Mounting evidence indicates that regular exercise programs are associated with reduced risk of chronic disease and enhanced physical fitness. However, the important question “How much exercise is optimal?” remains unresolved. The intensity and duration of exercise seem to play a key role in determining responses to exercise. On one hand, the health benefits of exercise appear to accrue in approximate proportion to the total amount of activity performed. Clear associations between accumulated caloric expenditure, physical fitness, and cardiovascular disease mortality have been demonstrated (17,23,24). The minimum threshold level of improvement in fitness has been proposed to be approximately 50% of the maximal oxygen consumption (V̇O2max) (2). On the other hand, heavy exercise is known to have the potential to trigger the onset of acute myocardial infarction and premature ventricular depolarizations (15,20). Furthermore, strenuous exercise may induce inflammatory reactions and immune disturbances (5), which may manifest clinically as increased incidence of postexercise infection or muscle injury (32). Therefore, the risk and benefit of exercise merit further investigation.
Exercise causes complex changes in the distribution and function of a number of cellular and humoral immune parameters. Malfunctions of the immunocompetent cells are incriminated as part of the causes of these phenomena. Recently, a high-percentage lymphocyte apoptosis has been demonstrated after intense treadmill exercise and was associated with postexercise immunodepression (19). One of the potential pathomechanisms leading to this change is the alteration in mitochondrial energization status. Cellular energization status is crucial to the activity and vitality of cells. Mitochondria membrane potential (MTP), the driving force of cellular ATP formation, is an important determinant of the form of cell death (28). There are gathering indications that mitochondrial depolarization precedes nuclear signs of apoptosis and constitutes an obligate step in ongoing apoptotic death (25). Mitochondrial depolarization can potentially be triggered by glucocorticoids, tissue ischemia and reperfusion (29), oxidative stress (6), cytokines (16), and reactive nitrogen metabolites (14). All these factors show varying extents of changes during exercise (1,30). Therefore, it is tempting to speculate that exercise may induce leukocyte apoptosis by perturbing the maintenance of MTP.
Mitochondrial depolarization can be evaluated by demonstrating a disruption of MTP. To further investigate the effects of exercise on leukocyte function and vitality, we measured MTP and the propensity of apoptosis in peripheral blood polymorphonuclear (PMN) leukocytes, monocytes, and lymphocytes from 12 subjects who performed three separate, 3-d sessions of AE of different intensities (35%, 60%, and 85% of V̇O2max). Time-sequence changes of mitochondrial alterations and apoptosis during each session of AE were gauged to study potential accumulative effects of exercise and to elucidate causal relationships among them.
Twelve trained male runners (mean age 21.1 ± 1.8 yr, mean body weight 61.4 ± 7.3 kg) volunteered to participate in this study. Questionnaires were used to exclude the presence of acute or chronic infectious, inflammatory, or immune disorders during the study period. The participants did not take antiinflammatory agents, steroid hormones, antioxidants, or vitamin supplements before the beginning of the study. The participants were asked to refrain from any form of trainings or vigorous physical activities 2 wk before study entry. Informed consents were obtained from all the subjects, and the research protocol was approved by the Committee for Research on Human Subjects of Taipei Physical Educational College.
Exercise mode and blood sampling.
All subjects performed a maximum AE test to exhaustion on a treadmill to determine their V̇O2max. The mean V̇O2max was 71.9 ± 4.7 mL·kg−1·min−1. Two weeks later, the subjects returned to perform the first session of AE at an intensity of 35% V̇O2max (mean metabolic equivalent (ME) 5.9 ± 0.8) on a treadmill for 30 min daily for 3 consecutive days. Three mm of venous blood samples were collected immediately before the first days (D1) of treadmill running and at sequential time points (D1′: immediately postexercise on the first day; D3: preexercise on the third day; D3′: immediately postexercise on the third day; D5: the fifth day; and D7: the seventh day) for subsequent assay. All AE and blood sampling were completed before 10:00 a.m. on each test day. Four weeks after completion of the first AE course, the subjects were randomly assigned to perform the second session of AE at an intensity of 60% V̇O2max or 85% V̇O2max). The third course of AE was performed 4 wk after the completion of the second AE course at a higher (85% V̇O2max) or lower (60% V̇O2max) intensity than the second session. The mean ME, corresponding to 60% V̇O2max and 85% V̇O2max, were 12.5 ± 1.0 and 17.5 ± 1.1, respectively. The participants were asked to refrain from any training or vigorous physical activity between individual AE sessions.
Approximately 200-μL heparinized blood was treated with 3 mL of 1:10 erythrocyte lysing buffer (PharMingen, San Diego, CA) for 10 min. The buffer did not contain a fixative agent, so leukocytes remained viable after red blood cell lysis (21). The supernatant was discarded and the cells were washed once with phosphate-buffered saline (PBS) at 200 g for 5 min. The cell pellets were resuspended with Hank’s balanced salt solution (Gibco BRL, Paisley, Scotland, UK) to approximately 105 cells·mL−1 for subsequent cell labeling. The viability of leukocytes was confirmed to be more than 95% as assessed by trypan blue exclusion test.
In separate experiments, PMN and peripheral blood mononuclear cells (PBMC) were isolated using the density gradient method and assayed for DNA fragmentation as described below. Briefly, 2 mL of heparinized blood was mixed with an equal volume of PBS. Three mL of Histopaque-1119 (Sigma, St. Louis, MO) was layered under 3 mL of Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) in 10-mL polypropylene conical tubes. The dilated blood was carefully layered over the gradient and then spun at 300 ×g for 15 min at room temperature. The opaque layer containing PBMC and the band, which formed at the interface containing PMN, was aspirated and transferred to separate siliconized glass tubes, and washed with 5 mL PBS. The suspensions were appropriately diluted with PBS to give a cell concentration of 106 cells·mL−1. The cells were fixed with 1% paraformaldehyde and 70% ethanol, and were stored under −70°C for further assay.
Because the mitochondria mass of different populations of leukocytes may vary greatly among different blood samples, a more reliable method to specify MTP, using the fluorochrome 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) (Molecular Probes, Eugene, OR), was employed in the present study. The leukocyte suspension was incubated with 5 μmol·L−1 JC-1 for 20 min at 37°C. After staining, JC-1 incorporates into mitochondria, where it either forms monomers (green fluorescence, 527 nm) or, at high transmembrane potential, aggregates (red fluorescence, 590 nm). The ratio between fluorescence intensity of JC-1 aggregates and monomers can reliably reflect MTP, independent of changes in the number and mass of mitochondria (8).
DNA fragmentation assay.
DNA fragmentation of nuclei, a characteristic of apoptotic leukocytes, was assessed using terminal deoxynucleotidyltransferase to incorporate fluorescein-12-dUTP into nuclei (TUNEL method), following the manufacturer’s protocol (APO-BRDUTM Kit, PharMingen, San Diego, CA) (18). The cells (PMN or PBMC) were labeled using a mixture containing terminal deoxynucleotidyltransferase and fluorescein isothiocyanate-dUTP, and the percentage of apoptosis was calculated as the number of cells in the high fluorescence intensity population divided by the total numbers of cells analyzed.
Fluorescence was analyzed by cytometry using a FACScan with CellQuest flow cytometric analysis software (Becton Dickinson, San Jose, CA). During analysis, separate electronic gates were set on the dot plot of forward and side scatter to include PMN, monocytesor lymphocytes, and to exclude red blood cells and debris. At the beginning of each analysis session, the identity of leukocyte subpopulations was confirmed by CD 45 and CD 14 immunophenotyping (27). The total number of events from each sample was made such that at least 5000 events were collected for the three leukocyte subpopulations. Mean fluorescence of each cell was determined to permit comparisons among the data. For the cells labeled with JC-1, the ratio of mean red fluorescence intensity (FL 2: 590 nm) over green fluorescence intensity (FL 1: 527 nm) was calculated and quoted as an index of MTP. Throughout all the measurements, the photomultiplier value of the detector in FL1 and FL2 remained constant and were set at 450 V. The whole flow cytometer assay was performed by the same investigator (T-G. Hsu), and was completed within 3 h after removal of the blood samples. The reproducibility of the assay was verified by a preliminary experiment conducting on 20 healthy human subjects, with an intra-assay coefficient of variation of 3.5% and an interassay coefficient of variation of 5.2%.
All continuous data were expressed as mean ± SE. Group comparison was judged by Mann-Whitney U-tests. Sequential changes were assessed by repeated measurement ANOVA. A P < 0.05 was considered statistically significant.
Figure 1 displays sequential alterations of leukocyte MTP during different intensities of AE. For three leukocyte subpopulations, MTP did not change significantly after a single bout of 35% V̇O2max AE, whereas its level began to rise on the third day of AE. MTP peaked on the fifth day and declined thereafter. The sequential changes of MTP were significant for monocytes and lymphocytes. In contrast, when higher intensity AE (60% and 85% V̇O2max) was performed, leukocyte MTP exhibited converse time-sequence alterations. Leukocyte MTP did not change after the first day of 60% V̇O2max AE, but its level declined significantly on the third day of running. When the intensity of AE was increased to 85% V̇O2max, MTP declined immediately postexercise on the first day, and the difference from the preexercise value reached statistically insignificance for monocytes and lymphocytes. Leukocyte MTP reached its lowest value upon completion of the 3-d AE session (D3′) and gradually normalized thereafter. The sequential changes of MTP were all statistically significant.
Figure 2 shows the propensity of apoptotic death, as indicated by DNA fragmentation assay, in PMN and PBMC during different intensities of AE. Before exercise, PMN showed a greater extent of apoptosis than PBMC. The propensity did not change significantly over the 3-d 35% V̇O2max running. In contrast, at an exercise intensity of 60% and 85% V̇O2max, both PMN and PBMC showed an increased propensity of apoptosis on the third day of AE. The leukocyte apoptosis peaked 2 d after completion of the AE (the fifth day), and declined thereafter.
The results in our study show that exercise at an intensity greater than 60% V̇O2max leads to impaired energization and vitality status of peripheral blood leukocytes in trained athletes, whereas similar findings are not seen when a more moderate exercise is performed. These findings may shed a new light on the pathomechanisms of exercise-induced immune disturbances and also confer evidences to justify the currently suggested intensity of “moderate” exercise (3–6 ME) for healthy adults (24).
There are several potential explanations for leukocyte mitochondrial depolarization after intense AE. First, exercise is known to activate endothelial nitric oxide productions due to increased shear stress (22). Nitric oxide is able to trigger thymocyte mitochondrial permeability transition (14), an important pathway leading to mitochondrial depolarization. Second, increased plasma glucocorticoids due to hypothalamic-pituitary axis stimulation may also contribute to the postexercise MTP changes. Physiological concentrations of glucocorticoids equivalent to blood levels measured after a single bout of AE is able to induce apoptosis of lymphocytes (4,12), and in vitro models have shown that the reduction of MTP is an early commitment step in glucocorticoid-induced apoptosis (9,33). It is likely that similar mechanisms might also operate in vivo. Third, intense AE may produce repetitive hypo- and reperfusion in the vascular compartment. Under such circumstances, inorganic phosphate and calcium ions will accumulate during the hypoperfusion stage and may subsequently induce mitochondria permeability transition during the reperfusion phase (10,26).
The delayed appearance of leukocyte apoptosis after the occurrence of mitochondrial depolarization was probably due to the temporal effect of the apoptotic program. Mitochondrial depolarization may initiate cellular apoptosis by disturbing cellular ATP production (28) or by releasing mitochondrial pro-apoptotic factors (31). The presence of these causal relationships in the present exercise model could be further supported by the synchronous changes of the propensity of apoptosis and the magnitude of MTP reduction, with higher intensity of AE resulting in more prominent changes. Alternatively, exercise-associated factors with potential to induce mitochondrial depolarization, such as the repetitive vascular hypo- and reperfusion, higher plasma glucocorticoids, catecholamines, and nitrogen metabolites, might also contribute to apoptosis through mechanisms other than mitochondrial disturbances (3,7,9,14,29). If this is the case, mitochondrial depolarization may merely act as an intermediate step in the apoptotic cell death programs rather than a causative factor.
Caution must be taken in interpreting the alterations of leukocyte function in the present clinical exercise model. It was measured on different leukocyte populations at different time points. Exercise is known to induce mobilization of PMN due to the stimulation of catecholamines or cortisol as a physiological response to tissue reparative processes (26). Leukocytes from other compartments, such as spleen or thymus, may have different mitochondrial functional status and different propensity of apoptosis (13). This may help explain the gradual increase in leukocyte MTP following moderate intensity (35% V̇O2max) AE, as the cell subpopulation mobilized by exercise may have higher MTP to cope with the increased ATP requirement for their active metabolism. Nevertheless, leukocyte populational changes did not account for the substantial mitochondrial depolarization and increased propensity of apoptosis after AE at higher intensities (60% and 85% V̇O2max). It is convincing that when the exercise intensity is higher, other plasma factors, such as glucocorticoids, may become a predominant factor in determining the functional status of peripheral leukocytes (12).
It is interesting to note that AE seems to have accumulative effects on leukocyte MTP and vitality. At the intensity of 60% V̇O2max, AE did not lead to reduction of MTP until the third day (D3) of exercise session. When higher intensity (85% V̇O2max) AE was performed, leukocyte MTP declined significantly after a single bout of AE, and its level declined further after daily, repetitive running. The lowest MTP level appeared after the third day of running (D3′). Similar changes were also noted in the propensity of apoptosis, which sustained over the AE course and peaked after completion of the entire running course. Similar findings have been demonstrated by Mars et al. (19), who found a link between repetitive exercise and sustained level of lymphocyte apoptosis.
The clinical significance of postexercise leukocyte mitochondrial alterations and apoptosis seems to be multi-fold. On one hand, the findings in the present study seem to support the “inverted J hypothesis” of postexercise immunosuppression and elevated risk for infectious disease (32) by showing that intense AE is detrimental to immunocompetent cells whereas moderate AE is not. In our study subjects, peripheral blood leukocytes suffered from impaired energization and survival status after intensive AE (60% and 85% V̇O2max). It is noticeable that, even at an intensity of 60% V̇O2max, previously considered “moderate” (11), AE still produced significant mitochondrial alterations in leukocytes. Therefore, for trained athletes, the intensity of exercise seems to be better defined in terms of multiples of ME rather than in percentages of V̇O2max, which will vary with different training status. On the other hand, the increased apoptosis of leukocytes may be one of the self-protective mechanisms in limiting the extent of tissue injury and systemic inflammation. Intensive exercise is known to induce inflammatory muscle injuries that resemble those seen in acute inflammation (5). Leukocyte infiltrations of sites of muscle damage seem to be essential to prepare the tissue for effective regeneration. It is plausible that leukocytes underwent late-onset apoptosis to prevent excessive inflammation, which is more prominent when more vigorous exercise is performed.
In conclusion, the present study suggests that intense exercise has accumulative effects on the leukocyte mitochondrial functional status. These changes are dependent on the intensity of exercise. The findings also suggest that leukocyte MTP is a potentially applicable indicator for monitoring immune distress due to overtraining. To what extent leukocyte mitochondrial depolarization may contribute to the exercise-associated disturbances in innate immunity and host defense demands further investigation.
Address for correspondence: Dr. Kelvin Tsai, Oxidative Stress Clinical Research Group, Division of Critical Care, Department of Medicine, Taipei Veterans General Hospital, 112, Taiwan; E-mail: firstname.lastname@example.org.
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