ml−1). After 18 hours, the supernatants were collected and stored at ≤−80° C before cytokine determination by ELISA (Quantikine) (13).
Cell Viability Assay (Proportion of Necrotic Cells)
Neutrophil viability was assessed using an FACSCalibur Cytometer (Becton Dickinson Systems, Franklin Lakes, NJ, USA). The percentage of viable cells in each sample was determined based on PI staining (solution at 0.05% in PBS). Ten thousand events were analyzed per sample. Fluorescence of the PI was measured using the FL2 channel (orange-red fluorescence = 585/42 nm) (15).
Proportion of Cells with DNA Fragmentation
The DNA fragmentation was analyzed by flow cytometry after DNA staining with PI. The presence of detergent in the solution permeabilized the cells, which promptly incorporated the dye into DNA. Briefly, after incubation, the cells were centrifuged at 1,000g for 15 minutes at 4° C. The resulting pellets were carefully resuspended in 300 μl hypotonic solution containing 50 μg·ml−1 PI, 0.1% sodium citrate, and 0.1% Triton X-100. The cells were then incubated for 30 minutes at 4° C. Ten thousand events were analyzed per sample. Fluorescence of the PI was measured using the FL2 channel (orange-red fluorescence = 585/42 nm) (15).
Annexin V Staining of Apoptotic Cells
Neutrophils (2.5 × 106 cells per milliliter) were harvested from culture plates and centrifuged at 4° C, 200g, for 10 minutes. The translocation of phosphatidylserine residues from the inner to the outer leaflet of plasma membrane, assessed by the reaction with AnnexinV–fluorescein isothiocyanate (FITC; Clontech Laboratories, Inc., Palo Alto, CA, USA) and 50 μl of PI (50 μg·ml−1), was used as a measure of apoptosis and scored in a FACScalibur flow cytometer (Becton Dickinson). Three cell subpopulations were identified: viable cells (Annexin V-FITC−/PI−), early apoptotic cells (Annexin V-FITC+/PI−), and late apoptotic cells (Annexin V-FITC+/PI+) (8).
Flow Cytometric Measurement of Reactive Oxygen Metabolites Using Hydroethidine
Hydroethidine (1 μM) was added to the neutrophil (2.5 × 106 cells per milliliter) incubation medium when required. Immediately afterward, the cells were treated with PMA (54 ng·ml−1). The ROS release was monitored for 30 minutes. The assays were run in PBS buffer supplemented with CaCl2 (1 mM), MgCl2 (1.5 mM), and glucose (10 mM), at 37° C, in a final volume of 0.3 ml. Fluorescence was measured using the FL3 channel of a FACSCalibur flow cytometer (Becton Dickinson). Ten thousand events were analyzed per experiment (11).
Neutrophils were suspended in PBS containing 100 mM CaCl2, 50 mM MgCl2, and 100 μM glucose to 1.0 × 106 cells·per milliliter with 1.0 × 107 particles of opsonized zymosan per milliliter. The reaction mixture was composed of 10:1 bacteria/neutrophils in a final total volume of 0.1 ml. The cells were kept at 37° C under moderate shaking. Phagocytosis was assessed by a fluorochrome assay using acridine orange, as previously described (4).
The values are presented as mean ± standard error of 16 players. The statistical analysis consisted of 1-way analysis of variance using the post hoc Student-Newman-Keuls multiple Comparison test (INStat; Graph Pad Software, San Diego, CA, USA). The significance level was set at p < 0.05. The degree of linear relationship between CK and LDH variables was established by Pearson's correlation.
The players' participation in a futsal match increased the serum activities of CK (2.5-fold) and LDH (1.3-fold) (Table 1). The serum CK activities were correlated with serum activities of LDH (p = 0.87) (Figure 1). As shown in Table 1, the futsal match increased the players' serum levels of CRP (1.6-fold) and IL-6 (1.6-fold). Under the conditions of this study, the players showed no difference in serum levels of TNF-α, IL-1β, IL-8, IL-10, and IL-1ra before and immediately after the futsal match (Table 1).
The futsal match induced phosphatidylserine externalization of neutrophils (6.0-fold) (Figure 2A). No alterations in loss of membrane integrity (Figure 2B) and DNA fragmentation (Figure 2C) were observed.
In the basal condition, that is, without PMA stimulation, neutrophils collected immediately after the futsal match spontaneously released higher amounts of ROS (by 1.3-fold, p = 0.02) than neutrophils collected from the players before the match (Figure 3). Also in the basal condition, that is, without LPS stimulation, neutrophils obtained postexercise released higher amounts of TNF-α (5.8-fold), IL-1β (4.8-fold) than in the preexercise condition (Figure 4). No alterations were observed in the basal release of IL-8 and IL-1ra by neutrophils before and after a futsal match (Figure 4).
In the stimulated condition, that is, with LPS stimulation, postexercise neutrophils released higher amounts of IL-8, IL-1β, TNF-α, and IL-1ra than in the preexercise condition. Cytokine production by LPS-stimulated neutrophils increased 1.6, 2.2, 4.0, and 1.8-fold in IL-8, IL-1β, TNF-α, and IL-1ra, respectively (Figure 4). In the stimulated condition, that is, neutrophils treated with PMA, we found that the release of ROS was diminished (1.5-fold) (Figure 4). These results were accompanied by decreased (1.6-fold) phagocytic capacity of neutrophils when exposed to zymosan (Figure 5).
Exercise-induced lesions cause the levels of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 to increase twofold to threefold (18). This increase in the levels of proinflammatory cytokines is accompanied by an increase in serum levels of CRP, a classic acute-phase protein. In the inflammatory state, the plasma concentration of CRP may increase 1,000-fold in relation to its basal level (9,14). The CRP synthesis is largely regulated by inflammation-associated cytokines such as TNF-α, IL-1β, and IL-6 (9,14). The biological function of CRP is associated with improved phagocytosis of bacterial pathogens by phagocytes. C-reactive protein interacts with Fc receptors in phagocytic cells and acts as an opsonin, contributing to the phagocytosis of microorganisms. Although playing a futsal match was found to increase the serum levels of CRP, we did not observe a concerted mode of action driving a more powerful response of innate host defense. Our results indicated that the phagocytic capacity of the players' postmatch neutrophils decreased when exposed to zymosan, which is a classical stimulus to study phagocytosis.
Neutrophils are the first leukocytes to migrate to the site of injury. Together with macrophages, they are responsible for local cleaning, phagocytizing pathogens and removing cellular debris (10–12,19). Under inflammatory conditions, ROS may be important signaling molecules for the release of cytokines and the activation and deactivation or death of leukocytes such as neutrophils. The initial release of proinflammatory mediators and neutrophil activation are important events of tissue repair; however, the inflammatory response must be a self-controlled event. The hypoproduction or hyperproduction of neutrophils and cytokines may lead to increased susceptibility to invasive microorganisms, impairing the athlete's performance. Specific knowledge about subclinical systemic inflammation and neutrophil function may help futsal professionals to devise strategies to control the processes that can result in infection and tissue injury. However, regardless of the origin, excessive or deficient neutrophil function in players must be controlled.
The observed increment in acute-phase proteins and cytokines may prime and activate neutrophils (12). An important aspect of neutrophil function is their ability to synthesize proinflammatory and antiinflammatory cytokines and growth factors that modulate the inflammatory response. The exercise described herein increased the basal responsiveness of neutrophils, increasing the release of ROS and TNF-α, IL-1β in the nonstimulated condition (absence of stimuli). The increased production of cytokines by cultured neutrophils observed after a futsal match implicates the match as a priming agent. Classical priming agents such as TNF-α and serum amyloid A alone cannot stimulate cell cytokine production, but they augment the maximal rate of cytokine release when a second stimulus occurs (12). In the LPS-stimulated condition, it was found that postexercise neutrophils collected from futsal players released higher amounts of IL-8, IL-1β, TNF-α, and IL-1ra than in nonexercised controls.
In inflammatory processes, increased levels of ROS, acute-phase proteins, proinflammatory cytokines, and fatty acids can prime neutrophils and other immune cells (12). Under these conditions, inflammatory mediators play a role in the process of resolution or progression of the damaged tissue. Whatever the origin, the hypoproduction or hyperproduction of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms, impairing the athlete's performance. Our findings also indicated that playing futsal induces neutrophil death, possibly by apoptosis, as indicated by phosphatidylserine externalization. Additionally, a single futsal match diminished neutrophil function in the stimulated condition, decreasing the release of ROS by PMA-stimulated neutrophils, and lowering the phagocytic capacity. Playing futsal reduced the efficiency of neutrophils to ward off infection resulting from exposure to pathogens (Scheme 1).
The current literature on futsal is devoid of information about the intensity and most beneficial type of training or the amount of time each player should participate actively in a match to maximize the benefit while maintaining minimal risk. This study performed an extensive measurement of systemic inflammation and neutrophil function in athletes after a futsal match. In conclusion, playing futsal induces inflammation, activates neutrophils, and reduces the efficiency of neutrophils against infection resulting from exposure to pathogens immediately after a match (Figure 6). These findings may be important for designing a strategy to protect futsal players against microorganism infection or to prevent reduced athletic performance because of subclinical inflammation immediately after a match.
Specific knowledge about subclinical systemic inflammation and neutrophil function may help athletic coaches, doctors, and nutritionists devise strategies for controlling processes that may result in infection and tissue injury. Whatever the origin, excessive or deficient neutrophil function in players must be controlled. Thus, if one considers that incremental levels of proinflammatory cytokine and acute-phase proteins are an important characteristic of acute-phase response, it is reasonable to assume that the administration of antioxidant supplements, for instance n-3 fatty acids and antiinflammatory drugs, may be a useful adjunct to athlete health management.
The authors are indebted to Sabrina da Silva Moura and Gustavo Barquilha Joel for their technical assistance. This research was supported by the Brazilian research funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2009/06039-0 and 2008/56105-7) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Conception and study design were done by N.R. de Moura and E. Hatanaka; data acquisition, analysis, and interpretation were performed by N.R. de Moura, E. Hatanaka, M.F. Cury-Boaventura, V.C. Santos, A.C. Levada-Pires, J.R. Bortolon, J. Fiamoncini, T.C. Pithon-Curi, and R. Curi; and drafting/revision was done by T.C. Pithon-Curi, R. Curi, and E. Hatanaka. The authors declare that they have no conflict of interest.
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Keywords:© 2012 National Strength and Conditioning Association
indoor soccer; leukocytes; inflammation; cytokines