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1School of Human Movement Studies, Queensland University of Technology, Kelvin Grove 4057; and 2Cellular Immunology Laboratory, Queensland Institute of Medical Research, Herston 4006, Australia
Submitted 20 February 2003 ; accepted in final form 7 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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fluorescent-cell tracking; mitogen-induced proliferation; long-duration exercise; immune function; glucose; cortisol
Although the ergogenic effect of CHO feeding during exercise has been acknowledged for some time, recent investigation has examined the potential role for CHO in maintaining immune function during and after intense exercise. Several studies (13, 24, 28) have identified a beneficial effect of CHO supplementation on immune function during and after exercise. Henson et al. (13) found that CHO ingestion, compared with placebo (Pl), prevented a decline in phytohemagglutinin (PHA)-induced T-lymphocyte proliferation after 2.5 h of treadmill running. There remains, however, a need to clearly identify the mechanism by which CHO may help maintain immune function during acute exercise.
One potential explanation for the role of CHO supplementation in attenuating the reduction in T-lymphocyte function after acute intensive exercise may be the stress hormone cortisol. There is a reported reduction in T-lymphocyte function (13, 25, 33) and an elevation in cortisol (7, 19, 43) after long-duration intense exercise, and the immunosuppressive effects of cortisol are widely recognized (9, 34). Several studies (13, 29) have found a relationship between postexercise cortisol concentration and lymphocyte function, whereas other studies (22) have not. Furthermore, CHO ingestion has been reported to attenuate the usual increase in cortisol concentration with long-duration intense exercise (13, 22).
The potential mechanism by which cortisol influences T-lymphocyte function after exercise is unknown. Cortisol may directly alter lymphocyte function via interaction with receptors on the surface of T lymphocytes (1), or it may indirectly alter proliferative function by inducing changes in composition of the cellular pool (16, 38). Although CHO ingestion has been shown to reduce cortisol release into the bloodstream and diminish changes to immune function (28), the mechanism behind CHO-induced changes in immune cell function and a causal relationship with cortisol has not been clearly demonstrated. Furthermore, understanding of the connection between exercise, cortisol, and T-lymphocyte function has been hindered by the use of techniques to assess lymphocyte responses to mitogen that do not provide an accurate account of cell division on a per cell basis.
We have previously shown that using carboxyfluorescein succinimidyl ester (CFSE) to quantify cell division can provide unique insights into cell function during and after exercise (11). In addition, we have shown that acute intensive exercise is associated with increased death of both CD4 and CD8 cells in culture in response to PHA (11). There is a strong association between glucocorticoids and T-lymphocyte apoptosis (36, 37) and the potential for CHO ingestion to reduce the cortisol response to exercise. This study investigated the effect of CHO ingestion during long-duration intense exercise on the function of the T-lymphocyte subsets CD4 and CD8 cells by using the CFSE division method (15). We hypothesized that CHO ingestion rather than Pl would result in an attenuation of the cortisol response to exercise and a reduction of the exercise-associated alterations in cell death.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Maximal incremental exercise test. Subjects completed a continuous
incremental cycling test on a computerized cycle ergometer (Lode BV Excalibur,
Groningen, The Netherlands) to volitional exhaustion. The incremental protocol
started at 0 W and increased by 2 W every 5 s until subjects could not
maintain cadence above 80 rpm. Breath-by-breath gas analysis was conducted on
expired pulmonary gases throughout the test (Medical Graphics CPX/D mobile
cart system, Medical Graphics, St. Paul, MN). Maximal oxygen consumption was
defined as the single highest 5-s average value attained during the test
coinciding with a respiratory exchange ratio of >1.10. Ventilatory
threshold was determined from a graph of the ventilation
(
E)-to-oxygen consumption
(O2) ratio plotted
against time. Threshold was defined as the inflection point of the
E/O2
curve and was identified independently by two researchers
(44).
Exercise trials. Subjects completed a 3-day food diary over the 3
days before both exercise trials that was analyzed to determine the amount of
habitual CHO consumption. Subjects reported to the laboratory at 6:30 AM,
having refrained from exercise for the previous 24 h and having only consumed
water since midnight. They were fitted with an indwelling venous cannula,
through which blood samples were drawn. After a 15-min rest, a blood sample
[6:45 AM (T1)] was taken. Subjects completed a standardized warm-up consisting
of 5 min of low-resistance cycling followed by stretching. At 7:00 AM,
subjects began 150 min of cycling at a constant power output adjusted to
elicit a O2 equivalent to
85% of ventilatory threshold. Subjects were encouraged to maintain a cycling
cadence between 80 and 100 rpm. Heart rate was monitored continuously
throughout both trials. After 60 min of exercise, subjects continued cycling
while a blood sample was taken [8:00 AM (T2)]. Subsequent blood samples were
taken immediately after exercise [9:30 AM (T3)] and after 60 min of recovery
[10:30 AM (T4)]. During the recovery period, subjects were seated quietly in
the laboratory.
CHO and Pl drinks. During the two exercise trials, each subject consumed an equal volume of fluid containing either CHO or Pl. The volume of the pre- and postexercise drinks was kept constant for all subjects. All subjects drank 0.75 liter after the first blood sample but before starting exercise and 0.5 liter during the recovery hour. During the exercise trials, subjects drank 0.28 ± 0.05 liter every 15 min. The volume consumed by each subject during exercise was adjusted individually to ensure all subjects were fed 3.2 g CHO/kg body wt in total over the testing period.
The CHO supplement was Polycose (Ross Products Division Abbott Laboratories, Columbus, OH), which consisted of glucose polymers derived from controlled hydrolysis of cornstarch (17 kJ/g). An appropriate amount of the product was dissolved in water to yield a 6% CHO solution. Pl was an artificial sweetener (Hermesetas Gold, 2:1 Aspartame-Acesulfame-K, Hermes Sweeteners, Zurich, Germany). A solution was prepared containing three tablets per liter of water (no CHO, 0.8 kJ/tablet).
Blood analyses. Whole blood (7 ml) was collected for determination of a full blood count. Full blood counts [white blood cells (WBCs), neutrophils, lymphocytes, and monocytes] were obtained by standard procedures from a clinical hematology laboratory using a Sysmex Se 9000 (Roche Diagnostics, Sydney, Australia). Serum was obtained from a further 8 ml of whole blood for measurement of cortisol and glucose. Cortisol was measured by using a commercial radioimmunoassay kit (Coat-A-Count, Bio-Mediq DPC, Victoria, Australia). Glucose was measured by using a commercial kit (Glucose Hexokinase, TR15003, Trace Scientific, Noble Park, Australia).
Lymphocyte separation. Whole blood was collected into sodium heparin tubes (8-ml vacutainer, Becton Dickinson, Lane Cove, Australia), diluted with an equal volume of phosphate-buffered saline (PBS), then layered over Ficoll-Paque (Sigma Chemical, St. Louis, MO) density gradient separation solution, and centrifuged at 300 g for 20 min at room temperature. The mononuclear cell layer (PBMC) was removed and washed twice in RPMI 1640 medium (Sigma Chemical) supplemented with 2 mM glutamine (Sigma Chemical) and gentamycin (Sigma Chemical). Cell viability and cell counts were assessed by Trypan blue exclusion; cells were then labeled with CFSE.
CFSE labeling of lymphocytes and culture. CFSE was stored frozen in a 1 mM stock solution until ready for use. A pellet of 1 x 107 cells was resuspended in 0.5 ml of PBS; 0.5 ml of CFSE labeling solution (10 µM) was added to yield a final CFSE concentration of 5 µM. After 1 min, samples were diluted with PBS and centrifuged at 400 g for 10 min before being resuspended in culture medium (RPMI-1640; 2 mM glutamine; 10% fetal calf serum) to yield a final cell concentration of 8 x 108 cells/l. Two hundred microliters of cell suspension were cultured with either 5 µg/ml PHA (stimulated) or RPMI (unstimulated). PHA titration experiments were conducted to determine the concentration that yielded maximum T-cell expansion. This optimal concentration (5 µg/ml) was used in all subsequent assays. Cultures were incubated at 37°C in a 5% CO2 incubator (Contherm Scientific Lower Hutt) for 72 h in Falcon tubes (Becton Dickinson).
Flow cytometry. After culture, cell suspensions were labeled with CD3 (allophycocyanin), CD8 (phycoerythrin) and Viaprobe (7-aminoactinomycin D) for 15 min in the dark at room temperature. Samples were then washed with 3 ml of PBS and resuspended in 200 µl of paraformaldehyde solution (2%) and placed in Trucount tubes (Becton Dickinson). Surface marker and CFSE analysis was conducted by using a FACScalibur flow cytometer (Becton Dickinson). The same forward- and side-scatter parameters were used for each trial as established for human peripheral leucocytes. Standard gating procedures were used to select mainly lymphocytes and to differentiate between labeled and unlabeled cells. Viable lymphocytes were identified by using Viaprobe staining, and forward- and side-scatter parameters. Fluorescent staining was used to further characterize T lymphocytes (CD3) and lymphocyte subsets CD3+CD8+ and CD3+CD8- (CD3+CD4+). Data were analyzed by using Flow Jo version 3.3 software (Treestar, San Carlos, CA).
Assessment of lymphocyte expansion. The absolute number of cells in each sample was calculated by determining the ratio of gated lymphocytes or lymphoblasts to gated Trucount beads, multiplied by the known number of beads in the Trucount tube. The increase in the number of viable cells in culture was calculated from the absolute number of cells in the PHA-stimulated sample, relative to the absolute number in the unstimulated sample. This was termed lymphocyte expansion and was completed separately on CD3+CD4+ and CD3+CD8+ lymphocyte populations. An expansion value of 1.0 or more indicated that there were a greater number of cells in the PHA-stimulated sample than in the unstimulated sample after 72 h of culture.
Estimation of lymphocyte mitosis and cell death. As CFSE fluorescence intensity halves with mitotic division, each generation of proliferated lymphocytes (G1,G2, etc.) appears as a distinct peak on a CFSE histogram plot (Fig. 1). With the use of separate histograms for CD3+CD8+ and CD3+CD4+ lymphocyte populations, and standard histogram gating procedures, the number of cells in each generation was determined for each cell type.
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The decrease in parent (G0) cells from unstimulated to PHA-stimulated samples can be attributed to cells undergoing mitosis or cell death. If it is assumed that no cell death occurs, all missing G0 cells must be assumed to have undergone mitosis and would be expected to generate two G1 cells. The same assumption can be made for each generation. There was considerable overestimation of cell numbers in the final generation if the assumption of no cell death was used. Iterative processes were then used to change the assumed cell death rate until all missing cells could be accounted for by mitosis or cell death. The mitosis rate for each generation was then calculated as the percentage of cells estimated to have undergone mitosis. The cell death rate was the percentage of cells estimated to have undergone cell death (apoptosis).
Statistical analysis. All data are expressed as means ± SE throughout and in all graphical presentations. In the case of glucose, cortisol, expansion, and cell death rates, a two-way ANOVA design, with repeated measures on both factors, was conducted. Main effects of time (T1-T4 sample points) and supplement (CHO and Pl) were fitted, as well as the interaction between time and supplement. For mitosis rates, the factors of time and supplement were considered in each generation (G0-G3) separately. To aid interpretation of significant interactions, a one-way ANOVA with main effect of time was carried out for CHO and Pl data separately. Where significant main effects or interactions were observed, Scheffé's post hoc analysis was carried out. Pearson's product-moment correlation analysis was carried out between cortisol, glucose, cell numbers, and cell death rates for each variable individual time points as well as averaged data. Statistical significance level was set at P < 0.05; Cohen's statistical power was calculated to be >90%.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Exercise trials. During the incremental maximal test, the mean
O2 peak value was 56
± 2 ml · kg-1 ·
min-1 and mean maximum heart rate was 181 ± 8
beats/min. Both exercise trials were completed at the power output identified
in the incremental maximal test to be 85% of individual anaerobic threshold
(228 ± 25 W), equivalent to 67 ± 2% of maximal
O2. There was no
difference between the mean heart rate during the CHO and Pl trials (145
± 5 beats/min). One subject failed to complete the 2.5 h ride in the Pl
trial because of hypoglycemia and stopped after 2 h of cycling; all data
points for this subject were included in the analysis. The total volume of
fluid consumed during each trial was 4.0 ± 0.46 liters and was
identical between trials for each subject. The total amount of CHO consumed
during the CHO trial was 240 ± 28 g.
Glucose and cortisol. There was a significant time x supplement interaction for both glucose and cortisol. Glucose concentration was significantly higher during recovery (T4) in the CHO trial compared with the Pl trial (Fig. 2). Cortisol concentration was significantly lower immediately postexercise (T3) and in recovery (T4) in the CHO trial compared with the Pl trial (Fig. 3).
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Circulating leukocyte numbers. There were significant time x supplement interactions for the number of circulating WBC and polymorphonuclear neutrophils. Immediately after exercise (T3), the numbers of WBC and polymorphonuclear neutrophils were significantly lower in the CHO trial compared with the Pl trial (Table 1). The numbers of WBC, polymorphonuclear neutrophils, and monocytes were all significantly lower during recovery (T4) in the CHO compared with the Pl trial. Although there was no time x supplement interaction for the number of lymphocytes or monocytes, significant main effects of time were observed in both the CHO and Pl trials. Compared with preexercise (T1), the number of lymphocytes in circulation was significantly higher during exercise (T2) and significantly lower during recovery (T4) in both trials. Similarly, there was no significant time x supplement interaction for the number of circulating CD3, CD4, or CD8 cells (Table 1). There was a significant main effect of time in both the CHO and Pl trials. The numbers of CD3, CD4, and CD8 cells were significantly higher during exercise (T2), and the numbers of CD3 and CD4 cells were significantly lower during recovery (T4) than the preexercise sample (T1) (time main effects not indicated in table).
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Lymphocyte expansion. There was a significant time x supplement interaction for CD4 lymphocyte expansion. For CD8 cells, the interaction approached significance (P = 0.057). There was a main effect of time in Pl only; compared with preexercise (T1), lymphocyte expansion of both CD4 and CD8 cells was significantly lower during exercise (T2). For CD4 cells, lymphocyte expansion was also significantly reduced immediately postexercise (T3) in Pl. Although the CHO trial followed the same pattern of change in cellular expansion, there were no significant changes from baseline in either cell type. In Pl, cellular expansion decreased between T1 and T2 from 1.32 ± 0.17 to 0.94 ± 0.25 for CD4 cells and from 1.97 ± 0.38 to 1.29 ± 0.29 for CD8 cells. At all time points, expansion of the CD8 cell population was larger than that of CD4 cells (Fig. 4).
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Lymphocyte mitosis. There was no significant interaction between time and supplement for lymphocyte mitosis in G0-G2 in either CD4 or CD8 cells, such that the percentage of CD4 or CD8 cells undergoing mitosis in each generation was not significantly different between CHO and Pl trials. The mitosis rate of CD4 cells in G0 was significantly decreased during exercise in both CHO and Pl trials. However, in all other generations for both cell types, there was no effect of time or condition. At all time points, the mitosis rates of CD8 cells were significantly higher compared with CD4 cells, and mitosis rates of each successive cell generation declined significantly (Fig. 5).
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Lymphocyte death. There was a significant time x supplement interaction for cell death rates of CD4 cells only. There was a significant main effect of time in the Pl trial only for both CD4 and CD8 cells; compared with preexercise (T1), the percentage of apoptotic CD4 and CD8 cells in cell culture was significantly increased during and immediately after exercise (T2 and T3). For CD8 cells, there was also a main effect of supplement because cell death rates were higher in Pl compared with CHO trials. In CHO trials, there was no significant change from baseline in the amount of cell death in cultures during exercise or recovery for either cell type. Cell death increased in Pl trials between T1 and T2 from 12 ± 6 to 28 ± 8% in CD4 cells and from 4 ± 6 to 24 ± 7% for CD8 cells. At all time points, CD4 cells exhibited higher cell death rates than CD8 cells (Fig. 6).
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Correlation analysis. In Pl trials, the number of WBCs at T3 (immediately postexercise) correlated significantly with the cortisol concentration at T2 (1 h of exercise) (r = 0.93). There were no significant correlations between the numbers of WBCs, lymphocytes, glucose, cortisol, cell death, and CD3, CD4, or CD8 cells at any time point in either condition.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This study confirmed previous reports that CHO compared with Pl ingestion is associated with higher blood glucose concentrations, reduced cortisol concentration, less perturbation of circulating leukocytes numbers, and an attenuation of exercise-induced decrease in T-cell proliferative function (13). The present study also supports our previous findings that the exercise-associated impairment of cellular expansion is due to an increase in cell death rather than to a decrease in mitosis (11). Furthermore, these data extend the understanding of how CHO ingestion acts to maintain T-lymphocyte function to suggest that CHO ingestion during exercise acts to prevent cell death (Fig. 6). CHO ingestion during exercise (compared with Pl) prevented any reduction in the expansion of the cell population by avoiding any exercise-associated increase in the death of cells in culture (Fig. 4).
We have previously proposed two possible explanations as to why cell death rates are increased with exercise (11). In this study, we have further explored these possibilities to examine the mechanism by which CHO ingestion is capable of preventing exercise-induced cell death. High cell death rates occurred during culture of cells taken during and immediately after exercise; these time points coincide with the greatest number of circulating lymphocytes. Previous studies have identified that CHO ingestion is connected with significant reductions in the extent of leukocytosis during exercise (30, 31). CHO ingestion acts to moderate fluctuations in lymphocyte numbers, in particular, CHO compared with Pl is linked to a reduction in the concentrations of lymphocytes immediately postexercise and an increase after 3 h of recovery (13). Because exercise-induced lymphocytosis is largely due to an influx of immature cells and cells from marginized pools, the composition of the lymphocyte pool is considerably different during and immediately after exercise. Increased cell death rates at these time points may potentially have been explained by an increased susceptibility to apoptosis of the new cells in the circulation. However, our present data do not support this speculation. CHO ingestion significantly reduced cell death rates, particularly at the 1-h exercise time point (T2), despite no difference in lymphocyte numbers between Pl and CHO trials. We also found no significant correlation between the numbers of cells in circulation and apoptosis rates.
It is widely recognized that glucocorticoids are capable of inducing apoptosis of lymphocytes (14, 39); therefore, an alternative explanation for the exercise-associated increase in cell death may be due to alterations in immunomodulators such as cortisol. It has previously been shown that, during and after exercise, the time course of suppression of lymphocyte proliferation is inversely related to that of plasma cortisol (33). One proposed mechanism by which CHO ingestion during exercise reduces changes in immune cell numbers and function is through blunting the cortisol response to exercise (28). However, in the present study, we found no relationship between cortisol concentrations and changes in T-lymphocyte numbers or function. We observed the highest rates of cell death in samples taken during exercise, an effect that was significantly blunted by CHO ingestion, despite no effect of CHO ingestion on serum cortisol concentration during exercise. CHO supplementation only manifested a reduction in cortisol concentration postexercise and during recovery (Fig. 3). The disparity between the time points of attenuating effects of CHO would indicate that CHO ingestion acted through some mechanism other than cortisol to reduce cell death associated with exercise.
Because data from the present study do not directly implicate cortisol in the mediation of lymphocyte death after exercise, the reduction in apoptosis with CHO ingestion may be related to other factors, such as glucose availability or adrenaline concentration. Glucose is well established as an essential nutrient for optimal immune cell function during exercise (2), and CHO ingestion has been shown to improve its availability (3, 4, 10, 42). Similarly, epinephrine has been shown to be elevated during exercise and to have immunosuppressive effects (5, 6, 40). There are also reports that epinephrine concentrations are suppressed by CHO ingestion during exercise (21, 23). Although we did not measure epinephrine in this study, it is worthy of investigation in future studies of the mechanism behind exercise changes in apoptosis of T lymphocytes.
This study confirms our previous finding that reductions in the mitogen response of T lymphocytes after exercise are related to an increase in cell death of both CD4 and CD8 cells in culture (11). We have also identified that CHO ingestion during exercise acts to reduce the impairment of T-lymphocyte function by decreasing death in cell cultures. In addition, our results indicate that mechanisms independent of changes in cell numbers and cortisol may be responsible for exercise-induced apoptosis. We suggest that other immunomodulatory factors, possibly the direct role of glucose availability or epinephrine, may provide a more consistent hypothesis regarding exercise-associated changes of in vitro immune cell function. Further research is also needed to establish the connection between in vitro cell death and susceptibility to infection in athlete groups.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and placebo (Pl; 
) conditions. *Significant
difference between Pl and CHO (P < 0.05).
) and Pl (
) conditions. *Significant difference between Pl and
CHO (P < 0.05).
