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00005768-200605000-0001000005768_2006_38_864_timmons_carbohydrate_5article< 121_0_22_10 >Medicine & Science in Sports & Exercise©2006The American College of Sports MedicineVolume 38(5)May 2006pp 864-874Puberty Effects on NK Cell Responses to Exercise and Carbohydrate Intake in Boys[BASIC SCIENCES: Original Investigations]TIMMONS, BRIAN W.1; TARNOPOLSKY, MARK A.2; SNIDER, DENIS P.3; BAR-OR, ODED11Children's Exercise and Nutrition Centre; 2Departments of Pediatrics and Medicine; and 3Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, CANADAAddress for correspondence: Mark A. Tarnopolsky, M.D., Ph.D., FRCP(C), 4U4 Department of Neurology, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5; E-mail: tarnopol@mcmaster.caSubmitted for publication July 2005.Accepted for publication November 2005.ABSTRACTPrevious research has demonstrated that younger versus older animals and humans experience smaller perturbations in natural killer (NK) cells in response to physiological stress.Purpose: To determine whether the smaller perturbations in NK cells induced by strenuous exercise and carbohydrate (CHO) intake, previously reported in children, are influenced by puberty.Methods: Twenty 12-yr-old boys, distinguished as prepubertal (Tanner (T) 1, N = 7), early pubertal (T2, N = 7), or pubertal (T3-5, N = 6), cycled for 60 min at 70% V̇O2max while drinking 6% CHO (CT) or flavored water (WT). Blood was collected at rest and during (30 and 60 min) and following (30 and 60 min) exercise to identify NK cells as CD3−CD56dim or CD3−CD56bright. CD69 expression on CD3−CD56+ cells was also determined.Results: A puberty × CHO × exercise interaction was found for the proportion, but not number, of CD56dim cells (P = 0.06). CD56dim cell counts were lower in CT versus WT (P < 0.001). Responses of CD56bright proportions (P = 0.007) and counts (P = 0.03) depended on pubertal status, but not CHO. The CD56bright:CD56dim ratio remained stable during exercise, but during recovery was higher in T1 and T3-5 versus T2 (P = 0.08) and in CT versus WT (P = 0.04). During recovery, CD3−CD56+ cells expressed higher levels of CD69 (P = 0.01), with no change in the proportion of CD69+ cells.Conclusion: These results confirm the influence of puberty on the distribution of NK cell subsets in response to exercise and CHO intake. Increased CD69 expression suggests that NK cells increase activation status during recovery from physiological stress.Natural killer (NK) cells are large granular lymphocytes with natural cytotoxicity (7) and play important innate roles in antiviral (2) and anticancer (6) defenses. The traditional phenotype of NK cells, based on cell surface markers, is the lack of expression of the T cell receptor polymeric complex, CD3 and the coexpression of the Fcγ receptor III (CD16) and an isoform of the human neural cell adhesion molecule (CD56) (7). As early as 1986, however, the existence of two unique and functionally distinct NK cell populations, based on the expression intensity of CD56, was noted (15). CD3−CD56dim cells, which express high levels of CD16, are more cytotoxic than CD3−CD56bright cells, which express low or no levels of CD16 (15). Mounting evidence suggests that the CD56bright subset, which comprises approximately 10% of NK cells, may be of particular relevance in the early events of immune challenge by coordinating "cross-talk" between innate and adaptive arms of immunity (9). Little is known, however, regarding the responses of CD56dim and CD56bright cells to various forms of physiological stress such as high-intensity exercise.In adults (26) and in children (32), a substantial increase in NK cells (i.e., CD3−CD16+CD56+) in the peripheral blood can be observed in response to aerobic exercise. Although NK cells present in the blood represent a very small proportion of the body's total NK cell pool at rest (35), the striking exercise-induced increase in the peripheral pool is thought to translate into enhanced immune surveillance (23). The majority of exercise studies, however, have not distinguished between CD56dim and CD56bright responses. Although it was reported (12) that exercise mobilized NK cells with greater intensity of CD16/CD56 expression, this study could not distinguish between the expression of CD16 and CD56 antigens. In another study (11), CD56bright cell counts determined 10 min following a 250-km cycling road race were actually lower than preexercise levels. To our knowledge, there are no studies that have distinguished CD56dim and CD56bright subset responses to exercise in children. Given the alterations that occur in the CD56bright:CD56dim ratio with aging (5) and some disease conditions (13) and the unique immunoregulatory properties of these subsets (7), it is important to clarify the potential effects of acute exercise on these NK cell populations.While few studies have investigated the influence of puberty per se on NK cells under resting conditions (1), the interaction of stress hormones and NK cells may be quite different during and following exercise in children. The growth hormone (GH) response to exercise, for example, increases with advanced pubertal development (36), and children demonstrate fewer GH receptors on lymphocytes as compared with adults (33). Moreover, there is some evidence that the catecholamine response to exercise is also lower in children as compared with adults (25). This latter observation is particularly important because epinephrine (EPI) is considered one of the main mediators of exercise-induced mobilization of NK cells (23,26). Indeed, NK cells (i.e., CD3−CD16+CD56+) are the most responsive cell type to exercise in children, but the magnitude of the response to strenuous exercise is lower in pre- and early-pubertal boys as compared with men (32). In response to anaerobic exercise, boys at more advanced stages of puberty also experience the largest NK cell response (4), and in prepubertal as compared with mature rats, it has been demonstrated that NK cells are less responsive to physiological stress (22). Together, the above observations suggest that puberty may be an important determinant of the NK cell response to exercise. The primary objective of this study, therefore, was to clarify the influence of puberty on NK cell responses (i.e., CD56dim and CD56bright) to exercise, by recruiting healthy boys at the same chronological age, but different biological ages (i.e., pubertal status), in an attempt to isolate possible effects of puberty. We hypothesized that the NK cell response to exercise would be greater with progressing physical maturity and that exercise would be an effective means to assess how the CD56dim and CD56bright subsets respond to physiological stress.Another aim of this study was to investigate the effect of carbohydrate (CHO) intake on NK cell responses to exercise, according to pubertal status. CHO, as compared with water, attenuates the exercise-induced rise in peripheral blood NK cells in adults (21). A similar effect can also be observed in young children, but it was found that NK cells were more sensitive to CHO intake in the children (32), suggesting that maturity status may also influence the interaction effects of CHO and exercise on NK cells. While it has been proposed that CHO-mediated effects on NK cell redistribution in adults are due to a blunted stress hormone (e.g., EPI, GH, and cortisol) response mediated by maintained or increased blood glucose concentrations (21), this explanation may not be adequate for the pediatric population, given their already smaller stress hormone response to exercise. In addition, we are aware of no study reporting the responses of the different CD56+ subsets to the combination of exercise and CHO intake in adults, and no study has investigated these factors in children, in whom the interaction of exercise and CHO intake may be different than in adults. Whether a nutritional blunting of NK cells in response to exercise is of significant health relevance is as yet unclear, but further clarification of puberty effects on NK cell responses to exercise and CHO intake seems warranted. Based on our previous findings that NK cell responses to exercise were more sensitive to CHO intake in boys, as compared with men (32), we hypothesized that CHO effects on NK cells would be more pronounced in prepubertal boys. To better understand potential mechanisms involved in NK cell recruitment with and without CHO intake, we measured a number of stress hormones implicated as key regulators of NK cell mobilization during exercise (23). We hypothesized that exercise would elevate hormone concentrations, but that CHO intake would attenuate this increase.METHODSSubjects.Twenty 12-yr-old boys volunteered for this study approved by the McMaster University research ethics review board. Pubertal status of the boys was self-assessed based on pubic hair development according to Tanner (30) and used to allocate the boys into three separate groups classified as prepubertal (Tanner stage 1, N = 7), early pubertal (Tanner stage 2, N = 7), or pubertal (Tanner stages 3-5, N = 6). The number of subjects in each group was based on a sample size calculation performed on data from our previous publication (32), yielding a statistical power > 80%. Serum testosterone levels (see below) were also determined, but only as a supplemental indication of pubertal status. Table 1 provides the subjects' characteristics. All subjects were healthy, recreationally active, and not taking medication. After the purpose, procedures, and risks of the study were explained, the boys agreed verbally to participate and their parent then signed a written informed consent.TABLE 1. Subject characteristics.Preliminary session.An initial visit was conducted at least 5 d prior to the experimental trials in order to measure body height (SECA 216 Accu-Hite Stadiometer, Creative Health Products, Plymouth, I), body mass (BM; BWB-800, Tanita, Tokyo, Japan), percent body fat (bioelectric impedance-101A, RJL Systems, Clinton Twp., MI) and to determine Tanner staging. Maximal O2 uptake (V̇O2max) was determined on a cycle ergometer (Ergomedic 818E, Monark, Sweden) using a progressive, continuous exercise test. Subjects began cycling at either 30 or 60 W, with pedaling rate constant at 60 rpm, and work rate was increased by 30 W every 2 min. A test was determined maximal when pedaling rate dropped below 50 rpm for 3 s despite strong encouragement, and the respiratory exchange ratio was greater than 1.1. Heart rate (HR) was continuously monitored during the test with a Polar HR monitor (Polar A1, Polar Electro, Kempele, Finland) and subjects breathed through a Hans Rudolph valve with an appropriately sized mouthpiece. Expired air was collected continuously and analyzed for O2 (Beckman O2 analyzer OM-11, Beckman Inc., CA) and carbon dioxide (HP47210A capnometer, Hewlett Packard, CA) with analyzers connected to a Vista PC interface with Turbofit software (VacuMed, Ventura, CA) on a personal computer. The highest 30-s V̇O2 was taken as the V̇O2max.Experimental sessions.All subjects recorded their nutrient intake and physical activity for 2 d prior to their first experimental session. Recorded food intake and physical activity were then repeated the 2 d prior to their next session. In addition, all subjects complied to avoid fast-food-type meals and strenuous physical activity during these days. To avoid effects of circadian rhythm on immune measures, subjects arrived to the laboratory at either 0730 or 0830 h on the day of testing in an approximately 10-h fasted state. They voided their bladder and a nude weight was taken to calculate fluid intake for the session. Subjects then rested supine for approximately 10 min, after which time an indwelling venous catheter (Becton Dickinson, NJ) was placed in either an arm or a hand. After a further 10 min of supine rest, a resting, preexercise blood sample was drawn. Subjects then consumed a small breakfast with their first drink (12 mL·kg−1 BM) to standardize preexercise nutrition. Volumes of 4 mL·kg−1 BM were subsequently consumed at 15-min intervals throughout exercise and at 20 min into recovery. This CHO feeding schedule has been shown to maintain body hydration, increase blood glucose levels, and influence NK cell responses to exercise in children (32). Thus, in one trial (CT) subjects consumed a 6% CHO-electrolyte solution (4% sucrose, 2% glucose, ~18 mmol·L−1 Na+, ~3 mmol·L−1 K+), and in another trial (WT), water (identical in flavor, sweetness, and electrolyte concentration, but without CHO) for a total of 40 mL·kg−1 BM. Forty minutes after the resting blood sample, subjects began cycling at a power output equivalent to 70% of their predetermined V̇O2max, with the target intensity achieved in the first 5 min by analysis of expired gas. Exercise consisted of two 30-min bouts separated by a 5- to 7-min rest period. Additional expired gas samples were collected at steady state from minutes 11-15 and 26-30 of each exercise bout to ensure the proper work intensity, with the power output adjusted accordingly. Additional blood samples were collected after 30 and 60 min of exercise and at 30 and 60 min of recovery. Blood samples were drawn while subjects remained seated on the cycle ergometer or quietly in the laboratory. The catheter was kept patent by flushing with approximately 1.5 mL of sterile saline (0.9% NaCl) after each blood sampling. Consequently, the first 2 mL of blood at each sampling time was discarded. During the recovery period, subjects sat quietly and were allowed to empty their bladder if necessary. The two experimental trials were conducted 1-2 wk apart in a double-blind and counterbalanced fashion.Glucose analysis.Whole blood treated with EDTA was centrifuged at 2000 × g for 10 min, and the plasma was stored at −50°C until analyzed. Plasma glucose was measured enzymatically (2300 L STAT, Yellow Springs Instruments, OH), and concentrations were corrected for exercise-induced changes in plasma volume (see below). The intra- and interassay CV for this assay were < 1.5%.Cortisol, growth hormone and testosterone analyses.Whole blood sampled at rest and at 60 min of exercise was allowed to clot and centrifuged at 2000 × g for 10 min. The serum was stored at −70°C until analyzed in duplicate for cortisol and growth hormone (GH) using commercially available RIA kits (Cat. No. TKCO1 and KGHD1, respectively, Diagnostic Products Corporation, CA). In our hands, the intra- and interassay CV, respectively, are ≤ 2.5 and 8% for cortisol and ≤ 5 and 12% for GH, and postexercise concentrations were corrected for exercise-induced changes in plasma volume (see below). Only resting serum samples from both experimental sessions were assayed in duplicate for total testosterone using a commercially available RIA kit (Cat. No. TKTT1, Diagnostic Products Corporation, CA). In our hands, the intra- and interassay CV, respectively, for this assay are ≤ 2 and 7%. Testosterone values presented in Table 1 represent the average value from both sessions.Catecholamine analysis.Whole blood collected at rest and at 60 min of exercise was treated with EGTA and reduced glutathione and centrifuged at 2000 × g for 10 min, and the plasma was stored at −70°C until analyzed for epinephrine (EPI) and norepinephrine (NEPI). Plasma catecholamines were analyzed by high-performance liquid chromatography with electrochemical detection as previously described (16). Recovery rates of catecholamines from plasma ranged between 80-85% and the intraclass correlation of this procedure is 0.96, representing very high reliability. All postexercise concentrations were corrected for exercise-induced changes in plasma volume (see below).Lymphocytes and NK cell subsets.Total lymphocyte counts were determined in whole blood treated with EDTA using an automated Coulter counter. Hemoglobin and hematocrit were also assessed in these samples to calculate changes in blood and plasma volume as described (32), with all immune cell counts corrected for exercise-induced changes in blood volume. EDTA-treated whole blood was used to determine NK cell subsets by two- and three-color immunophenotyping. Monoclonal antibodies directly conjugated with PerCP (CD3) or PE (CD56) were mixed (10 μL each) with 100 μL of whole blood. An additional 100 μL of blood were mixed with 10 μL each of CD3 (PerCP), CD56 (FITC), and CD69 (PE). CD69 was used as a marker of activation status of circulating NK cells. All blood samples were stained within 6 h of collection using standardized procedures, and all reagents were purchased from BD Biosciences. Mixed samples were briefly vortexed and incubated for 20 min at room temperature (RT) in the dark. After adding 2 mL of BD Pharm Lyse™ solution to lyse red blood cells, samples were vortexed and incubated a further 10 min at RT. Samples were centrifuged (300 × g for 5 min at RT), washed with 2 mL of BD Pharmingen™ stain buffer, centrifuged (200 × g for 5 min at RT), and fixed with 0.5 mL of BD Cytofix™ buffer. Samples were stored at 3°C for no more than 48 h before being run on a FACScan flow cytometer (Becton Dickinson, Mississauga, Canada) with CELLQuest software. A total of 10,000 events were collected in the lymphocyte gate based on forward (FSC)- versus side-scatter (SSC) characteristics and saved as flow cytometry standard files. Figure 1 outlines the gating procedures for the proportions of NK cell subsets, analyzed "offline" with WinMDI 2.8 software (Joseph Trotter, The Scripps Research Institute, CA). First, the lymphocyte population was gated using FSC versus SSC characteristics and a dot plot of CD3 and CD56 fluorescence was created from events within the lymphocyte gate. The expression of CD69 was determined in a similar fashion by creating a dot plot of CD3 and CD56 created from the lymphocyte gate. A histogram of CD69 fluorescence was then created from the gated CD3−CD56+ cell population. For each preexercise sample, one tube containing a cocktail of CD3 (PerCP), CD56 (FITC), and IgG1 (PE) served as an isotype control and was used to set the marker for CD69+ cells. The placement of this marker was held constant in the analysis of subsequent samples to detect shifts of events within the marked population over time (e.g., with exercise). Because the proportion of NK cells expressing CD69 was relatively low, no attempt was made to distinguish CD69 expression on CD56dim and CD56bright cells. Cell counts of each NK cell subset were calculated by multiplying the percentage of cells with appropriate fluorescence with the absolute lymphocyte count. Blood for CD69 analysis was collected at rest, after 60 min of exercise, and after 60 min of recovery only. The proportion of CD3−CD56+ cells expressing CD69 and the median fluorescence intensity (MFI) of CD69+ cells were determined. To facilitate intersubject comparisons, postexercise and recovery CD69 MFI were expressed as a percentage of the preexercise value.FIGURE 1-Flow cytometric analysis of CD56+ natural killer cells and expression of CD69 in whole blood. A) Gated lymphocyte population (R1) based on forward- vs side-scatter characteristics. B) CD3−CD56bright (R2) and CD3−CD56dim (R3) cell populations derived from events within the lymphocyte gate. C) Histogram of CD69 expression on CD3−CD56+ cells; the dark line represents isotype control.Statistical analyses.Data are presented as means ± SEM, unless stated otherwise. Group differences in physical and fitness characteristics were analyzed by one-way ANOVA. Dependent variables (glucose, hormones, immune cell proportions, and counts) were submitted to a group × trial × time mixed-factorial ANOVA. Where appropriate, a Tukey's post hoc test was used to determine significance among means. Pearson correlations were performed to determine associations between hormone concentrations and NK cell data. STATISTICA 5.0 (StatSoft, Tulsa, OK) was used for ANOVA, and GraphPad Prism 3.0 (GraphPad Software, San Diego, CA) was used for correlation analyses. The NK cell data were also assessed for a normal distribution, which was confirmed using STATISTICA. The threshold for statistical significance was set at P ≤ 0.05.RESULTSThere were no differences in exercise intensity, as a percentage of V̇O2max, between groups or trials (P ≥ 0.79); the average exercise intensity was 68.5 ± 0.8% of V̇O2max or 31.4 ± 0.4 mL·kg−1 BM·min−1.Plasma glucose.Glucose concentrations at rest were similar between groups and trials. Postexercise glucose was higher in CT versus WT in all groups (trial × time, P < 0.001) and remained higher at 30 min of recovery (Fig. 2).FIGURE 2-Plasma glucose concentrations before, during, and after exercise in water and carbohydrate trials in boys of different pubertal status. Values are means ± SEM. The shaded box represents exercise disregarding 5-min rest. T1, Tanner stage 1; T2, Tanner stage 2; T3-5, Tanner stages 3-5; CT, carbohydrate trial (closed symbols); WT, water trial (open symbols). § Significant difference between CT and WT, P < 0.05.Catecholamines, cortisol, and GH.EPI increased with exercise (time effect, P < 0.001), with no influence of pubertal group or trial. There was a group × trial × time interaction (P = 0.03) for NEPI. NEPI increased with exercise in T1 and T2 in both CT and WT, whereas T3-5 increased NEPI levels only in CT. Cortisol decreased over time (time effect, P < 0.001) with no influence of pubertal group or trial. There were group × time (P = 0.02) and trial × time interactions (P < 0.001) for GH. T3-5 and T1 boys increased GH levels with exercise, but T2 boys did not, and postexercise GH levels were lower in CT versus W(Table 2).TABLE 2. Stress hormone concentrations before, during, and after exercise in carbohydrate and water trials in boys of different pubertal stages.Lymphocytes.Circulating lymphocyte counts increased with exercise for all groups in both trials (time effect, P < 0.001). The increase in WT, however, tended to be greater than in CT (trial × time interaction, P = 0.08). In both trials, lymphocyte counts remained below resting levels at 30 and 60 min of recovery (Table 3).TABLE 3. Total lymphocyte counts before, during, and after exercise in carbohydrate and water trials in boys of different pubertal stages.NK cell populations.Consistent with the literature, the CD56dim subset, pooled across pubertal groups and trials, comprised 89 ± 1% of total CD3−CD56+ cells under resting conditions. However, the CD56dim subset comprised a greater proportion of total CD3−CD56+ cells in boys at T2 (93 ± 1%), compared with boys at T1 (86 ± 2%) and T3-5 (87 ± 2%) (group effect, P = 0.01).A strong trend for a three-way interaction was found for CD56dim cells as a proportion of lymphocytes (group × trial × time interaction, P = 0.06, Fig. 3). In WT, cell proportions at 30 min of exercise were significantly higher than at rest in T1 and T3-5, but not in T2. In CT, only T3-5 demonstrated a significant increase in cell proportion at 30 min of exercise versus rest. By 60 min of exercise in WT, cell proportions in all pubertal groups were higher than resting values. However, the actual proportion at this time point was significantly higher in T3-5 versus T1 and T2, with no difference between the latter two groups. In contrast, cell proportions at 60 min of exercise were not significantly different from resting values in CT in all pubertal groups. Differences in cell proportions between WT and CT at the same time point were evident in T1 by 30 min of exercise, whereas in T3-5 intertrial differences were only evident by 60 min of exercise, and not at all in T2. In both trials, cell proportions had returned to resting levels by 60 min in T1, whereas in T2 and T3-5 values at 30 and 60 min of recovery remained below resting levels in CT.FIGURE 3-CD3−CD56dim cell proportions (A,B,C) and CD3−CD56dim cell counts (D,E,F) before, during, and after exercise in water and carbohydrate trials in prepubertal (A,D), early-pubertal (B,E), and pubertal (C,F) boys. Values are mean ± SEM. Shaded boxes represent exercise disregarding 5-min rest. CT, carbohydrate trial; WT, water trial. For cell proportions, § significant difference between CT and WT, P < 0.05; ∥ significantly different from −40 min within WT, P < 0.05; ¶ significantly different from −40 min within CT, P < 0.05; ** significantly different than T3-5 in WT, P < 0.05. For cell counts, CHO × trial interaction, P < 0.001: CT < WT at 30 and 60 min exercise; +30 and +60 min values < rest in CT.Although there was no three-way interaction for CD56dim cell counts (group × trial × time interaction, P = 0.23), a significant effect of CHO intake was found (trial × time interaction, P < 0.001, Fig. 3). Values at 30 and 60 min of exercise but not at 30 or 60 min of recovery were lower in CT versus WT. In WT, values at 30 and 60 min of recovery were not different than rest, whereas in CT the 30- and 60-min recovery values were below resting levels.CHO intake did not influence exercise-induced changes in CD56bright cells, but there was a significant group × time interaction for both their proportion and number (P = 0.007 and P = 0.03, respectively, Fig. 4). With respect to cell proportions, values for both T1 and T2 did not change over time, whereas T3-5 had higher values at 60 min of exercise versus rest. At all time points, T3-5 had higher values than either T1 or T2. With respect to cell counts, values for T2 did not change over time. Only at 60 min of exercise were values significantly higher than at rest in T1, whereas T3-5 demonstrated higher values at 30 and 60 min of exercise versus rest. In all pubertal groups, recovery of CD56bright cell counts was complete by 30 min.FIGURE 4-CD3−CD56bright cell proportions (A) and counts (B) before, during and after exercise in boys of different pubertal status. Values are mean ± SEM. Shaded boxes represent exercise disregarding 5-min rest. T1, Tanner stage 1; T2, Tanner stage 2; T3-5, Tanner stages 3-5. †† T3-5 significantly different from T1 and T2, P < 0.05. ‡‡ T3-5 significantly different from T2, P < 0.05. §§ Significantly different from −40 min in T3-5, P < 0.05. ∥∥ Significantly different from −40 min in T1 and T3-5, P < 0.05.To highlight the influence of exercise on the relationship between the CD56bright and CD56dim subsets, the ratio of CD56bright to CD56dim cells was calculated (Fig. 5). This ratio remained stable during exercise in all groups, but tended to increase during recovery more in T1 and T3-5 than in T2 (group × time interaction, P = 0.08). This ratio was also higher during recovery in CT versus WT (trial × time interaction, P = 0.04).FIGURE 5-Ratio of CD3−CD56bright cells to CD3−CD56dim cells before, during, and after exercise in boys of different pubertal status (A) and in carbohydrate and water trials (B). Values are mean ± SEM. Shaded boxes represent exercise disregarding 5-min rest. T1, Tanner stage 1; T2, Tanner stage 2; T3-5, Tanner stages 3-5; CT, carbohydrate trial; WT, water trial. § Significant difference between CT and WT, P < 0.05. ∥ Significantly different from −40 min in WT, P < 0.05. ¶ Significantly different from −40 min in CT, P < 0.05. ∥∥ Significantly different from −40 min in T1 and T3-5, P < 0.05. ¶¶ T1 and T3-5 significantly different from T2, P < 0.05.CD69+ cells.CD69 levels were undetectable in two of the 20 subjects (both T2) and data for the remaining 18 boys are reported (Fig. 6). There was no effect of group, exercise, or CHO intake on the proportion of CD3−CD56+ cells expressing CD69. However, the number of circulating CD3−CD56+CD69+ cells did increase with exercise and returned to resting levels by 60 min of recovery (time effect, P < 0.001), with no effect of pubertal group or CHO intake. The MFI of CD69+ cells, as a percentage of preexercise values, was not influenced by pubertal group or CHO intake, but was greater at 60 min of recovery compared with after 60 min of exercise and rest (time effect, P = 0.01). The absolute MFI of CD69+ cells at rest after 60 min of exercise and after 60 min of recovery were 16.3 ± 0.8, 16.9 ± 0.8, and 19.6 ± 1.0 fluorescence intensity units, respectively (time effect, P < 0.001) (Fig. 6).FIGURE 6-Expression of CD69 on circulating CD3−CD56+ cells: Proportion of CD3−CD56+ cells expressing CD69 (A); CD3−CD56+CD69+ cell counts (B); and median fluorescence intensity (MFI) of CD69 on CD3−CD56+ cells as a percentage of preexercise values (C). Values are mean ± SEM. Pre, 40 min before exercise; post, after 60 min of exercise; recovery, 60 min after exercise. ‡ Significantly different from pre, P < 0.05. *** Significantly different from post, P < 0.05.Correlations.When data from all subjects were considered, only GH concentrations at 60 min of exercise in both trials correlated with the proportion of CD56dim (r = 0.49, P = 0.002) and CD56bright cells (r = 0.31, P = 0.055) at the same time point (Table 4).TABLE 4. Pearson correlation coefficients between postexercise stress-hormone concentrations and postexercise proportions of CD56dim and CD56bright cells in carbohydrate and water trials.DISCUSSIONThis study investigated the influence of puberty on NK cell responses to high-intensity exercise and CHO intake in healthy boys. The novelty of this work is the distinction between responses of CD56dim and CD56bright NK cell subsets in boys at the same chronological age, but varying biological ages (i.e., pubertal status). The main findings are that: 1) boys at more advanced stages of physical maturity demonstrated larger increases in the proportion of CD56dim and CD56bright cells; 2) the CD56bright:CD56dim ratio during recovery was lowest in early-pubertal boys and highest with CHO intake; 3) CHO intake attenuated exercise-induced increases in the CD56dim subset, but not in the CD56bright subset; and 4) circulating CD3−CD56+ cells during recovery, compared with rest, expressed higher levels of the activation marker, CD69.Studies in adults (26) and in children (32) have reported on NK cell responses to acute aerobic exercise. NK cells are rapidly mobilized into the peripheral circulation most likely by a catecholamine-induced downregulation of adhesion molecule expression (20). The significance of these transient changes is thought to reflect an enhanced state of readiness against potentially harmful pathogen (23). Therefore, a blunted peripheral infiltration of NK cells, particularly of the more cytotoxic (i.e., CD56dim) subset, might reflect a reduced ability to defend against pathogen. In this study, boys at the most advanced stages of puberty demonstrated the greatest increase in the proportion of CD56dim during WT in the absence of puberty-related differences in EPI responses and, in fact, with lower NEPI responses compared with the other pubertal groups. Moreover, T3-5 boys also had the greatest increases in the proportion of CD56bright cells. This maturity-related effect is consistent with our previous findings in 9- and 10-yr-old boys who exhibited a smaller increase in CD3−CD16+CD56+ cell proportion, compared with men (32). Boas et al. (4) also found that the increase in CD3−CD16+CD56+ cell counts following 30 s of "all-out" cycling was smallest in prepubertal and largest in postpubertal boys. Given the direct relationship between chronological age and lymphocyte β-adrenergic receptor density (24), smaller NK cell responses in pre- and early-pubertal individuals could be due to a lower density of β-adrenergic receptors on NK cells. However, boys in the current study were the same chronological age, suggesting that expression and/or activity of β-adrenergic receptors may also vary with pubertal status. Indeed, NK cells of prepubescent rats, compared with mature animals, are resistant to β-adrenergic stimulation (22), but to our knowledge similar findings in humans are lacking. Based on these observations, therefore, one might conclude that less mature organisms are less capable of generating an enhanced state of readiness in response to an acute physiological stressor. Alternatively, it could be argued that smaller increases in peripheral blood NK cells are beneficial because more cells remain in marginated pools and tissue (e.g., spleen) where the likelihood of contact with pathogen is greatest. Unfortunately, our study cannot establish which of these interpretations is closer to the true state of the organism.In contrast to their relative proportion, the absolute number of CD56dim cells did not vary statistically by pubertal group due to a slightly greater increase in total lymphocyte counts in pre- and early-pubertal boys versus pubertal boys. Thus, smaller perturbations in the redistribution of CD56dim cells in pre- and early-pubertal boys were offset by a slightly more pronounced overall lymphocytosis. Considering that the CD56dim subset is the more cytotoxic NK cell subset (15), an overall greater lymphocytosis might serve to maintain cytotoxic activity in the face of physiological stress. CD56bright cell counts, however, did retain a graded pubertal effect. These findings highlight the need to report both cell proportions and cell counts in response to exercise and suggest that mobilization of the CD56dim and CD56bright subsets may be differentially regulated, at least in children. Further evaluation of subset-specific expression of adhesion molecules may shed some light on this issue. In this regard, it is noteworthy that the CD56bright subset expresses higher levels of the adhesion molecule L-selection (10), as compared with the CD56dim subset, suggesting that the former cell population may be particularly capable of recruitment to sites of infection or tissue damage.A well-described phenomenon in the exercise immunology literature is a period of relative immune suppression following high-intensity exercise, consistently associated with suppression of NK cell function and termed the "open window": a period of time when the host may be at increased susceptibility to infection (23). A recent study (29) tracked changes in CD56dim and CD56bright cells over 1 month of competitive sports training in healthy adult females and found that the time during training with the lowest NK cell cytotoxicity corresponded to the time when CD56bright cell counts were highest and CD56dim cells remained unchanged (i.e., when CD56bright:CD56dim ratio was highest). In the current study, the CD56bright:CD56dim ratio remained unchanged during exercise, but was highest during recovery due to unchanged CD56bright levels and a decrease in CD56dim levels. Thus, the current and previous findings support the hypothesis that reduced NK cell function during recovery from high-intensity acute exercise (23) and periods of intensified training (29) may be due to disproportionate changes in NK cell subsets. A high proportion of CD56bright cells, which have low unstimulated cytotoxicity (7), may effectively "dampen" overall in vitro killing capacity. Our findings are, however, limited because we did not measure NK cell function per se, although we did measure an activation marker (CD69) closely associated with NK cell cytotoxicity (14) and found this to be elevated during recovery (see below). We suggest that future exercise studies including assays of NK cell cytotoxicity should account for the distribution of CD56dim and CD56bright cells.In adults, CHO intake has consistently resulted in attenuation of exercise-induced increases in NK cell counts and activity (21) and maintenance of stimulated NK cell cytotoxicity (17), with little impact on recovery of NK cell counts following exercise (21,17). CHO intake also attenuates the rise in the proportion and number of NK cells during exercise in 9- and 10-yr-old boys, with no influence during recovery (32), but the mechanisms for this effect may be different between children and adults. In this study, CHO intake attenuated the increase in the relative proportion of CD56dim cells earlier in the prepubertal boys, compared with more physically mature boys, in the absence of higher plasma glucose concentrations. Our findings also present dissociations between changes in cell populations and stress hormones with and without CHO intake (Table 4). Taken together, the data question the proposed roles of blood glucose levels per se and changes in stress hormones as mediators of the redistribution of NK cells observed with exercise and CHO intake, at least in children. Furthermore, the CD56bright subset was resistant to the effects of CHO intake, suggesting that factors mediating CHO effects on NK cell responses to exercise are specific to the CD56dim subset. Thus, previous studies reporting CHO effects on traditional CD3−CD16+CD56+ NK cells may have been confounded by not distinguishing between CD56+ subsets. While CHO intake might have had a larger effect if the exercise duration was 90 min or longer, we believe that our previous and current findings highlight a maturity-related sensitivity to the effects of CHO on NK cells, and it will be interesting to further clarify the mechanisms by which age and/or puberty might influence this interaction. One possible mechanism may be related to GH. CHO intake blunted the GH response to exercise and postexercise GH concentrations in both trials were positively correlated with the proportions of CD56dim and CD56bright cells at the same time point. Interestingly, GH induces T cell migration in vitro, possibly due to altered affinity of adhesion molecules (31), and children express lower levels of GH receptors on CD2+ lymphocytes (i.e., T and NK cells) than do adults (33). Whether GH receptor expression and/or activity on NK cells vary with puberty per se is unknown. Based on our findings, it is possible that GH may be an important mediator of the NK cell response to exercise and CHO in growing children. However, the overall health significance of CHO effects on NK cell responses to exercise is unclear. By attenuating the number of NK cells entering the peripheral pool, we might be leaving more of these cells in tissue where they can serve their normal function (as suggested earlier). On the other hand, we could be attenuating the number of NK cells trying to arrive at some destination in order to proceed with their effector or regulatory functions and effectively dampening the immune surveillance afforded by these cells. To further clarify the health significance of these changes, it would be interesting, for example, to describe the relationship between CHO-induced attenuation of NK cells during exercise and the susceptibility to infection over subsequent days or weeks.An interesting observation in this study was that high glucose concentrations during recovery in CT were associated with a concomitant suppression in CD56dim cell counts, with no effect on CD56bright counts. High glucose concentrations promote leukocyte adhesion to endothelium through upregulation of adhesion proteins (19), and CD56dim cells are more rapidly adherent than CD56bright cells under certain experimental conditions (34). In this regard, the higher than normal glucose levels during recovery in our boys may have led to a selective margination of CD56dim cells to the endothelium and out of the peripheral circulation. Further investigation of such a phenomenon in healthy children may provide a useful model to study the interaction between exercise, hyperglycemia, and atherosclerotic processes.Another novel finding in this study was the increased CD69 expression on circulating CD3−CD56+ cells during recovery from exercise. CD69 is a sensitive marker of lymphoid activation (28) and correlates with cytotoxicity of NK cells (14). Previous work in adults reported that exercise with and without CHO intake increased the number of CD69+ NK cells (17), but the current study is the first, to our knowledge, that reports the same effect in children. Although the CD3−CD56+CD69+ cell count returned to preexercise levels by 60 min of recovery, and there were no changes in the proportion of CD3−CD56+ cells positive for CD69, circulating CD3−CD56+ cells at this time expressed higher levels of CD69 than at rest or postexercise. Bishop et al. (3) recently demonstrated that caffeine supplementation in humans was associated with an increased percentage of T cells expressing CD69 and an enhanced EPI response to exercise, suggesting that increases in EPI during the preceding exercise could have activated cellular pathways leading to induction of CD69 expression. However, these authors did not measure NK cells, and it has been shown that EPI actually reduces CD69 expression on these cells (27). Therefore, some other unknown factors are likely to mediate the effect of exercise on NK cell expression of CD69. Although the increase in CD69 expression was a consistent finding in 15 of 18 subjects, the biological significance of an average 22% increase in CD69 on NK cells is unknown. Given its costimulatory role in cytotoxicity (18), an increase in CD69 expression on NK cells may "prime" these cells for effector function during the recovery period.In summary, the results of this study highlight maturity-related differences in the NK cell response to high-intensity exercise and the differential response of CD56dim and CD56bright NK cells to exercise and CHO intake in healthy boys. Physical maturity also mediated the effects of CHO intake on the redistribution of NK cell subsets during and following exercise. Finally, recovery from high-intensity exercise was associated with a modest increase in the expression of CD69 on CD3−CD56+ cells. Although the biological significance of these findings requires further investigation, NK cells are an important first line of defense against tumor growth, and the unique immunoregulatory properties of the CD56dim and CD56bright subsets mark them as candidates for immunotherapy of cancer (7). Whether the redistribution of CD56dim and CD56bright cells in response to exercise could be of therapeutic benefit in children recovering from cancer (8), for example, remains to be determined.We thank Melanie De Jonge, Marta Kubacki, Raymond Trott, Jae-Hunn Lee, Mazen Hamadeh, and Amy Mark for assistance with this experiment. We also thank Dr. Craig Horswill for valuable comments on an earlier version of this manuscript. The extraordinary effort and time of our subjects are gratefully acknowledged.Acquisition of flow cytometry data was performed in the McMaster Flow Cytometry Facility, supported by the Canadian Institutes of Health Research.The financial support of the Gatorade Sports Science Institute and BD Biosciences is gratefully acknowledged.REFERENCES1. Bartlett, J. A., A. R. Goldklang, S. J. Schleifer, and S. E. Keller. Immune function in healthy inner-city children. Clin. Diagn. Lab. Immunol. 8:740-746, 2001. [CrossRef] [Medline Link] [Context Link]2. Biron, C. A., K. B. 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Carbohydrate Intake in BoysTIMMONS, BRIAN W.; TARNOPOLSKY, MARK A.; SNIDER, DENIS P.; BAR-OR, ODEDBASIC SCIENCES: Original Investigations538