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Effects of a Simulated Tennis Match on Lymphocyte Subset Measurements a
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Mark Schafer , Holly Kell , James Navalta , Ramires Tibana , Scott Lyons & Scott Arnett a
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Western Kentucky University
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Catholic University of Brasilia Published online: 21 Feb 2014.
To cite this article: Mark Schafer , Holly Kell , James Navalta , Ramires Tibana , Scott Lyons & Scott Arnett (2014) Effects of a Simulated Tennis Match on Lymphocyte Subset Measurements, Research Quarterly for Exercise and Sport, 85:1, 90-96, DOI: 10.1080/02701367.2013.872219 To link to this article: http://dx.doi.org/10.1080/02701367.2013.872219
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Research Quarterly for Exercise and Sport, 85, 90–96, 2014 Copyright q AAHPERD ISSN 0270-1367 print/ISSN 2168-3824 online DOI: 10.1080/02701367.2013.872219
Effects of a Simulated Tennis Match on Lymphocyte Subset Measurements Mark Schafer, Holly Kell, and James Navalta Western Kentucky University
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Ramires Tibana Catholic University of Brasilia
Scott Lyons and Scott Arnett Western Kentucky University
Tennis is an activity requiring both endurance and anaerobic components, which could have immunosuppressive effects postexercise. Purpose: The purpose of this investigation was to determine the effect of a simulated tennis match on apoptotic and migratory markers on lymphocyte subsets. Method: Male high school (n ¼ 5) and college (n ¼ 3) tennis players (Mage ¼ 18.9 ^ 3.3 years) completed 10 sets of a tennis protocol including serves, forehand strokes, and backhand groundstrokes with 1-min rest periods between sets. Apoptosis antigen 1 receptor (CD95) and chemokine receptor fractalkine (CX3CR1) expression was analyzed on helper T lymphocytes (CD4 þ ), cytotoxic T lymphocytes (CD8 þ ), and B lymphocytes (CD19 þ) twice, at resting baseline and immediately after all 10 sets of the tennis protocol. Results: An increase was observed in each lymphocyte subtype ( p , .02, effect size ¼ .41), and comparison of absolute changes revealed increases in CD4 þ /CD95 þ , CD8 þ /CD95 þ , and CD8 þ /CX3CR1 lymphocytes following the tennis protocol ( p , .01, effect size ¼ .43), but not in CD19 þ cells. Conclusions: A simulated tennis match has adequate intensity to induce immune modulations in terms of increased cell death and cellular migration in T lymphocyte subsets. Lymphocytopenia following tennis play is influenced by both apoptotic and migratory mechanisms. Keywords: apoptosis, cell migration, lymphocytopenia
Continuous endurance exercise has been shown to modulate the immune system (Kendall, Hoffman-Goetz, Houston, MacNeil, & Arumugam, 1990; Nieman, 1997; Patlar, 2010). As little as 30 min of walking has been reported to increase plasma interleukin-6 concentration and lymphocyte proliferative response (Nieman, Henson, Austin, & Brown, 2005), whereas 2 hr of cycling increases neutrophil degranulation and oxidative burst (Li & Cheng, 2007). Anaerobic and short periods of exercise have been typically
Submitted May 4, 2012; accepted May 30, 2013. James Navalta is now at the University of Nevada, Las Vegas. Correspondence should be addressed to Mark Schafer, Department of Kinesiology, Recreation, and Sport, Western Kentucky University, 1059 Smith Stadium, 1906 College Height Blvd., Bowling Green, KY 42101. Email: [email protected]
less investigated, but recent research has shown changes in lymphocyte viability following 5 min of moderate-intensity swimming (Prestes et al., 2008), while cytotoxic T cell volume was found to increase significantly with repetitive supramaximal anaerobic cycle tests (Friedman et al., 2012). As active tennis play is an activity that requires a significant endurance component and also consists of periodic anaerobic bursts, it is possible that this mode of exercise could have specific immunoregulatory effects. A limited amount of research exists with respect to the sport of tennis and associated modulations on the immune system. Novas, Rowbottom, and Jenkins (2003) found that increases in training load or elevated competition level were directly correlated with the incidence of upper-respiratory tract infections in elite female tennis players. In an investigation comparing a group of elite male and female
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tennis athletes to matched nonathletic controls, it was reported that neutrophil count was 16% lower and natural killer cells were 53% elevated in the elite athletes, and that all other immune parameters were not significantly different between groups (Henson et al., 2001). A 2-hr bout of tennis drills in elite adolescent tennis players produced significant increases in neutrophil and monocyte concentrations along with a decrease in salivary immunoglobulin A secretion rate (Nieman, Kernodle, Henson, Sonnenfeld, & Morton, 2000). It is of interest to note that this investigation also revealed a significant reduction in the overall lymphocyte count measured at 1 hr postexercise compared with the baseline measurement (Nieman et al., 2000). A depressed lymphocyte cell count following physical activity is termed exercise-induced lymphocytopenia and has been hypothesized to lead to a period of immunosusceptibility (Nieman, 1997; Walsh et al., 2011). Emerging research from our laboratory and from others has attributed the lymphocytopenia response in the postexercise period to either cell death (via apoptosis; Mars, Govender, Weston, Naicker, & Chuturgoon, 1998; Mooren, Bloming, Lechtermann, Lerch, & Volker, 2002; Navalta, McFarlin, & Lyons, 2010), cellular migration from the vascular compartment into the lymphoid pools (Friedman et al., 2012; Hong et al., 2005; Kruger, Lechtermann, Fobker, Volker, & Mooren, 2008; Simpson et al., 2007), or a combination of both (Navalta et al., 2013). Differential contributions from these mechanisms have varying consequences with regard to susceptibility in the period following exercise. Each mechanism has associated benefits and drawbacks; greater apoptosis could result in the permanent removal of potentially harmful cells at the cost of needing to fill the immune space with more viable cells, while greater migration could increase efficiency by maintaining access to lymphocyte subsets at the cost of retaining these cells, which have the potential for receptor cross-reactivity, which has been implicated as a mechanism underlying autoimmune disorders (Fujinami & Oldstone, 1989). Regardless of the etiology, a transient reduction of immune cells in the postexercise period can leave an athlete susceptible when presented with an immune challenge. Therefore, it is of interest to determine the lymphocytopenia response after various exercise modes, such as tennis, which requires both endurance and anaerobic components in a free-play model. We have recently shown that the apoptotic and migratory response of lymphocyte subfractions (i.e., helper T lymphocytes [CD4 þ ], cytotoxic T lymphocytes [CD8 þ ], and B lymphocytes [CD19 þ ]) respond differently in the postexercise period following an acute progressive treadmill bout to exhaustion (Navalta et al., 2013). To our knowledge, this response in lymphocyte subsets has not been characterized following an intermittent athletic activity such as tennis. In a previous investigation from our laboratory, we found that high-intensity intermittent cycling had no effect on lymphocyte apoptosis utilizing the early-
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phase apoptosis marker annexin V (Friedman et al., 2012). Therefore, in the current investigation we decided to utilize the apoptosis antigen 1 receptor (CD95) as an indicator of cell death (apoptosis), along with the chemokine receptor fractalkine (CX3CR1) as the indicator of lymphocyte extravasation (cell migration). We hypothesized that a tennis match would modulate significant increases in CD4 þ , CD8 þ , and CD19 þ lymphocytes with regard to cell concentration, the apoptotic marker CD95, and the migration marker CX3CR1. Therefore, the purpose of this investigation was to determine the effect of a simulated tennis match on the lymphocytopenia response in CD4 þ , CD8 þ , and CD19 þ lymphocytes.
METHODS Participants An a-priori power analysis was completed utilizing data for overall apoptotic lymphocytes from our previous work (Navalta, Sedlock, & Park, 2005). As a large effect size (.80) was expected, the sample size required to detect significant differences between means was determined to be six participants (a ¼ .05, b ¼ .80). In an attempt to utilize a conservative approach, eight male participants were recruited from area high schools and the university. Descriptive characteristics are presented in Table 1. In the current investigation, the participants included high school tennis players (n ¼ 5) and college tennis players (n ¼ 3). Participants had participated in regular scheduled tennis practice 4 to 6 days per week for 1 hr and 30 min to 2 hr and played competitively in the 3 months prior to participating in the investigation. At the time of recruitment and prior to the study protocol, each participant completed the Physical Activity Readiness Questionnaire and a medical history and signed an informed consent. The participants were apparently healthy tennis players with no musculoskeletal limitations or diagnosed cardiovascular or metabolic disease. The study was approved by the Western Kentucky University Human Subjects Review Board and all participants provided informed consent prior to participation in the investigation. For participants younger than the age of 18, parental consent was obtained prior to data collection.
TABLE 1 Participant Anthropometric Characteristics Variables Age (years) Height (cm) Weight (kg) % Body Fat
M
SD
18.9 178.1 66.7 10.9
3.3 6.8 8.1 3.5
Note. M ¼ mean; SD ¼ standard deviation.
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Protocol Initial measurements of age, height, weight, and skinfold analysis were recorded for each player upon reporting to the indoor tennis facility where the temperature ranged from 708F to 748F (218C to 238C), and percent humidity was less than 60%. Height and weight were measured using a DetectMedic Scale and attached stadiometer (Detecto Scales Inc., Webb City, MO). Skinfold measurements were taken at the chest, abdomen, and thigh using calibrated Lange skinfold calipers (Beta Technology, Santa Cruz, CA). Body composition was estimated using the Jackson and Pollock (1985) equations. Prior to the simulated tennis match, a 10-min to 15-min warm-up was performed including three laps around the two-court tennis facility and static and dynamic stretching according to the personal preference of each participant. In addition, 5 serves to the deuce court, 5 serves to the ad court, 10 crosscourt forehand ground strokes, and 10 crosscourt backhand ground strokes returned from a Play Mate Grand Slam oscillating ball machine (Metaltek, Morrisville, NC) were performed prior to beginning the simulated tennis match. Subsequent to the warm-up period, the participants began the simulated tennis match protocol with 5 serves to the deuce court and 5 serves to the ad court. Following the service trials, the participants hit 480 ground strokes fed at a constant speed, spin, and placement by the Play Mate Grand Slam ball machine. Participants hit 24 forehand strokes and 24 backhand strokes against the oscillating ball machine to complete one hitting session. The simulated tennis match protocol consisted of a total of 10 hitting sessions preceded by the service trials, with 1-min rest intervals between each hitting session. The participants completed the simulated tennis match protocol in approximately 90 min. Heart rate (HR) was measured using a using a wireless Polar Monitoring System (Woodbury, NJ), and the rating of perceived exertion (RPE) was measured using the Borg 6 to 20 RPE scale (Borg, 1998). HR and RPE were measured immediately upon completing each hitting session. Blood samples (120 mL) were obtained by finger stick using an antiseptic technique in the seated position at rest prior to the warm-up and immediately following the final
simulated tennis set. Processing for cell count and markers of apoptosis and cellular migration was carried out in duplicate as previously described (Navalta et al., 2011). Unless specified otherwise, antibodies were supplied by eBioscience (San Diego, CA). In brief, whole blood (10 mL) was added to a mixture containing Flow Cytometry Staining Buffer and antihuman antibodies specific for cell type (phycoerythrin: CD4, CD8, or CD19) and a marker for apoptosis (fluorescein isothiocyanate [FITC]: CD95) or migration (FITC: CX3CR1; BioLegend, San Diego, CA; see Table 2). Samples were incubated for 30 min in the dark at room temperature, then were centrifuged for 5 min and decanted. Cells were added to red blood cell lysis buffer and were allowed to incubate for 15 min. Following the incubation period, phosphate-buffered saline (SigmaAldrich, St. Louis, MO) was added to stop the lysis reaction, and the samples were centrifuged for 5 min, decanted, and vortexed thoroughly prior to analysis by flow cytometry (Accuri C6, Ann Arbor, MI). Statistical Analysis Data were analyzed using a paired-samples t test with the Statistical Package for the Social Sciences software program (IBM SPSS Statistics 18.0, Somers, NY). Absolute changes from rest (D baseline) values were calculated according to the following formula: ([measure – baseline] £ baseline – 1) £ 100. Assuming no change in absolute measures between changes in cell volume, apoptosis, and cell migration, we utilized the x2 test to determine differences between the expected and observed outcomes. For all statistical tests, significance was accepted at the p , .05 level.
RESULTS The simulated tennis match elicited an exercise intensity of 79 ^ 10% of the participants’ age-predicted maximum HR and an RPE of 14.3 ^ 1.8 over the 10 hitting sessions. The cardiovascular and perceptual responses for each of the 10 hitting sessions resulting from the simulated tennis match are presented in Table 3.
TABLE 2 Per-Tube Breakdown of Mixtures Containing Specific Antihuman Antibodies and Flow Cytometry Staining Buffer Tube 1 2 3 4 5 6
Buffer (250 mL)
FITC-Conjugated Antibody (0.25 mL)
PE-Conjugated Antibody (0.25 mL)
Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer
CD95 CX3CR1 CD95 CX3CR1 CD95 CX3CR1
CD4 CD4 CD8 CD8 CD19 CD19
Note. FITC ¼ fluorescein isothiocyanate; PE ¼ phycoerythrin; CD95 ¼ apoptosis antigen 1 (APO-1); CX3CR1 ¼ fractalkine receptor; CD4 ¼ helper T lymphocytes; CD8 ¼ suppressor/cytotoxic T lymphocytes; CD19 ¼ B lymphocytes.
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Cell Counts
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A simulated tennis match induced significant increases in the concentration of each lymphocyte subset compared with resting values (see Figure 1; CD4 þ , p ¼ .013, effect size ¼ .45; CD8 þ , p ¼ .011, effect size ¼ .57; CD19 þ , p ¼ .015, effect size ¼ .41). While the relative change in the apoptotic marker CD95 was not significant for any lymphocyte subfraction (CD4 þ , p ¼ .082, effect size ¼ .73; CD8 þ , p ¼ .47, effect size ¼ .35; CD19 þ , p ¼ .435, effect size ¼ .41), the tennis protocol induced significant relative changes in the expression of the migration receptor CX3CR1 in CD4 þ ( p ¼ .042, effect size ¼ .31) and CD8 þ ( p ¼ .002, effect size ¼ .19; see Figure 2).
Apoptosis/Migration Markers When assessing the impact of the tennis protocol to influence the exercise-induced lymphocytopenia response, the absolute change from baseline was determined for cell count, a TABLE 3 Cardiovascular and Perceptual Responses for Each of the Hitting Sessions From the Simulated Tennis Match Variable Hitting Session 1 Heart rate (bpm) RPE Hitting Session 2 Heart rate (bpm) RPE Hitting Session 3 Heart rate (bpm) RPE Hitting Session 4 Heart rate (bpm) RPE Hitting Session 5 Heart rate (bpm) RPE Hitting Session 6 Heart rate (bpm) RPE Hitting Session 7 Heart rate (bpm) RPE Hitting Session 8 Heart rate (bpm) RPE Hitting Session 9 Heart rate (bpm) RPE Hitting Session 10 Heart rate (bpm) RPE
M
SD
159.7 12.5
21.8 2.2
170.7 13.2
25.4 1.9
166.4 13.4
25.4 1.8
165.4 14.0
23.7 1.8
162.5 14.8
28.7 1.5
162.4 14.5
28.9 1.5
163.5 14.7
33.5 1.0
159.1 15.0
32.4 1.0
162.2 15.2
30.9 1.6
165.8 15.5
27.2 1.9
Note. M ¼ mean; SD ¼ standard deviation; bpm ¼ beats per minute; RPE ¼ rating of perceived exertion.
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marker of apoptosis (CD95), and a marker for lymphocyte migration (CX3CR1) as we have described previously (Navalta et al., 2013). We found that CD4 þ T cells displayed a significantly greater increase in CD95 membrane expression ( p ¼ .01, effect size ¼ .43), which was not matched with changes in cell concentration or migration membrane expression (see Figure 3). In addition, for CD8 þ lymphocytes, the observed increase of both CD95 ( p ¼ .001, effect size ¼ .57) and CX3CR1 membrane expression was significantly increased above the absolute change in cell volume ( p ¼ .001, effect size ¼ .44; see Figure 3).
DISCUSSION This investigation was designed to determine the lymphocyte subset response to a simulated tennis match. Specifically, we wished to determine the apoptotic and migratory responses of CD4 þ , CD8 þ , and CD19 þ to a tennis protocol. It was hypothesized that an intermittent activity with a combination of endurance capacity and anaerobic bursts such as tennis would modulate discrete changes in individual lymphocyte subfractions. Based on the results of the present investigation, we reject the null hypothesis and observed a significant increase in each lymphocyte type with tennis play and an increase in the relative expression of the migration marker on both T cell subtypes. In addition, when compared with the absolute expected rise in cell volume above baseline values, CD4 þ T cells expressed the CD95 apoptotic marker to a greater extent, whereas CD8 þ T lymphocytes displayed an elevation in both apoptotic and migratory markers. To our knowledge, this is the first investigation to report the lymphocyte subset response following a simulated tennis match. Henson et al. (2001) compared resting T cell counts in a group of tennis athletes who were in the midst of an intense training period to the resting blood
FIGURE 1 Lymphocyte subset concentration measured in participants (N ¼ 8) at rest (Pre) and upon completion of a simulated tennis match (Post; mean ^ SE). Note. CD19 þ ¼ B lymphocytes, CD8 þ ¼ cytotoxic T lymphocytes, CD4 þ ¼ helper T lymphocytes. *Indicates statistically significant difference from Pre at the p # .02 level.
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samples of age-matched controls. They reported no differences in resting concentrations of total lymphocytes (tennis athletes ¼ 2.53 ^ 0.13 106 cells·mL – 1 , 6 –1 controls ¼ 2.73 ^ 0.13 10 cells·mL ), CD3 þ T cells (tennis athletes ¼ 1.35 ^ 0.11 10 6 cells·mL – 1 , 6 –1 controls ¼ 1.39 ^ 0.12 10 cells·mL ), or CD19 þ B cells (tennis athletes ¼ 0.42 ^ 0.04 106 cells·mL – 1, controls ¼ 0.42 ^ 0.05 106 cells·mL – 1; Henson et al., 2001). Nieman et al. (2000) reported that a 2-hr bout of tennis drills was insufficient to change the total lymphocyte concentration compared with measured baseline values (rest ¼ 2.53 ^ 0.13 106 cells·mL – 1, postexercise ¼ 2.44 ^ 0.15 106 cells·mL – 1). In the current study, a significant increase in each lymphocyte subset was observed following the simulated tennis protocol (see Figure 1). A possible explanation for the difference between our findings compared with those of Nieman et al. (2000) is the training status of the participants. In the current investigation, the participants were local high school and college-level players, and they did not achieve the volume of practice of the elite tennis players in the Nieman et al. (2000) investigation. Also, the amount of time that the tennis protocol employed in the current investigation utilized much shorter rest periods (1 min vs. 4 –5 min). It is possible that the training status and the intensity of tennis match play with minimal rest periods can significantly increase the lymphocyte concentration in the circulation. Previous research revealed an exercise-induced lymphocytopenia that was evident for up to 1 hr following a 2-hr bout of tennis drills (Nieman et al., 2000). Various authors have contributed this lymphocytopenia response to either apoptosis (Mars et al., 1998; Mooren, Lechtermann, & Volker, 2004; Navalta et al., 2005), cellular migration (Friedman et al., 2012; Hong et al., 2005; Simpson et al., 2007), or a combination of both (Navalta et al., 2010, 2013). As far as we are aware, this is the first study to report the potential influence of an intense tennis protocol on measures of lymphocyte apoptosis and migration in T cell subsets. We
FIGURE 2 Relative expression of the migration receptor CX3CR1 on lymphocyte subfractions obtained from participants at baseline (Pre) and immediately following a simulated tennis protocol. *Indicates statistically significant difference from Pre at the p # .05 level.
observed that tennis play, which incorporated both endurance and anaerobic burst activities, significantly elevated the absolute CD4 þ apoptotic response above the expected rise in cell count. This response is similar to what we reported for continuous treadmill running at 76% maximal oxygen consumption (VO2max), but not at the higher exercise intensities of 87% or 100% VO2max (Navalta et al., 2013). In addition, a relative increase in the percent of expressed migration receptor CX3CR1 was noted in CD4 þ cells following the tennis protocol. Regarding the lymphocytopenia response specific to CD4 þ cells, these results indicate that the postexercise decrease in cell volume is due to a combination of cellular death as well as extravasation into the lymphoid pools. Simpson et al. (2007) have presented compelling evidence that high-intensity exercise (in the form of level treadmill running at 80% of aerobic capacity, or downhill running on a treadmill at 2 10% grade) significantly increases cellular adhesion molecules (CD54 þ , or Intercellular Adhesion Molecule 1; CD18 þ , or b-integrin) on lymphocytes that serve to facilitate the movement of white blood cells out of the circulation. Future investigations are warranted to determine the direct contribution of each mechanism as well as the practical consequences of immunoregulation in this lymphocyte subfraction that is charged with regulating the cell-mediated immune response (Walsh et al., 2011). In the present investigation, an intense tennis protocol successfully elevated an absolute change in CD8 þ lymphocytes with regard to both apoptotic as well as migratory marker expression. This response is different compared with any intensity of continuous treadmill running reported previously, which reported a significant apoptotic increase at 76% VO2max that was replaced by a much greater rise in migration at 87% VO2max (Navalta et al., 2013). It is likely that the difference is due to the apoptotic marker utilized (CD95 in the present investigation vs. annexin V previously) and the nature of the protocol (intermittent
FIGURE 3 Absolute change from baseline in cell count, apoptosis (CD95 þ ), and lymphocyte migration (CX3CR1 þ ) in CD4 þ and CD8 þ lymphocyte subsets from participants (N ¼ 8) who completed a simulated tennis protocol. *Indicates statistically significant increase compared with the expected change in cell volume at the p # .01 level.
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tennis vs. continuous treadmill running). It has previously been reported that CD8þ cells are influenced by exercise to a greater extent than the CD4þ subset, presumably due to the interaction of epinephrine with b2-adrenergic receptors (Ibfelt, Petersen, Bruunsgaard, Sandmand, & Pedersen, 2002; Pedersen & Hoffman-Goetz, 2000). The current investigation provides evidence that this phenomenon extends to tennis play, as greater influences were noted in both the absolute and relative CX3CR1 response, as well as the absolute apoptotic response of CD8þ lymphocytes. The current investigation could have been strengthened by the inclusion of a resting control group. In addition, another limitation is that our simulated tennis match protocol has restricted application to actual tennis play, where athletes have no prior knowledge of where an opposing player will hit a particular shot. From this standpoint, the level of intensity in the present investigation may have been lower than what is found during actual tennis competition. Finally, with regards to characterizing the lymphocytopenia response following tennis play, extending the blood-sampling time points for as long as 3 hr postexercise would be appropriate, as would the determination of plasma or serum measures that could explain the cell death/migration mechanisms such as catecholamines, cytokines, and/or caspases. In summary, we found that an intermittent tennis protocol designed to mimic actual match play had significant effects on the lymphocyte subset response. An increase in each lymphocyte subfraction (CD4 þ , CD8 þ , and CD19 þ ) was observed postexercise. In addition, exercise significantly increased the expression of both apoptotic and migratory markers on T cell subsets, helping to explain the decrease in overall lymphocytes reported 1 hr following tennis drills (Nieman et al., 2000). As it is usual for tennis athletes to compete in multiple matches during a short timeframe in tournament play, future investigations should be directed toward the practical implications of this noted immunomodulated period. Additionally, the influence of cell death and migration should be further investigated, and the consequences of each mechanism should be characterized. Although speculative, it is possible that cells removed from the circulation due to apoptosis could take longer to replace and increase the transient period of susceptibility, whereas cells that have migrated out of the circulation could be more readily recruited back and available in the case of an immune challenge. Thus, it may be possible to reduce potential negative impacts with modulations in nutritional intake or training program design leading up to a tournament.
WHAT DOES THIS ARTICLE ADD? The findings of the present investigation indicate for the first time that tennis play can have adequate intensity to
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induce immune modulations in terms of increased cell death and cellular migration in both T lymphocyte subsets. Transient decreases in CD4 þ could decrease an athlete’s ability to dictate the immune response to specific antigens, whereas a depressed cytotoxic volume would reduce cellto-cell killing capability in the period following a match. From a clinical standpoint, increased lymphocytopenia in the postexercise period could transiently open tennis athletes to susceptibility to respiratory infections. Future investigations should track the responses that we report here utilizing actual tennis play, particularly in terms of the number of groundstrokes hit during a rally and the time interval between rallies and sets. In theory, this knowledge would help to direct coaches and athletes to utilize timeouts or pace-of-play tactics to minimize the immunomodulations we report here, particularly in the framework of a multiple match tournament that extends over several days. REFERENCES Borg, G. (1998). Borg’s perceived exertion and pain scales. Champaign, IL: Human Kinetics. Friedman, R. A., Navalta, J. W., Fedor, E. A., Kell, H. B., Lyons, T. S., Arnett, S. W., & Schafer, M. A. (2012). Repeated high-intensity wingate cycle bouts influence markers of lymphocyte migration but not apoptosis. Applied Physiology, Nutrition, and Metabolism, 37, 241 – 246. doi:10.1139/h11-156. Fujinami, R., & Oldstone, M. (1989). Molecular mimicry as a mechanism for virus-induced autoimmunity. Immunologic Research, 8, 3 –15. Henson, D. A., Nieman, D. C., Kernodle, M. W., Sonnenfeld, G., Morton, D., & Thompson, M. M. (2001). Immune function in adolescent tennis athletes and controls. Sports Medicine, Training and Rehabilitation, 10, 235–246. Hong, S., Johnson, T. A., Farag, N. H., Guy, H. J., Matthews, S. C., Ziegler, M. G., & Mills, P. J. (2005). Attenuation of T-lymphocyte demargination and adhesion molecule expression in response to moderate exercise in physically fit individuals. Journal of Applied Physiology, 98, 1057– 1063. doi:10.1152/Japplphysiol.00233.2004 Ibfelt, T., Petersen, E. W., Bruunsgaard, H., Sandmand, M., & Pedersen, B. K. (2002). Exercise-induced change in type 1 cytokine-producing CD8 þ T cells is related to a decrease in memory T cells. Journal of Applied Physiology, 93, 645–648. doi:10.1152/japplphysiol.01214.2001 Jackson, A. S., & Pollock, M. L. (1985). Practical assessment of body composition. Physician and Sportsmedicine, 13(3), 76–90. Kendall, A., Hoffman-Goetz, L., Houston, M., MacNeil, B., & Arumugam, Y. (1990). Exercise and blood lymphocyte subset responses: Intensity, duration, and subject fitness effects. Journal of Applied Physiology, 69, 251–260. Kruger, K., Lechtermann, A., Fobker, M., Volker, K., & Mooren, F. C. (2008). Exercise-induced redistribution of T lymphocytes is regulated by adrenergic mechanisms. Brain, Behavior, and Immunity, 22, 324– 338. doi:10.1016/j.bbi.2007.08.008 Li, T. L., & Cheng, P. Y. (2007). Alterations of immunoendocrine responses during the recovery period after acute prolonged cycling. European Journal of Applied Physiology, 101, 539 –546. doi:10.1007/ s00421-007-0529-1 Mars, M., Govender, S., Weston, A., Naicker, V., & Chuturgoon, A. (1998). High intensity exercise: A cause of lymphocyte apoptosis? Biochemical and Biophysical Research Communications, 249, 366–370. doi:10.1006/ bbrc.1998.9156
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Mooren, F. C., Bloming, D., Lechtermann, A., Lerch, M. M., & Volker, K. (2002). Lymphocyte apoptosis after exhaustive and moderate exercise. Journal of Applied Physiology, 93, 147–153. doi:10.1152/japplphysiol.01262.2001 Mooren, F. C., Lechtermann, A., & Volker, K. (2004). Exercise-induced apoptosis of lymphocytes depends on training status. Medicine and Science in Sports and Exercise, 36, 1476–1483. doi: 10.1249/01. MSS.0000139897.34521.e9 Navalta, J. W., Lyons, S., Prestes, J., Arnett, S. W., Schafer, M., & Sobrero, G. L. (2013). Exercise intensity and lymphocyte subset apoptosis. International Journal of Sports Medicine, 34, 268–273. Navalta, J. W., McFarlin, B. K., & Lyons, T. S. (2010). Does exercise really induce lymphocyte apoptosis? Frontiers in Bioscience, 2, 478 –488. Navalta, J. W., McFarlin, B. K., Simpson, R., Fedor, E. A., Kell, H. B., Lyons, T. S., . . . Schafer, M. A. (2011). Finger-stick blood sampling methodology for the determination of exercise-induced lymphocyte apoptosis. Journal of Visualized Experiments, 48, e2595. doi:10.3791/2595 Navalta, J. W., Sedlock, D. A., & Park, K. S. (2005). Blood treatment influences the yield of apoptotic lymphocytes after maximal exercise. Medicine and Science in Sports and Exercise, 37, 369 – 373. doi:10.1249/01.MSS.0000155433.08698.C1 Nieman, D. C. (1997). Immune response to heavy exertion. Journal of Applied Physiology, 82, 1385–1394. Nieman, D. C., Henson, D. A., Austin, M. D., & Brown, V. A. (2005). Immune response to a 30-minute walk. Medicine and Science in Sports and Exercise, 37, 57– 62. doi:10.1249/01.MSS.0000149808.38194.21
Nieman, D. C., Kernodle, M. W., Henson, D. A., Sonnenfeld, G., & Morton, D. S. (2000). The acute response of the immune system to tennis drills in adolescent athletes. Research Quarterly for Exercise and Sport, 71, 403–408. Novas, A. M. P., Rowbottom, D. G., & Jenkins, D. G. (2003). Tennis, incidence of URTI and salivary IgA. International Journal of Sports Medicine, 24, 223–229. Patlar, S. (2010). Effects of acute and 4-week submaximal exercise on leukocyte and leukocyte subgroups. Isokinetics and Exercise Science, 18, 145–148. Pedersen, B. K., & Hoffman-Goetz, L. (2000). Exercise and the immune system: Regulation, integration, and adaptation. Physiological Reviews, 80, 1055–1081. Prestes, J., de Ferreira, C. K., Dias, R., Frollini, A. B., Donatto, F. F., CuryBoaventura, M. F., . . . Cavaglieri, C. R. (2008). Lymphocyte and cytokines after short periods of exercise. International Journal of Sports Medicine, 29, 1010–1014. doi:10.1055/s-2008-1038737 Simpson, R. J., Florida-James, G. D., Whyte, G. P., Black, J. R., Ross, J. A., & Guy, K. (2007). Apoptosis does not contribute to the blood lymphocytopenia observed after intensive and downhill treadmill running in humans. Research in Sports Medicine, 15, 157– 174. doi:10.1080/15438620701405339 Walsh, N. P., Gleeson, M., Shephard, R. J., Gleeson, M., Woods, J. A., Bishop, N. C., . . . Simon, P. (2011). Position statement. Part one: Immune function and exercise. Exercise Immunology Review, 17, 6 –63.
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Effects of a Simulated Tennis Match on Lymphocyte Subset Measurements a
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a
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Mark Schafer , Holly Kell , James Navalta , Ramires Tibana , Scott Lyons & Scott Arnett a
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Western Kentucky University
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Catholic University of Brasilia Published online: 21 Feb 2014.
To cite this article: Mark Schafer , Holly Kell , James Navalta , Ramires Tibana , Scott Lyons & Scott Arnett (2014) Effects of a Simulated Tennis Match on Lymphocyte Subset Measurements, Research Quarterly for Exercise and Sport, 85:1, 90-96, DOI: 10.1080/02701367.2013.872219 To link to this article: http://dx.doi.org/10.1080/02701367.2013.872219
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Research Quarterly for Exercise and Sport, 85, 90–96, 2014 Copyright q AAHPERD ISSN 0270-1367 print/ISSN 2168-3824 online DOI: 10.1080/02701367.2013.872219
Effects of a Simulated Tennis Match on Lymphocyte Subset Measurements Mark Schafer, Holly Kell, and James Navalta Western Kentucky University
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Ramires Tibana Catholic University of Brasilia
Scott Lyons and Scott Arnett Western Kentucky University
Tennis is an activity requiring both endurance and anaerobic components, which could have immunosuppressive effects postexercise. Purpose: The purpose of this investigation was to determine the effect of a simulated tennis match on apoptotic and migratory markers on lymphocyte subsets. Method: Male high school (n ¼ 5) and college (n ¼ 3) tennis players (Mage ¼ 18.9 ^ 3.3 years) completed 10 sets of a tennis protocol including serves, forehand strokes, and backhand groundstrokes with 1-min rest periods between sets. Apoptosis antigen 1 receptor (CD95) and chemokine receptor fractalkine (CX3CR1) expression was analyzed on helper T lymphocytes (CD4 þ ), cytotoxic T lymphocytes (CD8 þ ), and B lymphocytes (CD19 þ) twice, at resting baseline and immediately after all 10 sets of the tennis protocol. Results: An increase was observed in each lymphocyte subtype ( p , .02, effect size ¼ .41), and comparison of absolute changes revealed increases in CD4 þ /CD95 þ , CD8 þ /CD95 þ , and CD8 þ /CX3CR1 lymphocytes following the tennis protocol ( p , .01, effect size ¼ .43), but not in CD19 þ cells. Conclusions: A simulated tennis match has adequate intensity to induce immune modulations in terms of increased cell death and cellular migration in T lymphocyte subsets. Lymphocytopenia following tennis play is influenced by both apoptotic and migratory mechanisms. Keywords: apoptosis, cell migration, lymphocytopenia
Continuous endurance exercise has been shown to modulate the immune system (Kendall, Hoffman-Goetz, Houston, MacNeil, & Arumugam, 1990; Nieman, 1997; Patlar, 2010). As little as 30 min of walking has been reported to increase plasma interleukin-6 concentration and lymphocyte proliferative response (Nieman, Henson, Austin, & Brown, 2005), whereas 2 hr of cycling increases neutrophil degranulation and oxidative burst (Li & Cheng, 2007). Anaerobic and short periods of exercise have been typically
Submitted May 4, 2012; accepted May 30, 2013. James Navalta is now at the University of Nevada, Las Vegas. Correspondence should be addressed to Mark Schafer, Department of Kinesiology, Recreation, and Sport, Western Kentucky University, 1059 Smith Stadium, 1906 College Height Blvd., Bowling Green, KY 42101. Email: [email protected]
less investigated, but recent research has shown changes in lymphocyte viability following 5 min of moderate-intensity swimming (Prestes et al., 2008), while cytotoxic T cell volume was found to increase significantly with repetitive supramaximal anaerobic cycle tests (Friedman et al., 2012). As active tennis play is an activity that requires a significant endurance component and also consists of periodic anaerobic bursts, it is possible that this mode of exercise could have specific immunoregulatory effects. A limited amount of research exists with respect to the sport of tennis and associated modulations on the immune system. Novas, Rowbottom, and Jenkins (2003) found that increases in training load or elevated competition level were directly correlated with the incidence of upper-respiratory tract infections in elite female tennis players. In an investigation comparing a group of elite male and female
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tennis athletes to matched nonathletic controls, it was reported that neutrophil count was 16% lower and natural killer cells were 53% elevated in the elite athletes, and that all other immune parameters were not significantly different between groups (Henson et al., 2001). A 2-hr bout of tennis drills in elite adolescent tennis players produced significant increases in neutrophil and monocyte concentrations along with a decrease in salivary immunoglobulin A secretion rate (Nieman, Kernodle, Henson, Sonnenfeld, & Morton, 2000). It is of interest to note that this investigation also revealed a significant reduction in the overall lymphocyte count measured at 1 hr postexercise compared with the baseline measurement (Nieman et al., 2000). A depressed lymphocyte cell count following physical activity is termed exercise-induced lymphocytopenia and has been hypothesized to lead to a period of immunosusceptibility (Nieman, 1997; Walsh et al., 2011). Emerging research from our laboratory and from others has attributed the lymphocytopenia response in the postexercise period to either cell death (via apoptosis; Mars, Govender, Weston, Naicker, & Chuturgoon, 1998; Mooren, Bloming, Lechtermann, Lerch, & Volker, 2002; Navalta, McFarlin, & Lyons, 2010), cellular migration from the vascular compartment into the lymphoid pools (Friedman et al., 2012; Hong et al., 2005; Kruger, Lechtermann, Fobker, Volker, & Mooren, 2008; Simpson et al., 2007), or a combination of both (Navalta et al., 2013). Differential contributions from these mechanisms have varying consequences with regard to susceptibility in the period following exercise. Each mechanism has associated benefits and drawbacks; greater apoptosis could result in the permanent removal of potentially harmful cells at the cost of needing to fill the immune space with more viable cells, while greater migration could increase efficiency by maintaining access to lymphocyte subsets at the cost of retaining these cells, which have the potential for receptor cross-reactivity, which has been implicated as a mechanism underlying autoimmune disorders (Fujinami & Oldstone, 1989). Regardless of the etiology, a transient reduction of immune cells in the postexercise period can leave an athlete susceptible when presented with an immune challenge. Therefore, it is of interest to determine the lymphocytopenia response after various exercise modes, such as tennis, which requires both endurance and anaerobic components in a free-play model. We have recently shown that the apoptotic and migratory response of lymphocyte subfractions (i.e., helper T lymphocytes [CD4 þ ], cytotoxic T lymphocytes [CD8 þ ], and B lymphocytes [CD19 þ ]) respond differently in the postexercise period following an acute progressive treadmill bout to exhaustion (Navalta et al., 2013). To our knowledge, this response in lymphocyte subsets has not been characterized following an intermittent athletic activity such as tennis. In a previous investigation from our laboratory, we found that high-intensity intermittent cycling had no effect on lymphocyte apoptosis utilizing the early-
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phase apoptosis marker annexin V (Friedman et al., 2012). Therefore, in the current investigation we decided to utilize the apoptosis antigen 1 receptor (CD95) as an indicator of cell death (apoptosis), along with the chemokine receptor fractalkine (CX3CR1) as the indicator of lymphocyte extravasation (cell migration). We hypothesized that a tennis match would modulate significant increases in CD4 þ , CD8 þ , and CD19 þ lymphocytes with regard to cell concentration, the apoptotic marker CD95, and the migration marker CX3CR1. Therefore, the purpose of this investigation was to determine the effect of a simulated tennis match on the lymphocytopenia response in CD4 þ , CD8 þ , and CD19 þ lymphocytes.
METHODS Participants An a-priori power analysis was completed utilizing data for overall apoptotic lymphocytes from our previous work (Navalta, Sedlock, & Park, 2005). As a large effect size (.80) was expected, the sample size required to detect significant differences between means was determined to be six participants (a ¼ .05, b ¼ .80). In an attempt to utilize a conservative approach, eight male participants were recruited from area high schools and the university. Descriptive characteristics are presented in Table 1. In the current investigation, the participants included high school tennis players (n ¼ 5) and college tennis players (n ¼ 3). Participants had participated in regular scheduled tennis practice 4 to 6 days per week for 1 hr and 30 min to 2 hr and played competitively in the 3 months prior to participating in the investigation. At the time of recruitment and prior to the study protocol, each participant completed the Physical Activity Readiness Questionnaire and a medical history and signed an informed consent. The participants were apparently healthy tennis players with no musculoskeletal limitations or diagnosed cardiovascular or metabolic disease. The study was approved by the Western Kentucky University Human Subjects Review Board and all participants provided informed consent prior to participation in the investigation. For participants younger than the age of 18, parental consent was obtained prior to data collection.
TABLE 1 Participant Anthropometric Characteristics Variables Age (years) Height (cm) Weight (kg) % Body Fat
M
SD
18.9 178.1 66.7 10.9
3.3 6.8 8.1 3.5
Note. M ¼ mean; SD ¼ standard deviation.
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Protocol Initial measurements of age, height, weight, and skinfold analysis were recorded for each player upon reporting to the indoor tennis facility where the temperature ranged from 708F to 748F (218C to 238C), and percent humidity was less than 60%. Height and weight were measured using a DetectMedic Scale and attached stadiometer (Detecto Scales Inc., Webb City, MO). Skinfold measurements were taken at the chest, abdomen, and thigh using calibrated Lange skinfold calipers (Beta Technology, Santa Cruz, CA). Body composition was estimated using the Jackson and Pollock (1985) equations. Prior to the simulated tennis match, a 10-min to 15-min warm-up was performed including three laps around the two-court tennis facility and static and dynamic stretching according to the personal preference of each participant. In addition, 5 serves to the deuce court, 5 serves to the ad court, 10 crosscourt forehand ground strokes, and 10 crosscourt backhand ground strokes returned from a Play Mate Grand Slam oscillating ball machine (Metaltek, Morrisville, NC) were performed prior to beginning the simulated tennis match. Subsequent to the warm-up period, the participants began the simulated tennis match protocol with 5 serves to the deuce court and 5 serves to the ad court. Following the service trials, the participants hit 480 ground strokes fed at a constant speed, spin, and placement by the Play Mate Grand Slam ball machine. Participants hit 24 forehand strokes and 24 backhand strokes against the oscillating ball machine to complete one hitting session. The simulated tennis match protocol consisted of a total of 10 hitting sessions preceded by the service trials, with 1-min rest intervals between each hitting session. The participants completed the simulated tennis match protocol in approximately 90 min. Heart rate (HR) was measured using a using a wireless Polar Monitoring System (Woodbury, NJ), and the rating of perceived exertion (RPE) was measured using the Borg 6 to 20 RPE scale (Borg, 1998). HR and RPE were measured immediately upon completing each hitting session. Blood samples (120 mL) were obtained by finger stick using an antiseptic technique in the seated position at rest prior to the warm-up and immediately following the final
simulated tennis set. Processing for cell count and markers of apoptosis and cellular migration was carried out in duplicate as previously described (Navalta et al., 2011). Unless specified otherwise, antibodies were supplied by eBioscience (San Diego, CA). In brief, whole blood (10 mL) was added to a mixture containing Flow Cytometry Staining Buffer and antihuman antibodies specific for cell type (phycoerythrin: CD4, CD8, or CD19) and a marker for apoptosis (fluorescein isothiocyanate [FITC]: CD95) or migration (FITC: CX3CR1; BioLegend, San Diego, CA; see Table 2). Samples were incubated for 30 min in the dark at room temperature, then were centrifuged for 5 min and decanted. Cells were added to red blood cell lysis buffer and were allowed to incubate for 15 min. Following the incubation period, phosphate-buffered saline (SigmaAldrich, St. Louis, MO) was added to stop the lysis reaction, and the samples were centrifuged for 5 min, decanted, and vortexed thoroughly prior to analysis by flow cytometry (Accuri C6, Ann Arbor, MI). Statistical Analysis Data were analyzed using a paired-samples t test with the Statistical Package for the Social Sciences software program (IBM SPSS Statistics 18.0, Somers, NY). Absolute changes from rest (D baseline) values were calculated according to the following formula: ([measure – baseline] £ baseline – 1) £ 100. Assuming no change in absolute measures between changes in cell volume, apoptosis, and cell migration, we utilized the x2 test to determine differences between the expected and observed outcomes. For all statistical tests, significance was accepted at the p , .05 level.
RESULTS The simulated tennis match elicited an exercise intensity of 79 ^ 10% of the participants’ age-predicted maximum HR and an RPE of 14.3 ^ 1.8 over the 10 hitting sessions. The cardiovascular and perceptual responses for each of the 10 hitting sessions resulting from the simulated tennis match are presented in Table 3.
TABLE 2 Per-Tube Breakdown of Mixtures Containing Specific Antihuman Antibodies and Flow Cytometry Staining Buffer Tube 1 2 3 4 5 6
Buffer (250 mL)
FITC-Conjugated Antibody (0.25 mL)
PE-Conjugated Antibody (0.25 mL)
Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer Flow Cytometry Staining Buffer
CD95 CX3CR1 CD95 CX3CR1 CD95 CX3CR1
CD4 CD4 CD8 CD8 CD19 CD19
Note. FITC ¼ fluorescein isothiocyanate; PE ¼ phycoerythrin; CD95 ¼ apoptosis antigen 1 (APO-1); CX3CR1 ¼ fractalkine receptor; CD4 ¼ helper T lymphocytes; CD8 ¼ suppressor/cytotoxic T lymphocytes; CD19 ¼ B lymphocytes.
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Cell Counts
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A simulated tennis match induced significant increases in the concentration of each lymphocyte subset compared with resting values (see Figure 1; CD4 þ , p ¼ .013, effect size ¼ .45; CD8 þ , p ¼ .011, effect size ¼ .57; CD19 þ , p ¼ .015, effect size ¼ .41). While the relative change in the apoptotic marker CD95 was not significant for any lymphocyte subfraction (CD4 þ , p ¼ .082, effect size ¼ .73; CD8 þ , p ¼ .47, effect size ¼ .35; CD19 þ , p ¼ .435, effect size ¼ .41), the tennis protocol induced significant relative changes in the expression of the migration receptor CX3CR1 in CD4 þ ( p ¼ .042, effect size ¼ .31) and CD8 þ ( p ¼ .002, effect size ¼ .19; see Figure 2).
Apoptosis/Migration Markers When assessing the impact of the tennis protocol to influence the exercise-induced lymphocytopenia response, the absolute change from baseline was determined for cell count, a TABLE 3 Cardiovascular and Perceptual Responses for Each of the Hitting Sessions From the Simulated Tennis Match Variable Hitting Session 1 Heart rate (bpm) RPE Hitting Session 2 Heart rate (bpm) RPE Hitting Session 3 Heart rate (bpm) RPE Hitting Session 4 Heart rate (bpm) RPE Hitting Session 5 Heart rate (bpm) RPE Hitting Session 6 Heart rate (bpm) RPE Hitting Session 7 Heart rate (bpm) RPE Hitting Session 8 Heart rate (bpm) RPE Hitting Session 9 Heart rate (bpm) RPE Hitting Session 10 Heart rate (bpm) RPE
M
SD
159.7 12.5
21.8 2.2
170.7 13.2
25.4 1.9
166.4 13.4
25.4 1.8
165.4 14.0
23.7 1.8
162.5 14.8
28.7 1.5
162.4 14.5
28.9 1.5
163.5 14.7
33.5 1.0
159.1 15.0
32.4 1.0
162.2 15.2
30.9 1.6
165.8 15.5
27.2 1.9
Note. M ¼ mean; SD ¼ standard deviation; bpm ¼ beats per minute; RPE ¼ rating of perceived exertion.
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marker of apoptosis (CD95), and a marker for lymphocyte migration (CX3CR1) as we have described previously (Navalta et al., 2013). We found that CD4 þ T cells displayed a significantly greater increase in CD95 membrane expression ( p ¼ .01, effect size ¼ .43), which was not matched with changes in cell concentration or migration membrane expression (see Figure 3). In addition, for CD8 þ lymphocytes, the observed increase of both CD95 ( p ¼ .001, effect size ¼ .57) and CX3CR1 membrane expression was significantly increased above the absolute change in cell volume ( p ¼ .001, effect size ¼ .44; see Figure 3).
DISCUSSION This investigation was designed to determine the lymphocyte subset response to a simulated tennis match. Specifically, we wished to determine the apoptotic and migratory responses of CD4 þ , CD8 þ , and CD19 þ to a tennis protocol. It was hypothesized that an intermittent activity with a combination of endurance capacity and anaerobic bursts such as tennis would modulate discrete changes in individual lymphocyte subfractions. Based on the results of the present investigation, we reject the null hypothesis and observed a significant increase in each lymphocyte type with tennis play and an increase in the relative expression of the migration marker on both T cell subtypes. In addition, when compared with the absolute expected rise in cell volume above baseline values, CD4 þ T cells expressed the CD95 apoptotic marker to a greater extent, whereas CD8 þ T lymphocytes displayed an elevation in both apoptotic and migratory markers. To our knowledge, this is the first investigation to report the lymphocyte subset response following a simulated tennis match. Henson et al. (2001) compared resting T cell counts in a group of tennis athletes who were in the midst of an intense training period to the resting blood
FIGURE 1 Lymphocyte subset concentration measured in participants (N ¼ 8) at rest (Pre) and upon completion of a simulated tennis match (Post; mean ^ SE). Note. CD19 þ ¼ B lymphocytes, CD8 þ ¼ cytotoxic T lymphocytes, CD4 þ ¼ helper T lymphocytes. *Indicates statistically significant difference from Pre at the p # .02 level.
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samples of age-matched controls. They reported no differences in resting concentrations of total lymphocytes (tennis athletes ¼ 2.53 ^ 0.13 106 cells·mL – 1 , 6 –1 controls ¼ 2.73 ^ 0.13 10 cells·mL ), CD3 þ T cells (tennis athletes ¼ 1.35 ^ 0.11 10 6 cells·mL – 1 , 6 –1 controls ¼ 1.39 ^ 0.12 10 cells·mL ), or CD19 þ B cells (tennis athletes ¼ 0.42 ^ 0.04 106 cells·mL – 1, controls ¼ 0.42 ^ 0.05 106 cells·mL – 1; Henson et al., 2001). Nieman et al. (2000) reported that a 2-hr bout of tennis drills was insufficient to change the total lymphocyte concentration compared with measured baseline values (rest ¼ 2.53 ^ 0.13 106 cells·mL – 1, postexercise ¼ 2.44 ^ 0.15 106 cells·mL – 1). In the current study, a significant increase in each lymphocyte subset was observed following the simulated tennis protocol (see Figure 1). A possible explanation for the difference between our findings compared with those of Nieman et al. (2000) is the training status of the participants. In the current investigation, the participants were local high school and college-level players, and they did not achieve the volume of practice of the elite tennis players in the Nieman et al. (2000) investigation. Also, the amount of time that the tennis protocol employed in the current investigation utilized much shorter rest periods (1 min vs. 4 –5 min). It is possible that the training status and the intensity of tennis match play with minimal rest periods can significantly increase the lymphocyte concentration in the circulation. Previous research revealed an exercise-induced lymphocytopenia that was evident for up to 1 hr following a 2-hr bout of tennis drills (Nieman et al., 2000). Various authors have contributed this lymphocytopenia response to either apoptosis (Mars et al., 1998; Mooren, Lechtermann, & Volker, 2004; Navalta et al., 2005), cellular migration (Friedman et al., 2012; Hong et al., 2005; Simpson et al., 2007), or a combination of both (Navalta et al., 2010, 2013). As far as we are aware, this is the first study to report the potential influence of an intense tennis protocol on measures of lymphocyte apoptosis and migration in T cell subsets. We
FIGURE 2 Relative expression of the migration receptor CX3CR1 on lymphocyte subfractions obtained from participants at baseline (Pre) and immediately following a simulated tennis protocol. *Indicates statistically significant difference from Pre at the p # .05 level.
observed that tennis play, which incorporated both endurance and anaerobic burst activities, significantly elevated the absolute CD4 þ apoptotic response above the expected rise in cell count. This response is similar to what we reported for continuous treadmill running at 76% maximal oxygen consumption (VO2max), but not at the higher exercise intensities of 87% or 100% VO2max (Navalta et al., 2013). In addition, a relative increase in the percent of expressed migration receptor CX3CR1 was noted in CD4 þ cells following the tennis protocol. Regarding the lymphocytopenia response specific to CD4 þ cells, these results indicate that the postexercise decrease in cell volume is due to a combination of cellular death as well as extravasation into the lymphoid pools. Simpson et al. (2007) have presented compelling evidence that high-intensity exercise (in the form of level treadmill running at 80% of aerobic capacity, or downhill running on a treadmill at 2 10% grade) significantly increases cellular adhesion molecules (CD54 þ , or Intercellular Adhesion Molecule 1; CD18 þ , or b-integrin) on lymphocytes that serve to facilitate the movement of white blood cells out of the circulation. Future investigations are warranted to determine the direct contribution of each mechanism as well as the practical consequences of immunoregulation in this lymphocyte subfraction that is charged with regulating the cell-mediated immune response (Walsh et al., 2011). In the present investigation, an intense tennis protocol successfully elevated an absolute change in CD8 þ lymphocytes with regard to both apoptotic as well as migratory marker expression. This response is different compared with any intensity of continuous treadmill running reported previously, which reported a significant apoptotic increase at 76% VO2max that was replaced by a much greater rise in migration at 87% VO2max (Navalta et al., 2013). It is likely that the difference is due to the apoptotic marker utilized (CD95 in the present investigation vs. annexin V previously) and the nature of the protocol (intermittent
FIGURE 3 Absolute change from baseline in cell count, apoptosis (CD95 þ ), and lymphocyte migration (CX3CR1 þ ) in CD4 þ and CD8 þ lymphocyte subsets from participants (N ¼ 8) who completed a simulated tennis protocol. *Indicates statistically significant increase compared with the expected change in cell volume at the p # .01 level.
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tennis vs. continuous treadmill running). It has previously been reported that CD8þ cells are influenced by exercise to a greater extent than the CD4þ subset, presumably due to the interaction of epinephrine with b2-adrenergic receptors (Ibfelt, Petersen, Bruunsgaard, Sandmand, & Pedersen, 2002; Pedersen & Hoffman-Goetz, 2000). The current investigation provides evidence that this phenomenon extends to tennis play, as greater influences were noted in both the absolute and relative CX3CR1 response, as well as the absolute apoptotic response of CD8þ lymphocytes. The current investigation could have been strengthened by the inclusion of a resting control group. In addition, another limitation is that our simulated tennis match protocol has restricted application to actual tennis play, where athletes have no prior knowledge of where an opposing player will hit a particular shot. From this standpoint, the level of intensity in the present investigation may have been lower than what is found during actual tennis competition. Finally, with regards to characterizing the lymphocytopenia response following tennis play, extending the blood-sampling time points for as long as 3 hr postexercise would be appropriate, as would the determination of plasma or serum measures that could explain the cell death/migration mechanisms such as catecholamines, cytokines, and/or caspases. In summary, we found that an intermittent tennis protocol designed to mimic actual match play had significant effects on the lymphocyte subset response. An increase in each lymphocyte subfraction (CD4 þ , CD8 þ , and CD19 þ ) was observed postexercise. In addition, exercise significantly increased the expression of both apoptotic and migratory markers on T cell subsets, helping to explain the decrease in overall lymphocytes reported 1 hr following tennis drills (Nieman et al., 2000). As it is usual for tennis athletes to compete in multiple matches during a short timeframe in tournament play, future investigations should be directed toward the practical implications of this noted immunomodulated period. Additionally, the influence of cell death and migration should be further investigated, and the consequences of each mechanism should be characterized. Although speculative, it is possible that cells removed from the circulation due to apoptosis could take longer to replace and increase the transient period of susceptibility, whereas cells that have migrated out of the circulation could be more readily recruited back and available in the case of an immune challenge. Thus, it may be possible to reduce potential negative impacts with modulations in nutritional intake or training program design leading up to a tournament.
WHAT DOES THIS ARTICLE ADD? The findings of the present investigation indicate for the first time that tennis play can have adequate intensity to
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induce immune modulations in terms of increased cell death and cellular migration in both T lymphocyte subsets. Transient decreases in CD4 þ could decrease an athlete’s ability to dictate the immune response to specific antigens, whereas a depressed cytotoxic volume would reduce cellto-cell killing capability in the period following a match. From a clinical standpoint, increased lymphocytopenia in the postexercise period could transiently open tennis athletes to susceptibility to respiratory infections. Future investigations should track the responses that we report here utilizing actual tennis play, particularly in terms of the number of groundstrokes hit during a rally and the time interval between rallies and sets. In theory, this knowledge would help to direct coaches and athletes to utilize timeouts or pace-of-play tactics to minimize the immunomodulations we report here, particularly in the framework of a multiple match tournament that extends over several days. REFERENCES Borg, G. (1998). Borg’s perceived exertion and pain scales. Champaign, IL: Human Kinetics. Friedman, R. A., Navalta, J. W., Fedor, E. A., Kell, H. B., Lyons, T. S., Arnett, S. W., & Schafer, M. A. (2012). Repeated high-intensity wingate cycle bouts influence markers of lymphocyte migration but not apoptosis. Applied Physiology, Nutrition, and Metabolism, 37, 241 – 246. doi:10.1139/h11-156. Fujinami, R., & Oldstone, M. (1989). Molecular mimicry as a mechanism for virus-induced autoimmunity. Immunologic Research, 8, 3 –15. Henson, D. A., Nieman, D. C., Kernodle, M. W., Sonnenfeld, G., Morton, D., & Thompson, M. M. (2001). Immune function in adolescent tennis athletes and controls. Sports Medicine, Training and Rehabilitation, 10, 235–246. Hong, S., Johnson, T. A., Farag, N. H., Guy, H. J., Matthews, S. C., Ziegler, M. G., & Mills, P. J. (2005). Attenuation of T-lymphocyte demargination and adhesion molecule expression in response to moderate exercise in physically fit individuals. Journal of Applied Physiology, 98, 1057– 1063. doi:10.1152/Japplphysiol.00233.2004 Ibfelt, T., Petersen, E. W., Bruunsgaard, H., Sandmand, M., & Pedersen, B. K. (2002). Exercise-induced change in type 1 cytokine-producing CD8 þ T cells is related to a decrease in memory T cells. Journal of Applied Physiology, 93, 645–648. doi:10.1152/japplphysiol.01214.2001 Jackson, A. S., & Pollock, M. L. (1985). Practical assessment of body composition. Physician and Sportsmedicine, 13(3), 76–90. Kendall, A., Hoffman-Goetz, L., Houston, M., MacNeil, B., & Arumugam, Y. (1990). Exercise and blood lymphocyte subset responses: Intensity, duration, and subject fitness effects. Journal of Applied Physiology, 69, 251–260. Kruger, K., Lechtermann, A., Fobker, M., Volker, K., & Mooren, F. C. (2008). Exercise-induced redistribution of T lymphocytes is regulated by adrenergic mechanisms. Brain, Behavior, and Immunity, 22, 324– 338. doi:10.1016/j.bbi.2007.08.008 Li, T. L., & Cheng, P. Y. (2007). Alterations of immunoendocrine responses during the recovery period after acute prolonged cycling. European Journal of Applied Physiology, 101, 539 –546. doi:10.1007/ s00421-007-0529-1 Mars, M., Govender, S., Weston, A., Naicker, V., & Chuturgoon, A. (1998). High intensity exercise: A cause of lymphocyte apoptosis? Biochemical and Biophysical Research Communications, 249, 366–370. doi:10.1006/ bbrc.1998.9156
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