Exercise modulates immune function depending on its frequency, duration, and intensity (25). An intense long-duration exercise is associated with an increased risk of infections in the upper respiratory tract (25). This has been thought to be a consequence of postexercise immunosuppression, which is characterized by suppression of natural killer cell activity, lymphocyte proliferation, neutrophil phagocytosis (17), and microbicidal activity. Studies in humans and rats have shown that intense exercise causes lymphocyte apoptosis (22,23,31). Recently, our group showed that a single bout of intense exercise causes apoptosis of rat neutrophils (20).
Leukocyte apoptosis plays an important role to maintain lymphoid tissue homeostasis and to avoid immune-activation involving diseases (14,40). The morphologic features of apoptosis include condensation and marginalization of the nuclear chromatin, DNA fragmentation, phosphatidylserine (PS) externalization, plasma membrane blebbing, and cell shrinkage (13). The PS externalization occurs because the cells lose the membrane phospholipid asymmetry, resulting in exposure of phosphatidylserine at the cell surface (16). Apoptosis can be induced via receptor death (intrinsic pathway) or mitochondria-mediated pathway (extrinsic pathway) (19).
Plasma-free fatty acids are the main fuel used by skeletal muscle during endurance exercise being responsible for up to 65% maximal oxygen consumption (V˙O2max) (12). During a physical effort, fatty acids are released from adipose tissue being used by working muscles. On discontinuing exercise, utilization by skeletal muscles ceases and so plasma fatty acid levels rapidly increase (38).
High levels of free fatty acids reduce lymphocyte proliferation and induce lymphocyte apoptosis (5). At doses close to physiological concentrations, fatty acid-induced cell death has been reported to occur by apoptosis as assessed by internucleosomal DNA cleavage and PS externalization (15). High levels of fatty acids preferentially cause necrosis, with a rapid loss of membrane integrity and cell swelling. Previous research from our laboratory has shown that treatment with oleic and linoleic acid induces apoptosis and necrosis of human lymphocytes (4). The occurrence of apoptosis is high in lymphocytes from fasting and diabetic states (26,28) that present increased plasma free fatty acid levels. The toxicity of fatty acids to Jurkat (human T lymphocyte) and Raji (human B lymphocyte) cell lines cultured for 24 h increases with the carbon chain length and the number of double bonds (21). The cytotoxicity of the fatty acids seems to be associated with oxidative stress, because they can be partially prevented by antioxidant agents such as tocopherol (29).
The Half Ironman is a category of long-duration triathlon that consists of 2-km swimming, 80-km cycling, and 20-km running. A high-intensity sport competition, as in triathlon, leads to a marked increase in plasma free fatty acid levels (10) but the occurrence of neutrophil death under this condition remains to be studied. In this study, the effect of a Half Ironman competition, performed by elite male athletes, on neutrophil death was examined. The proposition was to evaluate the involvement of plasma free fatty acids on neutrophil death induced by the competition.
MATERIALS AND METHODS
In Vivo Experiments
Eleven healthy male sedentary volunteers and12 healthy male elite triathlon athletes participated in the study. They were not taking medication at the time of blood donation. All human blood donors signed written informed consent. The study was approved by the ethical committee of the Institute of Biomedical Sciences, São Paulo University (691).
Anthropometric and V˙O2max measurements.
Measurements of the total body mass (kg), height (m), and skinfold (Table 1) were performed according to the International Society for the Advancement of Kinanthropometry (ISAK) (32). Aerobic power and aerobic capacity tests were performed on a treadmill (ATL-10.200; Imbramed, Brazil) to determine V˙O2max of the triathletes. An ECG was carried out to detect possible cardiac pathologies during the physical effort. The subjects performed a graded exercise test with increasing oxygen consumption V˙O2max (Model V˙max 29; SensorMedics, Yorba Linda, CA; with variation uphill and of velocity) (Table 1). A similar protocol of exhaustion was used by Heck et al. (9).
The athletes performed a triathlon competition (Half Ironman) in Ubatuba, São Paulo, Brazil. The sedentary group did not perform physical activity. Blood samples (25 mL) were collected from the antecubital vein of the triathletes at rest and immediately after the competition, whereas from the sedentary group samples were obtained at rest under similar conditions.
Human neutrophils were isolated from peripheral blood of the sedentary volunteers and triathletes. Blood was diluted in phosphate-buffered saline (PBS, 1:1; pH 7.4 containing 100 mmol·L−1 CaCl2, 50 mmol·L−1 MgCl2) and carefully layered on Histopaque (d= 1.077). The tubes were then centrifuged at 400g and 4°C for 30 min. The supernatant, rich in mononuclear cells, was separated. Neutrophils were prepared from the inferior sediment that was submitted to hypotonic treatment with 10-mL solution containing 150 mmol·L−1 NH4Cl, 10 mmol·L−1 NaHCO3, 0.1 mmol·L−1 ethylenediaminetetraacetic acid (EDTA) to promote lysis of contaminating erythrocytes. The preparation was homogenized and maintained in ice for 10 min to allow erythrocyte lysis. Afterward, the tubes were centrifuged at 400g and 4°C for 10 min, and this procedure was repeated twice. Neutrophils obtained from sedentary volunteers and triathletes were counted in a Neubauer chamber under an optical microscope (Nikon, Melville, NY).
Cell viability assay.
Neutrophils (1 × 106 cells·mL−1) were resuspended in 500 μL of PBS and 50 μL of propidium iodide (PI) solution (20 μg·mL−1 in PBS) was added. Percentage of viable cells in each sample was determined by using a FACSCalibur flow cytometer (Becton Dickinson, San Juan, CA). The flow cytometer was always revised, calibrated, and tested for quality control. PI is a highly water-soluble fluorescent compound that cannot passthrough intact membranes and is generally excluded from viable cells. It binds to DNA by intercalating between the bases with little or no-sequence preference. Fluorescence was measured using the FL2 channel (orange-red fluorescence = 585/42 nm). Ten thousand events were analyzed per experiment. Cells with PI fluorescence were then evaluated by using the Cell Quest software (Becton Dickinson).
DNA fragmentation was analyzed by flow cytometry after DNA staining with PI according to the method described by Nicoletti et al. (24). Fluorescence was measured and analyzed as described above.
PS externalization was analyzed by flow cytometry after PS staining with fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC). PI is used to distinguish viable from non viable cells. Fluorescence of annexin V-FITC was measured in the FL1 channel (green fluorescence = 530/30 nm) and PI in FL2 channel (orange-red fluorescence = 585/42 nm). Cells stained with annexin V-FITC were then evaluated as described above.
Mitochondrial transmembrane potential.
Neutrophils (1 × 106) were incubated for 15 min at 37°C with rhodamine 123 (5 μmol·L−1) in the dark. Afterward, the cells were washed twice with cold PBS and incubated for 30 min at 30°C in the dark. Rhodamine 123 is a cell-permeable, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxic effects. Therefore, rhodamine 123 allows for quick and easy detection of changes in mitochondrial transmembrane potential. Fluorescence of rhodamine 123 was determined using the FL1 channel (green fluorescence = 530/30 nm). Fluorescence was then evaluated as described above.
Measurement of reactive oxygen species.
Dihydroethidium was used for the flow cytometric measurement of reactive oxygen species (ROS) production by neutrophils. Dihydroethidium is rapidly oxidized to ethidium bromide (a red fluorescent compound). The cells (1 × 106 cells·mL−1) were incubated for 30 min in the presence of phorbol myristate acetate (PMA; 20 nmol·L−1) and stained with dihydroethidium (1 μmol·L−1) at room temperature in the dark. Spontaneous production of ROS was measured in unstimulated neutrophils incubated under similar conditions without PMA. Fluorescence was measured using FL3 channel (670 nm). Histograms of 10000 events were analyzed per assay.
Determination of thiobarbituric acid reactive substances.
Lipid peroxidation was estimated through measurement of thiobarbituric acid reactive substances (TBARS) in plasma as previously described (39), using 1,1,3,3-tetraethoxypropane as standard.
Western blotting of anti- and proapoptotic proteins.
Neutrophils (2 × 107 cells·mL−1) obtained from sedentary volunteers and triathletes at rest and after competition were homogenized in 250 μL of extraction buffer (100 mmol·L−1 Trizma, pH 7.5, 10 mmol·L−1 EDTA, 10% sodium dodecyl sulfate, 100 mmol·L−1 NaF, 10 mmol·L−1 sodium pyrophosphate, 10 mmol·L−1 sodium orthovanadate, at 100°C) and immediately sonicated for 30 s. Samples were boiled for 5 min and centrifuged at 12000 rpm, for 40 min at 4°C. Aliquots of supernatants were used for the measurement of total protein content, as described by Bradford (2). Equal amounts of proteins of each sample were separated using 8% sodium dodecyl sulfate-polyacrylamide gel. The proteins of the gel were transferred to a nitrocellulose membrane at 120 V for 1 h. Nonspecific binding was blocked by incubating the membranes with 5% defatted milk in basal solution (10 mmol·L−1 Trizma, pH 7.5, 150 mmol·L−1 NaCl, 0.05% Tween 20) at room temperature for 2 h. Membranes were washed three times with basal solution for 10 min each and then incubated with anti-bcl-xL (1:1000) and anti-bax (1:2000) antibodies in basal solution containing 3% defatted milk, at room temperature, for 3 h. Membranes were washed again (three times for 10 min each) and incubated with anti-IgG antibody linked to horseradish peroxidase (1:10,000) in basal solution containing 1% defatted milk, at room temperature, for 1 h. After washing once more, the membranes were incubated with substrate for peroxidase and chemiluminescence enhancer (ECL Western Blotting System Kit) for 1 min and immediately exposed to x-ray film for 3-10 min. The film was then developed in the conventional manner. Quantitative analysis of blots was performed using Image J software (National Institutes of Health, Bethesda, MD).
Measurement of intracellular neutral lipid accumulation.
Cells were centrifuged at 1000g for 15 min at 4°C and the pellet obtained was resuspended in 500 μL of PBS. Nile red (0.1 μg·mL−1), a selective fluorescent stain for neutral lipid, was added, and fluorescence was analyzed as described above in the FL1 channel (green fluorescence = 530/30 nm).
Determination of free fatty acids.
Plasma free fatty acid concentration was determined by an enzymatic colorimetric assay, NEFA C Kit (Wako Chemical, Neuss, Germany), following the manufacturer's instructions. The concentration of each fatty acid was calculated using the values of the proportion of fatty acids (oleic, linoleic, and stearic) found by the high-performance liquid chromatography (HPLC) analysis and the concentration of total fatty acids in the plasma.
Lipid extraction and determination of plasma fatty acid composition by HPLC.
According to the method of Folch et al. (6) lipids were extracted from plasma and saponified using 2 mL of an alkaline methanol solution(1 mol·L−1 NaOH in 90% methanol), at 37°C, for 2 h in a shaking water bath. Afterward, the alkaline solution was acidified to pH 3.0 with HCl solution (1 mol·L−1). Fatty acids were then extracted three times with 2 mL of hexane. After the extraction procedure and saponification, the fatty acids were derivatized with 4-bromomethyl-7 coumarin, and the analysis was performed in a liquid chromatographer (Model LC-10A; Shimadzu). The samples were eluted using a C8 column (25 cm × 4.6 internal diameter, 5-μm particles) with precolumn (2.5 cm × 4.6 internal diameter, 5-μm particles), 1 mL·min−1 of acetonitrile/water (77:23, v/v) flow and fluorescence detector (325 nm excitation and 395 nm emission). The standard fatty acids and margaric acid (C17:0) were obtained from Sigma Chemical Co. (St. Louis, MO). This latter fatty acid was used to calculate recovery. The capacity factor (K′), elution sequence, linearity, recovery, precision, interference, and limit of detection were determined. The minimum limit of quantification of the fatty acids ranged from 1 to 10 ng. Curve of calibration for each standard fatty acid was prepared to determine coefficients of correlation and regression.
In Vitro Experiments
Neutrophils were maintained in RPMI-1640 medium containing 10% fetal calf serum for 3 h. This medium was supplemented with glutamine (2 mmol·L−1), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (20 mmol·L−1), streptomycin (100 μg·mL−1), penicillin (100 U·mL−1), and sodium bicarbonate (24 mmol·L−1). Cells (1 × 106 cells·mL−1) were incubated in 2 mL per well of the plate. The cells were kept in a humidified atmosphere, at 37°C, containing 5% CO2.
Fatty acid treatment for cytotoxicity evaluation.
The cells were treated with various concentrations (50, 100, 200, 300, 400, 500, and 600 μmol·L−1) of oleic, linoleic, and stearic acids for 3 h. The fatty acids were dissolved in ethanol, the final concentration of which did not exceed 0.5%. This concentration of ethanol was not toxic to the cells. After incubation, the proportion of cells with loss of membrane integrity and/or DNA fragmentation was determined as described above. The presence of at least one sign of cell death was considered as indicating cytotoxicity.
Lucigenin-enhanced chemiluminescence assay.
Lucigenin is extensively used to measure production of oxygen reactive species by chemiluminescence. After being excited by a superoxide anion, lucigenin releases energy in the form of light. Lucigenin-amplified chemiluminescence provides information on both intra- and extracellular ROS content, whereas dihydroethidium method is used to determine the intracellular ROS content only (7). Lucigenin is the recommended assay for kinetic measurement of superoxide production. In this method, the response to xanthine-xanthine oxidase presents a positive correlation with light measurement and does not show augmentation of chemiluminescence when myeloperoxidase is added to the assay medium. Neutrophil chemiluminescence raised by fMet-Leu-Phe treatment is dose-dependently inhibited by scavengers of superoxide anions; and so this is the indicated method to measure superoxide anion production. Chemiluminescence response was monitored during 3 h, at 37°C, in the presence of diphenyleneiodonium chloride (DPI) (20 μmol·L−1), an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, using a Microplate Luminometer (Spectra Max Gemini XS; Molecular Devices, Sunnyvale, CA).
Results are presented as means ± SEM. The results were tested for normal distribution before analysis. Differences were assessed by ANOVA and Tukey-Kramer multiple comparisons test, using GraphPad Prism (GraphPad Software, Inc, San Diego, CA). The significance level was set for P < 0.05.
In vivo experiments.
The loss of plasma membrane integrity was low in neutrophils from both sedentary volunteers and triathletes at rest. The triathlon competition did not change the proportion of neutrophils with intact plasma membrane (data not shown).
DNA fragmentation (Fig. 1A) and PS externalization (Fig. 1B) were increased by threefold and twofold, respectively, in neutrophils obtained immediately after the triathlon competition when compared with sedentary and triathletes at rest. There was no difference in DNA fragmentation and PS externalization in neutrophils from sedentary volunteers and triathletes at rest. There was no change in mitochondrial transmembrane potential in neutrophils from triathletes after competition when compared with cells at rest; however, there was an increase in the proportion of neutrophils with mitochondrial transmembrane depolarization by 1.5- and 1.7-fold when cells from sedentary volunteers were compared with those of triathletes at rest and after competition, respectively (Fig. 1C).
After exposure to PMA, neutrophils from sedentary volunteers and triathletes at rest showed an increase of ROS production by fourfold and 2.5-fold, respectively. The PMA-induced increase of ROS production by neutrophils was not observed after the competition. The triathlon competition per se raised ROS production by neutrophils when compared with values at rest. After competition, the values obtained were close to those observed by PMA stimulation (3.3-fold; Fig. 2). The levels of TBARS in blood from triathletes after the competition were increased by 2.4- and 2.2-fold compared with sedentary volunteers and triathletes at rest, respectively (Fig. 3).
The triathlon competition decreased the content of antiapoptotic protein bcl-xL (by 69%; Fig. 4A) and increased that of the proapoptotic protein bax (by 61%; Fig. 4B) in neutrophils. Concomitantly, a decrease (by 65%) of intracellular neutral lipid content was observed in neutrophils obtained from athletes after the triathlon competition compared to rest (Fig. 5).
Plasma concentration of free fatty acids was increased after the triathlon competition by 3.5-fold when compared with sedentary volunteers and triathletes at rest (Fig. 6). A positive correlation between DNA fragmentation and the increase in plasma free fatty acid levels was found (r=0.688, P < 0.05; data not shown).
The proportion of lauric, myristic, palmitic, and γ-linolenic acids in plasma was decreased due to the triathlon competition when compared with sedentary volunteers. In contrast, the proportion of oleic, linoleic, and stearic acids was increased (Fig. 7). The proportion of palmitoleic, aralinolenic acids in plasma was decreased due to the triathlon competition when compared to sedentary volunteers. In contrast, the proportion of oleic, linoleic, and stearic acids was increased (Fig. 7). The proportion of palmitoleic, arachidonic, eicosapentaenoic, and docosahexaenoic acids was not changed. The plasma concentration of oleic, linoleic, and stearic acids was 0.102, 0.147, and 0.257 mmol·L−1, respectively, in the sedentary volunteers and 0.163, 0.328, and 0.540 mmol·L−1, respectively, in the triathletes after competition.
In vitro experiments.
The maximal tolerable (nontoxic) concentration of oleic and linoleic acids by neutrophils was 100 μmol·L−1 and of stearic acid was 200μmol·L−1 as indicated by the loss of membrane integrity (Fig. 8A) and/or DNA fragmentation (Fig. 8B). Oleic (Fig. 9A) and linoleic (Fig. 9B) acids markedly stimulated ROS production by neutrophils after 10 min of exposure either at nontoxic (100 μmol·L−1) or at toxic (200 μmol·L−1) concentrations.
Several studies have shown that high-intensity exercise induces apoptosis of lymphocytes from rats and athletes (22,23,30) and of neutrophils from rats (20). Evidence is presented herein that a triathlon competition leads to apoptosis of neutrophils obtained from elite athletes as indicated by DNA fragmentation and PS externalization.
In the present study, the triathlon competition markedly raised the plasma free fatty acid levels (by 3.5-fold), as also observed by Holly et al. (10) after an Ironman Triathlon (2.9-fold). The increase in plasma free fatty acid levels was positively correlated with the proportion of neutrophils with DNA fragmentation after the competition. Therefore, the toxicity of the plasma fatty acids to leukocytes may be involved in the results obtained. Other mediators, however, cannot be ruled out such as tumor necrosis factor α, glucocorticoids, and catecholamines (31,33,35).
Oxidative stress and lipid peroxidation have been postulated to be involved in the triggering of cell death induced by fatty acids. Intense exercise increases plasma levels of lipid peroxidation products (34,37), whereas provision of antioxidant compounds (N-acetyl-l-cysteine) decreases lymphocyte apoptosis observed in mice after a treadmill exercise (31). Spontaneous ROS production by unstimulated neutrophils and plasma levels of TBARS were increased after the competition. Levels of oleic, linoleic, and stearic acids found in plasma from the triathletes after competition were toxic to cultured neutrophils obtained from sedentary volunteers. Concurrently, these fatty acids led to ROS production by neutrophils after 10 min of treatment. These fatty acids probably stimulated nicotinamide adenine dinucleotide phosphate oxidase as indicated by the results with DPI (Figs. 9A and B).
Reactive oxygen metabolites, such as superoxide, can regulate the expression of anti- and proapoptotic genes (18). Accordingly, the triathlon competition decreased the expression of bcl-xL (antiapoptotic) and increased that of bax (proapoptotic). Decreased levels of antiapoptotic proteins and/or increased levels of proapoptotic ones result in neutrophil apoptosis. High-intensity exercise decreases the expression of the antiapoptotic protein bcl-2 in mice lymphocytes (30). Our group showed that a single session of a high-intensity exercise increased the expression of the bcl-xS and bax (proapoptotic) and reduced that of the bcl-xL (antiapoptotic) in rat neutrophils (20).
In addition to oxidative stress, other mechanisms have been shown to be involved in cell death induced by fatty acids; for example, increase in nitric oxide production (1), change in lipid rafts (36), decreased synthesis of mitochondrial phospholipid cardiolipin (1), phosphorylation and dephosphorylation of kinases and phosphatases (11), and de novo synthesis of ceramide (3). Of particular importance for the toxic effect of fatty acids is the intracellular accumulation of neutral lipids, mainly triglycerides (5,8). Some authors postulated that lipid accumulation promotes an increase in ceramide synthesis, which may induce apoptosis (27). Others, however, have shown that intracellular lipid accumulation protects the cells against death (4,8). In the present study, the triathlon competition led to a decrease of neutral lipid accumulation in neutrophils. This observation confirms the proposition that neutral lipid accumulation might reduce the occurrence of cell death.
In summary, evidence is presented herein that a triathlon competition induces apoptosis of neutrophils. Our findings suggest that high plasma levels of fatty acids (oleic, linoleic, and stearic acids), oxidative stress, decreased neutral lipid content, changes in expression of antiapoptotic (bcl-xL: decrease) and proapoptotic (bax: increase) may contribute to apoptosis after exercise.
The authors are indebted to the technical assistance of J.R. Mendonça, G. de Souza, E. Portiolli, and T.C. Alba-Loureiro. The authors thank S.T. Levada for his constant support and encouragement. This research has been supported by FAPESP, CNPq, and CAPES. The results of the present study do not constitute endorsement by ACSM.
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Keywords:©2008The American College of Sports Medicine
APOPTOSIS; LEUKOCYTE; FATTY ACIDS; TOXICITY; TRIATHLETES