Exercise is a well-known stress factor and is able to induce an inflammatory-like status. The cell counts of circulating leukocyte subsets change drastically during exercise and in the postexercise period. Whereas during and at the end of the exercise bout an increase of both lymphocytes and granulocytes can be observed, in the postexercise period a characteristic lymphopenia occurs (24). Changes in cell phenotype as measured by altered surface receptor expression, for example, adhesion molecules, document a cellular activation. Gabriel et al. (6) found increased CD45RA+-45RO+ cells after endurance exercise reflecting most probably an activation of T cells. Such an exercise-induced T cell activation needs to be shut off adequately similar to an antigen driven immune response. Otherwise the loss of immunological competence is imminent. Apoptosis is the immune system’s major mechanism to maintain lymphoid homeostasis and avoid disease (2). Recent investigations provide cumulative evidence that apoptosis is involved in the regulation of leukocyte count after exercise. Using the TUNEL method, Mars et al. (16) were the first to document lymphocyte apoptosis after strenuous exercise. We and others (12,19) found that apoptosis seemed to be related to exercise intensity. However, much information about the mechanisms responsible for exercise induced cell death is still lacking. Apoptosis might be induced on the one hand via the interaction of death ligands and receptors, namely Fas receptor (CD95) and Fas ligand (CD95L) (3). Fas receptor is a Type I integral receptor belonging to the tumor necrosis factor (TNF) superfamily. Upon binding to a ligand receptor, trimerization and clustering on the plasma membrane are required to initiate cell death (3). Fas ligand is a Type II membrane protein and is the natural ligand for Fas receptor. It is expressed either on the cell surface or in submembranous vesicles from which it can be released rapidly to the cell surface upon external stimuli (21). An up-regulation of Fas receptor, which serves as another indicator of cell activation, has been shown after treadmill exercise tests. However, the same study failed to determine an increase in surface expression of the pro-apoptotic Fas ligand (19). On the other hand, apoptosis can also be induced via a stress- or mitochondria-mediated pathway (2). Initial signals of that pathway may involve increases in cellular calcium transients or the increased formation of free radicals, which both have been shown to occur during exercise (20,26). These alterations are able to induce a loss of the mitochondrial transmembrane potential (MTP) (13,20,33). MTP loss in turn leads to a release of molecules from the intermembrane space such as cytochrome c or apoptosis-inducing factor (AIF), which participates in the execution of apoptosis (2,33).
The aim of the present study was to investigate the regulation of lymphocyte apoptosis after a long-lasting aerobic exercise such as running a marathon. By using subgroups of different performance levels, it was tested whether apoptosis was affected by the athlete’s training status. Moreover, the role of death receptors and ligands in this process should be determined.
Subjects and Experimental Design
A total of 38 male subjects were informed about the nature, purpose, and potential risks of the study and signed an informed consent statement approved by the University of Münster ethics committee. After a general medical check-up, the subjects were first tested for their maximal oxygen uptake (V O2max) during a continuous, progressive exercise test on a treadmill ergometer (Ergo XELG90 Spezial, Woodway, Weil am Rhein, Germany). The initial velocity was 8 km·h−1 increasing every 3 min by 2 km·h−1. Respiration parameters were analyzed using Quark b2 (Cosmed, Rome, Italy).
Depending on their V O2max, subjects were either excluded from the study or grouped into the high- or low-trained group. Athletes with a V O2maxof more than 60 mL·min−1·kg−1 were classified as highly trained (HT), whereas athletes with a V O2maxof less than 55 mL·min−1·kg−1 were classified as badly trained (BT). In total, 17 athletes (9 in the BT group; 8 in the HT group) were included into the study.
The anthropometric data as well as the performance parameters of the whole group and both subgroups are given in Table 1. Moreover, athletes were questioned about time and distance of their last training session before the marathon run.
All athletes participated then in the 2002 Münster marathon. Blood samples were taken after cannulation of the cubital vein. Resting blood samples were taken 2 d before the marathon at the same time of day because the athletes refused sampling initially before the marathon. After the marathon blood samples were taken immediately after, and 3 and 24 h after the run.
For the treadmill tests another 10 healthy, male subjects were included as reported previously (19). Their anthropometric data are presented in Table 1, too. All subjects passed through the same procedure as the marathon runners consisting of signing the consent statement, general medical check-up and performance test (see above). Then they performed first an exhaustive treadmill exercise test (ExT) at an intensity corresponding to 80% of the V O2max. About 10 –14 d later 5 of the 10 subjects performed a second exercise test at an intensity corresponding to 60% of the V O2max for the identical time as achieved during the strenuous exercise test (low intensity treadmill exercise test (LoT)). 5 athletes did not participate in the second exercise test due to different reasons (injury). Blood samples for both tests were taken before, after and 1 h after the tests.
Blood cell counts, hemoglobin and hematocrit determinations were performed on plasma anticoagulated with ethylenediaminetetraacetate (EDTA) using an semiautomated hematology analyzer (F-820, Sysmex, Norderstedt, Germany)
Cell Isolation Procedure
Lymphocytes were prepared by density gradient centrifugation. Briefly, 5 mL of a 50:50 mixture of whole blood anticoagulated with EDTA and 0.9% NaCl solution was carefully layered upon 3 mL of Lymphoprep (Nycomed, Oslo, Norway) and then centrifuged at 400 μg for 30 min at room temperature. After centrifugation the lymphocyte band between the sample layer and the Lymphoprep solution was removed. Cells were washed twice with buffer A of the following composition: NaCl 118 mM, KCl 5.4 mM, H-Hepes 10 mM, Na2HPO4 0.4 mM, KH2PO4, Glucose 5.5 mM adjusted to pH 7.4. Finally cells were resuspended in buffer B of the following composition: NaCl 140 mM, KCl 3 mM, H-Hepes 10 mM, Na2HPO4 0.4 mM, MgCl2 1 mM, CaCl2 0.8 mM, Glucose 5.5 mM adjusted to pH 7.4.
Cell viability was about 98% as demonstrated by trypan blue exclusion, whereas purity was about 95% as checked by flow cytometry in the forward and sideward scatter mode.
Analysis of Apoptosis-Related Cellular Surface Markers
Cell death was measured by flow cytometry (Epics XL, Coulter, Miami, FL) using annexin-V FITC and nuclear propidium iodide uptake for detection of apoptosis and necrosis, respectively (Roche Diagnostics, Mannheim, Germany). Lymphocytes (106) in 295-μL buffer B were incubated for 15 min at room temperature with 2.5 μL of each stock solution prepared according to the manufacturer’s instructions.
Furthermore, isolated lymphocytes (0.5 ×106) were labeled with either FITC-conjugated mouse antihuman CD95 monoclonal antibody (clone ANC95.1/5E2, Ancell Corp., Bayport, U.S.) or with phycoerythrin (PE)-conjugated mouse antihuman CD95 ligand monoclonal antibody (clone Alf-2.1a, Ancell Corp.) for 45 min at 4°C. Stock solutions of both antibodies were used according to the manufacturer’s instructions in a final working dilution of 1:50. CD95 and CD95L expression on the cell surface were analyzed by flow cytometry (Epics XL, Coulter).
All other chemicals were of the highest chemical grade available and were obtained from Sigma Chemical (St. Louis, MO).
If not indicated otherwise, data are presented as means ± SE. Differences between preexercise and postexercise values were tested using ANOVA. Statistical significance was set at the P < 0.05 level.
Physical and physiological characteristics.
Physical and physiological characteristics of all participants are listed in Table 1. There were only minimal differences between the groups and subgroups with respect to the physical data except for age. The classification of the marathon runners into two subgroups resulted in a V O2max difference of more than 30%. Moreover, the HT athletes showed a 24% increase in speed at the lactate threshold of 2.5 mM compared with the BT athletes, which resulted in a decrease of mean running time of about 21%. The training status of the treadmill group was more close to the BT group. Whereas the V O2max was slightly higher than the BT group, their speed at the lactate threshold of 2.5 mM was slightly lower. The mean running speeds of the ExT and LoT group were comparable to the values of the HT and BT group, respectively.
Leukocyte count alterations.
Resting lymphocyte count in all groups and subgroups were in a small range between 4800 and 6000 cells·μL−1 (Table 2). After the marathon run a leucocytosis of about 14,500 cells·μL−1 was observed that persisted at least 3 h; 24 h after the run, leukocyte levels were still enhanced compared with pre-marathon values. The leucocytosis in the HT and BT group was about 16,000 cells·μL−1 and 13,000 cells·μL−1, respectively. This difference was not significant because it resulted from one single athlete who reached a leucocytosis of about 30,000 cells·μL−1. After the treadmill tests leucocytosis was less pronounced and showed a relation to exercise intensity. Leukocytes were about 8050 cells·μL−1 after the ExT, whereas after LoT leukocytes increased only to about 5880 cells·μL−1; 1 h after the tests cell count returned to preexercise levels in both treadmill groups.
Exercise effects on lymphocyte apoptosis.
The percentage of basal lymphocyte apoptosis was similar in the whole marathon group and in the treadmill groups. However, analysis of the subgroups revealed that in the HT group basal apoptosis was significantly higher than in the BT group and in the treadmill groups (Fig. 1). For the marathon subgroups such difference couldn’t be attributed to a carryover effect from the premarathon training period as indicated by analysis of the questionnaires. HT athletes performed their last training session about 150 ± 49 h before the start of the marathon run, whereas BT athletes did so about 105 ± 41 h (not significant) before the race. Moreover, the last training distance was similar for both groups (HT: 16.1 ± 6.7 km vs BT: 12.0 ± 2.9 km; not significant). Lymphocyte apoptosis after the marathon run changed in a bivalent manner as displayed in Figure 2A. Initially after the run, the percentage of apoptotic cells was unchanged from preexercise levels; 3 h after the run a slight increase in apoptosis was observed, which turned into a significant decrease 1 d after the exercise. The mean increase in apoptosis of the marathon runners 3 h after the run was about 3.6 ± 2.0%. Interestingly, the subgroup analysis of the marathon runners revealed that the induction of apoptosis was dependent on the training status of the athletes. In the BT group, lymphocyte apoptosis was clearly enhanced by about 8.5 ± 2.4% 3 h after the run followed by a decline below resting levels about 1 d later (Fig. 2B). In contrast, apoptosis decreased in the HT group during the whole postexercise period investigated leading to a mean decrease of annexin-V positive cells of about 7.9 ± 1.6% 24 h after the run (Fig. 2B). Therefore, 3 h as well as 24 h after the run, mean changes of exercise induced apoptosis were different in the HT and BT group (Fig. 3A +B). A comparison of data with the treadmill tests revealed that the exercise-induced increases in apoptotic cells were similar for the BT group and the ExT group. For both groups, changes in annexin-V positive cells were significantly higher than in the HT group as well as in the LoT group. The results of the latter two groups were statistically not different from each other (Fig. 4).
Fas receptor and Fas ligand expression.
Next we determined the surface expression of the apoptosis-related membrane molecules, Fas receptor and Fas ligand. A sharp increase of Fas receptor bearing lymphocytes was observed immediately after marathon lasting for at least 3 h; 24 h after run the values were declined to preexercise levels. The curve of the Fas ligand changes after marathon was shifted to the right. Immediately after the run, values were not different from preexercise levels, whereas 3 h after the run an increase of about 200% was observed. Although the Fas ligand decreases during the next hour 1 d later, still a significant increase in Fas ligand of about 50% was found (Fig. 5).
The subgroup analysis demonstrated no difference in basal Fas receptor and Fas ligand expression between the HT and the BT athletes (Fig. 6, A and B). Likewise, marathon-induced alterations in Fas receptor expression were similar in both groups (Fig. 6, A and B). In contrast, after subgroup analysis Fas ligand expression displayed a higher response of the HT athletes (Figs. 6B and 7B).
Basal Fas receptor and ligand expression of the treadmill groups were in the same range as in the marathon group (data not shown). In contrast, the exercise induced changes of both surface molecules were significantly less pronounced after the treadmill tests than after the marathon. Marathon-induced increases in both death molecules were about 4– 6 times higher than after the treadmill tests. Comparison of the treadmill test data showed a similar increase of Fas receptor expression after both tests (Fig. 7A), whereas Fas ligand changes were significantly higher after ExT than after LoT (Fig. 7B).
There is growing evidence that apoptosis as a fundamental regulatory mechanism of the hematopoietic system is operative during exercise induced alterations of leukocyte count. This seems to be true for both granulocytes and lymphocytes and could be demonstrated for cells in the periphery as well as in lymphoid tissues (11,14,19). From a teleological point of view the involvement of apoptosis makes sense since it terminates and confines the inflammation-like activation of the immune system.
However, much information about mechanisms and parameters behind the exercise induced apoptosis is still lacking. The present data support the role of athlete’s training status as an important factor. HT and BT athletes demonstrated both a distinct basal lymphocyte apoptosis and different exercise-induced apoptosis changes. Basal lymphocyte apoptosis was higher in the HT group than in the BT group. In contrast, apoptosis after the marathon was substantially enhanced in the BT group, whereas a decline was observed in the HT group. In other words, lymphocytes of HT athletes seemed to be more resistant toward exercise-induced apoptosis than those from BT runners. Besides training status, other factors such as age, acute effects from previous training sessions, etc., might contribute to these differences. Apoptotic cell death has been shown to be age related. In elderly individuals, Schindowski et al. (29) showed that basal lymphocyte apoptosis rate was higher than in younger controls, which was different from our study. On the other hand, the sensitivity to oxidative stress induced apoptosis was enhanced in the senior group. It should, however, be emphasized that the age difference of both groups in the study by Schindowski et al. was much higher than in our present investigation (>25 vs 11.2 yr). Moreover, they reported higher surface expression of Fas receptors from the elderly, which was also not observed in our groups. Together, these data suggest that the slight age difference in the current study was most likely not responsible for the observed difference of exercise induced apoptosis between the HT and BT groups. Another factor to be concerned is a hang-over effect from the last training session. This possibility cannot be fully ruled out as the long-term kinetics of the exercise-induced changes of annexin-V and Fas receptor/ligand remain to be elucidated. However, time and distance of the last training session of both HT and BT group were not different from each other, making this point more unlikely. Indeed, our data coincide with recent studies about exercise induced DNA damage, which is supposed to be involved in apoptosis induction by exercise. DNA damage response pathways lead either to cell cycle arrest and DNA repair or to the apoptotic demise of the damaged cell. Recently, a number of studies demonstrated that exercise is followed by increased leukocyte DNA single strand breaks in an intensity-dependent manner (8,9,32). Moreover, genomic instability following exercise was less pronounced in endurance trained subjects (23,25,34).
In addition, the present data indicate that programmed cell death is regulated in an intensity and duration dependent manner. Apoptosis was higher after the ExT than after the LoT. Although the mean running time was rather small, the intensity of the ExT was obviously high enough to induce a substantial increase in lymphocyte apoptosis. On the other hand, exercise intensities of the BT and LoT group were comparable as indicated by their mean running speed, whereas exercise duration of both groups differed by a factor of 12. The longer duration had a significant impact on apoptosis, which suggests that high-intensity/short-duration exercise is equally effective in apoptosis induction than low-intensity/long-duration exercise.
The exercise-induced increase in lymphocyte cell death might be responsible at least in part for the well-known postexercise lymphopenia, which is therefore not supposed to be solely the result of a redistribution process (6,20,24). Instead, it is recognized as a clonal deletion process, which might therefore account for the temporal immune suppression in the postexercise period (24).
Cell death can be induced by several ways, one of which is via surface death receptors and ligands. Their cellular surface expression was differentially regulated by exercise. Fas receptor was enhanced after all types of exercise, indicating cellular activation as reported previously (19). However, there was a substantial difference of Fas receptor expression levels induced by the treadmill tests on the one hand and the marathon run on the other hand. In contrast, Fas ligand was unchanged after LoT, whereas a small upregulation of Fas ligand was observed after ExT. This was different from our previous study in which we couldn’t detect any Fas ligand changes after treadmill tests, which probably might be attributed to a gender effect as in the former study female subjects were included (19). Fas ligand upregulation was further enhanced after the marathon run, indicating the higher apoptotic potential of this long-lasting type of exercise. This was also emphasized by the coincidence of annexin-V and Fas ligand expression. Both surface markers reached their maximum at the same time 3 h after the marathon run. The interaction of Fas receptor/ligand is the main pathway for elimination of T cells during activation-induced cell death. This may occur either via fratricide or via suicide, depending whether receptor and ligand are expressed on different cells or on the same cell, respectively (2).
However, the subgroup analysis of Fas ligand expression favors also alternative explanations for the exercise-induced cell death. Although Fas ligand expression was higher in the HT athletes than in the BT group, this signal was not transduced into an enhanced lymphocyte apoptosis. In fact, apoptosis was lower in HT athletes, suggesting that Fas ligand up-regulation was not sufficient to induce apoptosis at least in the HT group and that other mechanisms may be involved.
Besides the Fas-mediated pathway (Type II pathway), cell death can be induced via the stress- or mitochondria-mediated pathway (Type I pathway) (2,33). Initial signals involve an increase in cellular calcium concentration and/or the formation of free radicals such as reactive oxygen and nitrogen species (RONS) (13,33). Exercise-linked alterations of cellular calcium signals have been recently demonstrated by us (20). Likewise, the enhanced formation of RONS during exercise is well documented, which seems to be inversely related to exercise intensity (7,26,32). Both altered calcium signals and RONS have the potential to depolarize the mitochondrial transmembrane potential (MTP), which seems to be an early step in the mitochondria-mediated apoptosis pathway (33). Recently, Hsu and colleagues (12) demonstrated alterations in MTP after repetitive exercise bouts of different intensities. Whereas exercise performed at an intensity corresponding to a VO2max of 35% lead to an increase in MTP, MTP decreased in an intensity-related manner after exercise at 60% and 85% of V O2max, respectively (12). Alterations in MTP are followed by the release of proteins from the intermembrane space such as cytochrome c and apoptosis-inducing factor (AIF) (2). These molecules trigger apoptosis either by activation of intracellular proteases such as capsases or by capsase-independent direct chromatin condensation, respectively.
To prevent and to confine the harmful effects of RONS, the cell is equipped with different mechanisms such as radical scavenging substances like glutathione or radical-depleting enzymes such as superoxide dismutase, glutathione peroxidase, or glutathione reductase. Intracellular glutathione is supposed to be an important switch leading to lymphocyte cell death if the GSH level falls below a certain threshold concentration (35). Several studies indicated that regular training is associated with an up-regulation of the radical depleting mechanisms (4,27). Yamamoto et al. (38) reported increased cellular levels of reduced glutathione (GSH) in physically active rats as well as an increase in antioxidant enzymes. Similar data in man have been reviewed by Sen et al. (30) for various tissues such as blood, skeletal muscle and liver. Therefore, it can be expected that after endurance training the exercise-induced oxidative stress is attenuated (18). Together, these findings would suggest that after exercise cell death is attenuated in trained athletes as documented in the present study.
However, it has to be admitted that exercise induced apoptosis is not necessarily harmful to the athlete but may have protective function against tumor development and progression. Malignant cells in general seem to be more vulnerable to damage by oxidative stress than normal cells (10). High redox state can serve as an activator/promoter of tumor suppressor genes, such as p53, thereby supporting the clearance of transformed cells and inhibiting tumor formation (5).
Finally, the analysis of apoptosis in the postexercise period revealed an unexpected finding. Exercise-induced apoptosis turned into a decreased percentage of apoptotic cells 24 h after the marathon. There is one study available that investigated late changes in lymphocyte apoptosis in humans after a single bout of exhaustive exercise. Mars et al. (16) revealed cell death in about 86% of lymphocytes 24 h after exercise using the TUNEL method. However, this part of the study was performed only with three subjects. Moreover, there are major drawbacks with the TUNEL method as it is not specific for apoptotic cells but also labels nonapoptotic viable cells that undergo DNA repair as well as necrotic cells. The method detects not only double-stranded DNA breaks (a hallmark of apoptosis) but also the free 3′-OH terminals of single-stranded DNA breaks as they may occur during DNA repair and cell necrosis (1,39). As described also by other authors, there seems to be no doubt about changes in DNA damage after exhaustive exercise (see above). But this does not necessarily mean that the cells go on to apoptosis. Cells may be rescued by DNA repair, which has been shown to be increased after training (25). Similar to our data, Mastaloudis et al. (17) reported recently bivalent changes of DNA damage after an ultramarathon. After an initial increase of the proportion of cells with DNA damage their proportion decreased below prerace levels at the days 1–6 postrace. Beside increased repair mechanisms the late decrease of apoptotic cells after marathon might be explained by an overshooting clearance of apoptotic cells. The clearance of apoptotic cells is the final step in the termination of a peripheral immune response (15). It depends on the phagocytic activity of monocytes/macrophages, but also other cell types such as hepatocytes or dendritic cells may be involved (28). The efficiency of this process seems to have a profound influence on the immune response in terms of enhancing or suppressing inflammation (36). If cell clearance is defective, autoimmunity or cancer can ensue (31). On the other hand, excessive cell clearance may cause lymphopenia and immunodeficiency, which are well known characteristics of the postexercise period (22,24,37). Unfortunately, in the present study, the latest time investigated was 24 h after the marathon. Further studies are therefore desirable to study apoptosis in the late postexercise period.
In summary, the present data indicate that exercise-induced lymphocyte apoptosis depends on the athlete’s training status. Cell death was induced only in badly trained athletes, whereas in highly trained athletes no changes occurred. Surface expression of the pro-apoptotic Fas ligand after the marathon run demonstrates the high apoptosis inducing potential of this massive aerobic exercise. However, there is evidence that also Fas ligand independent mechanisms are involved in exercise induced apoptosis.
The authors gratefully acknowledge the skilled technical assistance of Mrs. M. Lambrecht.
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Keywords:©2004The American College of Sports Medicine
MARATHON; CELL DEATH; FAS RECEPTOR; FAS LIGAND; ANNEXIN