Immune responses and increased training of the elite athlete TONY J. VERDE, SCOTT G. THOMAS, ROBERT W. MOORE, PANG SHEK, AND ROY J. SHEPHARD Department of Community Health, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S lA1, Canada VERDE,TONY J.,Scon, G. THOMAS,ROBERT W. MOORE, duced by heavy training would disappear after returning PANG SHEK, AND ROY J. SHEPHARD.Immune responses and to a normal regimen. increased training of the elite athlete. J. Appl. Physiol. 73(4): Studies that have investigated the effects of exercise 1494-1499, 1992.-Ten elite male runners (age, 29.8 t 1.7 yr; on immune function have employed a variety of immunomaximum oxygen consumption, 65.3 t 4.9 ml. kg-’ min-‘; lological assays (for review see Ref. 10). The measurekm times, 31 min 43 s & 1 min 46 s) deliberately increased ments of immune function chosen for this study were 1) training schedules by an average of 38% for 3 wk. Resting heart determination of the subpopulations of lymphocytes, 2) rate and maximal oxygen intake were unchanged, but the heart proliferation of lymphocytes in response to a mitogenic rate response to acute exercise was decreased. Following heavy stimulant, and 3) syntheses of immunoglobulins (Igs) foltraining, blood samples taken at rest showed trends to a delowing mitogenic stimulation. These immunological ascreased helper/suppressor cell ratio, an increased phytohemagglutinin (PHA) - and concanavalin (ConA)-stimulated lymsays are specifit measures of lymphocyte function, which phocyte proliferation, and a decreased production of immunois believed to be the integral-link between exercise and globulins IgG and IgM. Whereas PHA-stimulated lymphocyte immunity (17). Assessment of lymphocyte proliferation proliferation was initially unchanged by acute exercise, after 3 is a commonly used diagnostic test to reflect global cellwk of heavy training the same acute exercise caused an 18% mediated immunity, whereas mitogen-induced Ig synthesuppression of proliferation. Acute exercise following heavy sis is a diagnostic immunological laboratory test used to training did not alter pokeweed-stimulated IgG or IgM syntheassesshumoral immunity (3). sis. There was no correlation between changes in lymphocyte l
subpopulations, helper/suppressor ratios, and mitogen-induced cellular proliferation. The immune system of endurancetrained athletes at rest seemed to tolerate the stress of heavy training, but superimposition of a bout of acute exercise on the chronic stress of heavy training resulted in immunosuppression, which was transient and most likely not of clinical significance.
METHODS Subjects and Experimental Plan
The volunteers were 10 male distance runners aged 29.8 t 1.7 yr, who were recruited and tested in accordance with a protocol approved by the University Committee on Human Experimentation. To avoid difficulties acute exercise; heavy training; lymphocyte subpopulations; from age-related differences of immune function (2O), lymphocyte proliferation; immunoglobulin synthesis the age span was deliberately limited to 5 yr. All subjects were elite runners based on the following two criteria: 1) they had achieved a minimal performance standard of ACUTE EXERCISE may alter the counts and proportions of ~35 min over a lo-km distance within the previous 12 lymphocyte subpopulations in peripheral blood (11) and mo, and 2) they had a maximum oxygen consumption 2 perhaps suppress immune function (8, 21). These acute 60 ml kg-l min. alterations are attenuated by physical training of moderLaboratory visits were made after an initial 3 wk of ate intensity (18). However, the chronic stress associated baseline training (Bl), 3 wk of heavy training (HT), and 3 with the periods of heavy training required by a success- wk of reversion to the original baseline training schedule ful athlete may impair lymphocyte function (9). (B2). Training was limited to short distance and low-inThe differing response after heavy training may reflect tensity work in the 36 h immediately preceeding the laboan increase in the physical stress of exercise. Thus we ratory visits. During each of these three visits, measureasked whether endurance athletes would show a differments of body mass and skinfold thicknesses were made, ent immune response to acute exercise if they were examand subjects performed a bout of acute exercise. Venous ined before and after a period of heavy training that sur- blood samples were collected before, 5, and 30 min followpassed their habitual regimens. The hypotheses were I) ing the bout of acute exercise. that a bout of acute submaximal exercise would not supSubjects were allowed to increase their diets ad libitum press immune function while the athlete continued norduring the study, and nutritional analysis was not conmal training, 2) that the same bout of acute exercise ducted. Medical history inventories revealed that all subwould suppress immune function following heavy trainjects were medication free throughout the study. Plasma ing, and 3) that the suppression of immune function in- cortisol levels in conjunction with “Profile of Mood 1494 0161-7567192 $2.00 Copyright 0 1992 the American Physiological Society l
l
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States” were used in monitoring ported here. Maximal
Oxygen Intake
RESPONSE
stress
TO
ACUTE
but are not re-
l
Acute Exercise Acute exercise consisted of a treadmill run, during which subjects 0% slope. The speed was selected treadmill maximal oxygen intake, Dill (5). Gas exchange and heart throughout the test. Heavy
AND
HEAVY
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Cell viability (always >91%) was assessed by trypan blue exclusion. The final cell count was adjusted to 1 X lo6 cells/ml.
Determinations
During separate laboratory visits, immediately before and after HT, maximal oxygen intake was determined. Subjects who had not previously used a treadmill were habituated to this task by a preliminary lo-min run. Definitive tests began with a 3-min warm-up (9.6 km/h, 0% slope). The treadmill speed was then increased to a steady 14.5 km/h, and the slope was subsequently increased by 2% every 2 min to subjective exhaustion. Expired gas was collected and analyzed by means of a Beckman Metabolic Cart. The heart rate (CM5 electrode placement) was recorded during the final 10 s of each test minute. Three criteria were adopted to gauge maximal effort: 1) a plateau of oxygen consumption to within 2 ml kg-’ min-’ with a further increase of work rate, 2) a respiratory gas exchange ratio of 1.15, and 3) a plateauing of heart rate with further increase of work rate. l
EXERCISE
30-min submaximal ran on a treadmill at to elicit 80% of the using the formula of rate were monitored
Training
For the 3 wk of heavy training, subjects were asked to increase their training by 25% above their habitual volume of training. Training volume was monitored through personal diaries in which distance and speed were recorded. Blood Sampling Blood samples were collected from the antecubital vein before, 5, and 30 min following each bout of acute exercise. EDTA/heparin-coated vacutainers (BectonDickinson, Oakville, Ontario, Canada) were transported (l5- to 20-min walk) in a thermally insulated container to the laboratory where mononuclear cells were separated. As a precaution against contamination, all immunoassays were prepared under a level B laminar flow hood, and the counter was cleansed with 95% alcohol lo-15 min prior to use. Mononuclear cells were separated by mixing whole blood with an equal volume of a colloidal silica-based medium (Sepracell-MN, Sepratech, Oklahoma City, OK) prior to centrifuging for 20 min at 1,500 g. The mononuclear band was aspirated, mixed with four volumes of phosphate buffer [phosphate-buffered saline (PBS)-bovine serum albumin (BSA), pasteurized Cohn fraction V], and centrifuged at 300 g for 10 min. The cell pellet was resuspended in the same volume of PBS-BSA and recentrifuged at 300 g for 10 min. Finally, the cell pellet was again resuspended in 5 ml of RPM1 1640 medium (supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 1% L-glutamine; GIBCO).
Determination of Lymphocyte Subpopulations Cell suspensions were divided between six Eppendorf tubes. After centrifuging at 300 g for 5 min and removing the supernatant, 20-~1 volumes of monoclonal antibodies (Beckton-Dickinson) were added as follows: tube 1, monoclonal antibody for T-cells (anti-Leu-4) CD3; tube 2, monoclonal antibody for B-cells (anti-Leu-16) CD20; tube 3, monoclonal antibody for helper cells (anti-Leu3a) CD4; tube 4, monoclonal antibody for suppressor cells (anti-Leu-2a) CD8; tube 5, ascites mouse Ig fluorescence control for use with human cells (mouse IgGl); and tube 6, secondary control, containing PBS in place of fluorescent stain. The tubes were incubated in the presence of the fluorescent label for 30 min at 4°C. The stained cell suspensions were centrifuged, and the cell pellet was washed twice in PBS and fixed by adding 50 ~1 of 10% paraformaldehyde. The flow cytometer used light at 650 nm to activate the fluorochrome-labeled monoclonal antibodies and to determine the proportion of a 10,000 cell sample that carried a given fluorochrome label. The area under the measurable light profile for the mouse Ig was subtracted from other histograms if overlap occurred. Mitogen-Induced Proliferation Mononuclear Cells
of Peripheral Blood
Phytohemagglutinin (PHA-M form; GIBCO) and concanavalin A (ConA; ICN Biomedicals) are both specific stimulants of T-cell proliferation. Responses to PHA are resistant to oral cortisone, whereas responses to ConA are suppressed by cortisone (6). Mononuclear cells were incubated with PHA (10, 15, and 20 pglml) and ConA (1.0,2.0, and 4.0 pglml), with all assays being conducted in quadruplicate. Incubation in a humidified atmosphere containing 5% CO,-95% air continued for 72 h at 37°C with the cells being pulsed from 48 to 72 h with 1.0 &i of [methyl-3H]thymidine (Amersham). After specimens were harvested onto glass fiber filters and dried overnight, they were counted in 3 ml of scintillation fluid using a liquid scintillation counter (LKB Wallac 1217). Results were expressed as the difference between the average of counts from the three closest of the four assays and nonstimulated controls, with the coefficient of variation for this assessment averaging 2.8%. Mitogen-Induced
Ig Synthesis
Cell suspensions were mixed with various concentrations of pokeweed mitogen (GIBCO) and incubated for 7 days in a humidified atmosphere containing 5% CO,-95% air at 37OC. Samples were then centrifuged (300 g, 10 min) and analyzed by enzyme-linked immunosorbant assay. Culture plates were coated overnight at 4°C with goat anti-human IgG or IgM (Organon Teknika). Individual wells were incubated with the supernatant preparation or standard solutions of Ig for 1 h at 37°C and after washing by an automated processor (Behring ELISA Pro-
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1496
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TABLE
TO
ACUTE
1. Training practices during Bl, HT, and B2 Bl
% Increase Bl to HT
98.8t22.0
35&25
130.3k24.9
5,2002943
38&24
7,003-t877
Variable
Training kmlwk Training * units
RESPONSE
HT
EXERCISE
Variable VO 2 max, ml.
kg-‘. HRnax 9 beats/min TTE, s Body mass, kg
85.9+ 18.0
volume, 4,564+996
l
cessor II), an IgG or IgM peroxidase conjugate was added. Plates were incubated for another 1 h at 37”C, the washing was repeated, and there was then further incubation for 30 min in a darkened room with an o-phenylenediamine-2HCl substrate (Ortho-Diagnostic) catalyzed by 0.5% hydrogen peroxide. The reaction was halted by adding 50 ~1 of sulfuric acid to each well, and the absorbance was then determined at 492 nm. The average of the two closest optical density readings was interpreted relative to an appropriate calibration curve; the coefficient of variation between the two readings was 1.2%. Statistical Analysis Repeated-measures analysis of variance (ANOVA) in conjunction with Duncan’s post hoc multiple-range test were used to assessdifferences over time (Bl, HT, B2). Two-tailed paired t tests were used to analyze the responsesto acute exercise (before exercise compared with 5 min after exercise). The level of significance employed was P 5 0.05. Correlation analyses (Pearson correlation) were performed to assesspossible associations between lymphocyte subpopulations, helper/suppressor ratios, and mitogen-induced cellular proliferation. RESULTS
Subject Characteristics The subjects were elite distance runners, with an initial weekly baseline training volume of 98.8 t 22.0 km. Mean body mass for the group (66.1 t 4.7 kg) was low in relation to group mean height (176.8 t 4.4 cm), and there was relatively little subcutaneous fat (sum of biceps, triceps, subscapular, suprailiac, and medial calf folds, mean = 26.1 t 6.0 mm). The group mean maximal oxygen intake (65.3 t 4.9 ml. kg-’ min-‘) and the mean personal best lo-km time (31 min 43 s t 1 min 46 s) confirmed that the subjects were indeed elite runners. Despite the relative homogeneity of the sample, the personal best race times were significantly correlated with individual maximal oxygen intake values (r = -0.86, P < 0.002). l
TRAINING
2. Comparison of vo2-, at Bl and HT
B2
Values are means & SD. Bl, initial 3 wk of baseline training; HT, 3 wk of heavy training; B2, repeated 3 wk of baseline training. Training volume was approximated as a simple multiple of daily training distance and a speed-related assessment of the oxygen cost. For example, if an athlete ran a distance of 16 km at a pace of 4.47 m/s, the estimated oxygen cost would be 57.1 ml kg-’ min-’ (5), and the training volume would be 16 X 57.1 or 913.6 units; if there were five such sessions in a week, the total volume score for that individual would be 913.6 X 5, or 4,568 * units.
HEAVY
TABLE
distance,
l
AND
HR-,
and TTE
Bl
min-’
HT
65.3k4.9 186&g 650&83 66.1k4.7
65.1k4.5 181*8 664k90 65.9t4.6
Values are means + SD. VO, max, maximal 0, consumption; maximal heart rate; TTE, treadmill time to exhaustion.
Modification
HR,,
,
of Training
Each runner was individually consulted to establish a schedule of increased training that could be sustained for 3 wk without incurring gross orthopedic injuries. The average weekly distance was increased by 35%, and the estimate of training volume was increased by 38% during heavy training (Table 1). Oxygen Intake There was no significant impact of heavy training on maximal oxygen intake (Table 2). Likewise, the oxygen cost of running at a given submaximal pace was not significantly altered by 3 wk of heavy training (Table 3). The mean heart rate response to acute exercise was lower following heavy training (Table 3). Lymphocyte Subpopulations
At rest. The percentage of T lymphocytes in blood samples taken at rest were significantly lower following B2 compared with either Bl or HT percentages (Table 4). The percentage of helper cells in blood samples taken at rest tended to diminish after HT, with a further decrease evident following B2 (Table 5). The values observed at rest following B2 were significantly lower than those observed following Bl. In contrast, the percentage of suppressor cells in blood samples taken at rest tended to increase following HT, with a subsequent significant decrease observed following B2. The corresponding helper/ suppressor ratio at rest tended to be lowest following HT and significantly highest following B2 (Table 5). Acute exercise. There was a trend of a decrease in the percentages of both T and B lymphocytes 5 min after exercise, with recovery 30 min postexercise (Table 4). However, the decrease in the percentage of T lymphocytes 5 min after exercise was statistically significant only following HT. Following B2, the percentages of both T and B lymphocytes observed 30 min after exercise were higher (P < 0.05) than preexercise values. Under all three conditions, exercise tended to induce a lowering of the helper/suppressor cell ratio, a decrease that was statisti3. Comparison of submaximal responses to a 30-min treadmill run following Bl, HT, and B2 TABLE
Variable VO,,
HR,
ml kg-’ beats/min l
Bl l
min-’
51.6k3.0 163*10*
HT
B2
51.9k4.1 159+8t
Values are means t SD. Effects of training: values symbols differ significantly from each other (P < 0.05).
50.322.5 161+8* with
differing
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TO
ACUTE
EXERCISE
AND
HEAVY
1497
TRAINING
4. Effects of 30 min of exercise at 80% of maximal oxygen intake on lymphocyte populations following Bl, HT, and B2 TABLE
T Lymphocytes, Training Regimen
Before exercise
Bl HT B2
64.7&12.4* 62.6~13.4*” 55.2+12.9ta
%
B Lymphocytes,
5 min After exercise
30 min After exercise
57.5-tl3.6 54.3+13.gb 49.9kl2.8”
61.0+10.8 61.1k12.4” 62.2+11.1b
Before exercise
4.65k2.12 4.64k2.18 3.5lk1.23”
Values are means + SD. Data are percentages of the total lymphocyte count as seen in the peripheral Effects of acute exercise: values with differing letters differ significantly from each other (P < 0.05). Effects symbols differ significantly from each other (P < 0.05).
tally significant only following B2. Following HT, the helper/suppressor ratio was higher 30 min after exercise than observed preexercise (Table 5). Mitogen-Induced
Cell Proliferation
At rest. Following HT, the blood samples taken at rest showed a 24.6% increase of response to PHA and a 32.2% increase of response to ConA, the latter change being statistically significant (P < 0.05; Table 6). Following B2, the elevated proliferative response at rest remained significantly higher than observed before HT. Acute exercise. Following both Bl and B2, acute exercise induced no significant change in the proliferative response to mitogens (Table 6). In contrast, acute exercise induced a significant 17.9% suppression of the proliferative response to PHA following HT. Although not statistically significant, the proliferative response to ConA was suppressed 11.7%. After HT, preexercise PHA values were reestablished by 30 min after exercise. At B2, ConA values at 30 min after exercise were significantly greater than preexercise and 5 min postexercise values. Mitogen-Induced Ig Synthesis At rest. Data from resting subjects showed a trend to decreased IgG and IgM syntheses after HT, with significant increases in Ig production following B2 (Table 7). Acute exercise. Acute exercise did not alter Ig production following Bl, HT, or B2. In contrast to the data following Bl and B2, both IgG and IgM syntheses were significantly elevated 30 min postexercise after heavy training. DISCUSSION
Lymphocyte Subpopulations
The observed trend to an acute exercise-induced decrease in the percentage of T-cells, exacerbated by heavy
%
5 min After exercise
30 min After exercise
4.02k1.91 3.77zk2.21 2.89kO.99”
5.0122.58 5.28k2.53 4.68k1.48b
blood mononuclear cell suspension. of training (rest): values with differing
training, is in keeping with the larger responses previously described in less well-trained individuals (8,15,16). Nevertheless, there was no correlation between T-cell counts and the stimulation of T-cell activity by mitogens, emphasizing that simple blood counts (which fail to consider the responsiveness of the T-cells) can give very misleading information about immune function. Some previous authors have suggested that acute bouts of exercise induce an increase in the number and percentage of B-cells (8, 16, 19), whereas in the present study the trend was to a decrease in the proportion of B-cells under all three training conditions. This discrepancy may reflect differences of experimental technique. Earlier studies did not use monoclonal antibodies but rather by counting small samples (100-200 cells) identified B-cells as those that were non-T-cells (non-E-rosette-forming cells) or as those that possessed surface markers for Igs and/or Fc receptors, an approach that could lead to both counting errors and an inclusion of natural killer cells in the B-cell total (4). It is well recognized that natural killer cell populations increase with acute exercise (14). The trend to an acute exercise-induced decrease in the helper/suppressor cell ratio is in acccordance with the responses observed in less well-trained individuals (1, 2, 7, 12, 13). Although alterations in the helper/suppressor ratio have been suggested to influence alterations in mitogen-induced cellular proliferation (9, l2), no such correlation was found in the present study. Cell-Mediated Immune Function
There was extreme interindividual togen-induced proliferative response cytes, with the initial values at rest to 73,714 counts/min (cpm). Such plains the large deviations around
variation in the miof isolated lymphoranging from 18,503 large variation exthe reported means
TABLE 5. Response of helper and suppressor cells to 30 min of exercise at 80% of maximal oxygen intake following Bl, HT, and B2 Helper Training Regimen
Bl HT B2
Before exercise
38.2+ 14.0* 33.2+12.7*? 30.5+11.8t
Cells,
5 min After exercise
31.6k11.8 30.2k12.2 26.7k6.8
%
Suppressor 30 min After exercise
34.6k13.9 32.0k16.9 29.6k11.9
Cells,
%
Helper/Suppressor
Ratio
Before exercise
5 min After exercise
30 min After exercise
Before exercise
5 min After exercise
30 min After exercise
16.6+5.7*? 18.0+6.7* 12.5+7.6-f
19.2k7.0 17.6k6.8 14.5k8.1
14.0k5.2 14.2t8.8 11.6k9.8
2.91+2.25* 2.05&1.02*‘” 3.49+2.41ta
1.84kO.87 1.89kO.91” 2.53k1.62b
2.74kl.45 3.17k2.12b 3.9822.40”
Values are means k SD. Effects of acute exercise: values with differing (rest): values with differing symbols differ significantly from each other
letters differ (P < 0.05).
significantly
from
each other
(P < 0.05).
Effects
of training
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1498
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ACUTE
EXERCISE
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TRAINING
6. Effect of 30 min of exercise at 80% of maximal oxygen intake on mitogen-induced peripheral blood mononuclear cell proliferation following Bl, HT, and B2
TABLE
PHA, Training Regimen
Bl HT B2 Values significantly
Before exercise
cpm
ConA,
5 min After exercise
37,087+ 16,707* 46,218+14,04l*t” 48,680k15,202tab
30 min After exercise
34,979?20,153 38,162+13,49gb 44,610+17,750b
39,401+17,640 43,660+ 18,936ab 54,197+ 18,936”
are means k SE. PHA, phytohemagglutinin; ConA, concanavalin from each other (P < 0.05). Effects of training (rest): values with
(Table 6). Accordingly, there was also a large interindividual variation in the proliferative response (in absolute cpm) to acute exercise. At each end of the range, a similar 20% exercise-induced change would translate into a response that would range almost fourfold (3,700 and 14,743 cpm, respectively) in absolute numbers. A second source of variation around the exercise-induced changes in proliferation was a dichotomy of response, with a few subjects tending to show an exercise-induced enhancement and the remainder tending to show suppression. The dichotomy of response seemed to depend on the rested state of the individual, as discussed below. The statistical analysis employed in this study used each subject as their own control, thereby accounting for these interindividual variations. Both initially and after return to the normal regimen of training, the bout of acute exercise had no significant influence on the average proliferative response to either the cortisone-resistant (PHA) or the cortisone-suppressed (ConA) mitogen. However, when subjects performed the same bout of acute exercise immediately following heavy training, the proliferative response to PHA was significantly suppressed, with a similar trend in response to ConA. Our findings are in agreement with reported animal experimentation in which an acute exercise challenge is superimposed on the stress of longitudinal training (9). Analysis of the runners’ diaries further supports the suppressant effect of acute exercise when superimposed on heavy training. Well-rested individuals initially showed an exercise-induced enhancement of immune function, whereas those who were training harder relative to their usual habits tended to a negative response. However, even when the subjects were heavily trained, the functional impact of the acute exercise bout was very transient, with the mitogen response becoming normal within 30 min. It is thus most unlikely that the observed TABLE
Before exercise
29,060+ 38,433+ 39,609-t
16,735” 11,896t 11,268t”
cpm
5 min After exercise
30 min After exercise
28,525-t 18,335 33,810+13,595 38,876+12,756”
33,209-t 16,041 37,278+ 14,776 49,101k18,660b
A. Effects of acute exercise: values differing symbols differ significantly
with from
differing letters differ each other (P < 0.05).
change in immune function would have adverse clinical implications. Humoral Immune Function In contrast to a previous report (7), our data showed no acute exercise-induced reduction of the in vitro production of either IgG or IgM, regardless of the training regimen. The larger depression of immune response observed in the experiments of Hedfors et al. (7) may reflect their use of cell suspensions that were twice the concentration (2 X lo6 cells/ml) as those in the present study. Thus exercise-induced trends may have been magnified. Their subjects were also less well trained so that the bout of acute exercise may have been more stressful for them. Limitations One important limitation to this exercise immunology study, as in most other reports on human subjects, is that all observations of lymphocyte proportions and function were based on specimens of peripheral blood. Transient increases of peripheral lymphocyte counts could arise through an exercise-induced mobilization of sequestrated cells, and there can be no guarantee that either cell proportions or function as observed in peripheral venous blood are representative of other parts of the circulation. Moreover, measurements of function made in vitro provide only a general indication of responses in vivo. The importance of the transient changes observed in this and other exercise immunology studies has not been established. Increase of Training Volume The two methods of quantitating heavy training (distance and volume) ensured that the subjects did not increase distance at the expense of intensity. The 38% in* . 1
1
. 1
1 0.
1
7. Effect of 30 min of exercise at 80% of maximal oxygen vataRe on poReweed mttogen-vaaucea
Ig synthesis following Bl, HT, and B2 IgG, rig/ml Training Regimen
Bl HT B2
IgM,
mg/ml
Before exercise
n
5 min After exercise
n
30 min After exercise
n
Before exercise
n
5 min After exercise
n
30 min After exercise
n
644+656*t 537+412*a 884&433-j-
10 9 10
913k843 488k367” 847+555
8 10 10
7Olk851 904zk635b 828+475
9 9 10
73O-t600* 585k445*a 1,178+626”f
10 9 10
765+663 589+319” 1,052+360
8 10 10
836k581 908k617b 1,098+663
9 9 10
Values are means + SD; n, no. of men. Ig, immunoglobulin. Effects of acute exercise: values with differing letters differ other (P < 0.05). Effects of training (rest): values with differing symbols differ significantly from each other (P < 0.05).
significantly
from
each
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IMMUNE
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TO ACUTE
crease of average training volume was substantial, resulting in a significant increase in fatigue as assessed by psycho1 .ogical ev aluation (unpublished data). Because of fatigue, most of the runn ers were unable to repeat their initial training schedules, as reflected in the lower training volumes averaged during B2. For most of the runners, the 3 wk of heavy training was representative of a “peak” training schedule used in their ‘‘peak and taper” approach to competition. Conclusions
On the basis of this small sample and the specific in vitro measurements of immunity employed, individuals who are well trained can undertake a 30-min bout of acute exercise with at most a minimal and short-lived . suppression of cell-mediated immunity. Such changes are exacerbated by a deliberate increase of training, but they remain transient in nature and within the physiological rather than the pathological range of stress responses. I‘f the volume 0 If endu rance training is determined by the i.ndividua .l rather than an overambitious coach, there seems little danger of causi ng distu rbances of cell- media ted im munity that will have adverse 1clinical consequences. We thank Dr. Paul Corey for advice on statistical aspects of this study. We also thank the Defence and Civil Institute of Environmental Medicine and the Faculty of Medicine, University of Toronto, for the use of special laboratory facilities. This research was supported in part by a research grant from the United States Olympic Committee. Address for reprint requests: T. J. Verde, Graduate Hospital Human Performance and Sports Medicine Center, 200 W. Lancaster Ave., Wayne, PA 19087. Received 10 January 1991; accepted in final form 27 April 1992. REFERENCES 1. BERK, L. KRAMER,
S., D. NIEMAN, S. A. TAN, S. NEHLSEN-CANNARELLA, J. W. C. EBY, AND M. OWENS. Lymphocyte subset changes during acute maximal exercise (Abstract). Med. Sci. Sports Exercise
18: 706, 1986. 2. BRAHMI, Z., J.
E. THOMAS, M. PARK, AND I. R. G. DOWDESWELL. The effect of acute exercise on natural killer cell activity of trained and sedentary human subjects. J. Clin. Immunol. 5: 321-328,1985. 3. DESHAZO, R. D., M. LOPEZ, AND J. E. SALVAGGIO. Use and interpretation of diagnostic immunologic laboratory tests. J. Am. Med. Assoc. 258: 3011-3031, 1987. 4. DEUSTER, P. A., A. M. CURIALE,
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EXERCISE
AND HEAVY
TRAINING
1499
Exercise-induced changes in populations of peripheral blood mononuclear cells. Med. Sci. Sports Exercise 20: 276-280, 1988. DILL, D. B. Oxygen used in horizontal and grade walking and running on the treadmill. J. Appl. Physiol. 20: 19-22, 1965. FAUCI, A. S., AND D. C. DALE. The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J. Clin. Inuest. 53: 240MAN.
246, 1974. HEDFORS,
E., G. HOLM, M. IVANSEN, AND J. WAHREN. Physiological variation of blood lymphocyte reactivity: T-cell subsets, immunoglobulin production, and mixed lymphocyte reactivity. Clin. Immunol. Immunopathol. 27: 9-14, 1983. 8. HEDFORS, E., G. HOLM, AND B. OHNELL.
Variations of blood lymphocytes during work studied by cell surface markers, DNA syntheses and cytotoxicity. Clin. Exp. Immunol. 24: 328-335, 1976. 9. HOFFMAN-GOETZ, L., R. KEIR, R. THORNE, M. E. HOUSTON, AND C. YOUNG. Chronic exercise stress in mice depresses splenic T lymphocyte mitogenesis in vitro. Clin. Exp. Immunol. 66: 551-557, 1986. 10. KEAST,
D., K. CAMERON, AND A. R. MORTON. Exercise and immune response. Sports Med. 5: 248-267, 1988. 11. KENDALL, A., L. HOFFMAN-GOETZ, M. E. HOUSTON, B. MACNEIL, AND Y. ARUMUGAM. Exercise and blood lymphocyte subset responses: intensity, duration, and subject fitness effects. J. Appl. Physiol. 69: 251-260, 1990. 12. LANDMANN, R. M. A., F. B. MULLER, ERNE, AND F. R. BUHLER. Changes
13.
14. 15. 16.
C. H. PERINI, M. WESP, P. of immunoregulatory cells induced by psychological and physical stress: relationship to catecholamines. Clin. Exp. Immunol. 58: 127-135, 1984. LEWICKI, R., H. TCHORZEWSKI, E. MAJEWSKA, Z. NOWAK, AND Z. BAJ. Effect of maximal physical exercise on T-lymphocyte subpopulations and on interleukin 1 (ILl) and interleukin 2 (IL2) production in vitro. Int. J. Sports Med. 9: 114-117, 1988. MACKINNON, L. T. Exercise and natural killer cells: what is the relationship? Sports Med. 7: 141-149, 1989. MCCARTHY, D. A., AND M. M. DALE. The leucocytosis of exercise: a review and model. Sports Med. 6: 333-363, 1988. ROBERTSON, A. J., K. C. RAMESAR, R. C. POTTS, J. H. GIBBS, M. C. BROWNING, R. A. BROWN, P. C. HAYES, AND J. SWANSON-BUCK. The effect of strenuous physical exercise on circulating blood lymphocytes and serum cortisol levels. J. Clin. Lab. Immunol. 5: 53-57,
1981. 17. SIMON, H. B. Exercise and infection. Phys. Sports Med. 141,1987. 18. SOPPI, E., P. VARJO, J. ESKOLA, AND L. A. LAITINEN.
15: 134-
Effect of strenuous physical stress on circulating lymphocyte number and function before and after training. J. Clin. Lab. Immunol. 8: 43-46,
1982. 19. VISHNU-MOORTHY,
A., AND S. W. ZIMMERMAN. Human leukocyte response to an endurance race. Eur. J. Appl. Physiol. Occup. Phys-
iol. 38: 271-276, 1978. 20. WALFORD, R. L. Immunological theory of aging: current status. Federation Proc. 33: 2020-2027, 1974. 21. WATSON, R. R., S. MORIGUCHI, J. C. JACKSON, L. WERNER, J. H. WILMORE, AND B. J. FREUND. Modification of cellular immune
functions in human by endurance training during ,&adrenergic blockade with atenolol or propanolol. Med. Sci. Sports Exercise 18: 95-100,
1986.
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subpopulations, helper/suppressor ratios, and mitogen-induced cellular proliferation. The immune system of endurancetrained athletes at rest seemed to tolerate the stress of heavy training, but superimposition of a bout of acute exercise on the chronic stress of heavy training resulted in immunosuppression, which was transient and most likely not of clinical significance.
METHODS Subjects and Experimental Plan
The volunteers were 10 male distance runners aged 29.8 t 1.7 yr, who were recruited and tested in accordance with a protocol approved by the University Committee on Human Experimentation. To avoid difficulties acute exercise; heavy training; lymphocyte subpopulations; from age-related differences of immune function (2O), lymphocyte proliferation; immunoglobulin synthesis the age span was deliberately limited to 5 yr. All subjects were elite runners based on the following two criteria: 1) they had achieved a minimal performance standard of ACUTE EXERCISE may alter the counts and proportions of ~35 min over a lo-km distance within the previous 12 lymphocyte subpopulations in peripheral blood (11) and mo, and 2) they had a maximum oxygen consumption 2 perhaps suppress immune function (8, 21). These acute 60 ml kg-l min. alterations are attenuated by physical training of moderLaboratory visits were made after an initial 3 wk of ate intensity (18). However, the chronic stress associated baseline training (Bl), 3 wk of heavy training (HT), and 3 with the periods of heavy training required by a success- wk of reversion to the original baseline training schedule ful athlete may impair lymphocyte function (9). (B2). Training was limited to short distance and low-inThe differing response after heavy training may reflect tensity work in the 36 h immediately preceeding the laboan increase in the physical stress of exercise. Thus we ratory visits. During each of these three visits, measureasked whether endurance athletes would show a differments of body mass and skinfold thicknesses were made, ent immune response to acute exercise if they were examand subjects performed a bout of acute exercise. Venous ined before and after a period of heavy training that sur- blood samples were collected before, 5, and 30 min followpassed their habitual regimens. The hypotheses were I) ing the bout of acute exercise. that a bout of acute submaximal exercise would not supSubjects were allowed to increase their diets ad libitum press immune function while the athlete continued norduring the study, and nutritional analysis was not conmal training, 2) that the same bout of acute exercise ducted. Medical history inventories revealed that all subwould suppress immune function following heavy trainjects were medication free throughout the study. Plasma ing, and 3) that the suppression of immune function in- cortisol levels in conjunction with “Profile of Mood 1494 0161-7567192 $2.00 Copyright 0 1992 the American Physiological Society l
l
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IMMUNE
States” were used in monitoring ported here. Maximal
Oxygen Intake
RESPONSE
stress
TO
ACUTE
but are not re-
l
Acute Exercise Acute exercise consisted of a treadmill run, during which subjects 0% slope. The speed was selected treadmill maximal oxygen intake, Dill (5). Gas exchange and heart throughout the test. Heavy
AND
HEAVY
1495
TRAINING
Cell viability (always >91%) was assessed by trypan blue exclusion. The final cell count was adjusted to 1 X lo6 cells/ml.
Determinations
During separate laboratory visits, immediately before and after HT, maximal oxygen intake was determined. Subjects who had not previously used a treadmill were habituated to this task by a preliminary lo-min run. Definitive tests began with a 3-min warm-up (9.6 km/h, 0% slope). The treadmill speed was then increased to a steady 14.5 km/h, and the slope was subsequently increased by 2% every 2 min to subjective exhaustion. Expired gas was collected and analyzed by means of a Beckman Metabolic Cart. The heart rate (CM5 electrode placement) was recorded during the final 10 s of each test minute. Three criteria were adopted to gauge maximal effort: 1) a plateau of oxygen consumption to within 2 ml kg-’ min-’ with a further increase of work rate, 2) a respiratory gas exchange ratio of 1.15, and 3) a plateauing of heart rate with further increase of work rate. l
EXERCISE
30-min submaximal ran on a treadmill at to elicit 80% of the using the formula of rate were monitored
Training
For the 3 wk of heavy training, subjects were asked to increase their training by 25% above their habitual volume of training. Training volume was monitored through personal diaries in which distance and speed were recorded. Blood Sampling Blood samples were collected from the antecubital vein before, 5, and 30 min following each bout of acute exercise. EDTA/heparin-coated vacutainers (BectonDickinson, Oakville, Ontario, Canada) were transported (l5- to 20-min walk) in a thermally insulated container to the laboratory where mononuclear cells were separated. As a precaution against contamination, all immunoassays were prepared under a level B laminar flow hood, and the counter was cleansed with 95% alcohol lo-15 min prior to use. Mononuclear cells were separated by mixing whole blood with an equal volume of a colloidal silica-based medium (Sepracell-MN, Sepratech, Oklahoma City, OK) prior to centrifuging for 20 min at 1,500 g. The mononuclear band was aspirated, mixed with four volumes of phosphate buffer [phosphate-buffered saline (PBS)-bovine serum albumin (BSA), pasteurized Cohn fraction V], and centrifuged at 300 g for 10 min. The cell pellet was resuspended in the same volume of PBS-BSA and recentrifuged at 300 g for 10 min. Finally, the cell pellet was again resuspended in 5 ml of RPM1 1640 medium (supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 1% L-glutamine; GIBCO).
Determination of Lymphocyte Subpopulations Cell suspensions were divided between six Eppendorf tubes. After centrifuging at 300 g for 5 min and removing the supernatant, 20-~1 volumes of monoclonal antibodies (Beckton-Dickinson) were added as follows: tube 1, monoclonal antibody for T-cells (anti-Leu-4) CD3; tube 2, monoclonal antibody for B-cells (anti-Leu-16) CD20; tube 3, monoclonal antibody for helper cells (anti-Leu3a) CD4; tube 4, monoclonal antibody for suppressor cells (anti-Leu-2a) CD8; tube 5, ascites mouse Ig fluorescence control for use with human cells (mouse IgGl); and tube 6, secondary control, containing PBS in place of fluorescent stain. The tubes were incubated in the presence of the fluorescent label for 30 min at 4°C. The stained cell suspensions were centrifuged, and the cell pellet was washed twice in PBS and fixed by adding 50 ~1 of 10% paraformaldehyde. The flow cytometer used light at 650 nm to activate the fluorochrome-labeled monoclonal antibodies and to determine the proportion of a 10,000 cell sample that carried a given fluorochrome label. The area under the measurable light profile for the mouse Ig was subtracted from other histograms if overlap occurred. Mitogen-Induced Proliferation Mononuclear Cells
of Peripheral Blood
Phytohemagglutinin (PHA-M form; GIBCO) and concanavalin A (ConA; ICN Biomedicals) are both specific stimulants of T-cell proliferation. Responses to PHA are resistant to oral cortisone, whereas responses to ConA are suppressed by cortisone (6). Mononuclear cells were incubated with PHA (10, 15, and 20 pglml) and ConA (1.0,2.0, and 4.0 pglml), with all assays being conducted in quadruplicate. Incubation in a humidified atmosphere containing 5% CO,-95% air continued for 72 h at 37°C with the cells being pulsed from 48 to 72 h with 1.0 &i of [methyl-3H]thymidine (Amersham). After specimens were harvested onto glass fiber filters and dried overnight, they were counted in 3 ml of scintillation fluid using a liquid scintillation counter (LKB Wallac 1217). Results were expressed as the difference between the average of counts from the three closest of the four assays and nonstimulated controls, with the coefficient of variation for this assessment averaging 2.8%. Mitogen-Induced
Ig Synthesis
Cell suspensions were mixed with various concentrations of pokeweed mitogen (GIBCO) and incubated for 7 days in a humidified atmosphere containing 5% CO,-95% air at 37OC. Samples were then centrifuged (300 g, 10 min) and analyzed by enzyme-linked immunosorbant assay. Culture plates were coated overnight at 4°C with goat anti-human IgG or IgM (Organon Teknika). Individual wells were incubated with the supernatant preparation or standard solutions of Ig for 1 h at 37°C and after washing by an automated processor (Behring ELISA Pro-
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1496
IMMUNE
TABLE
TO
ACUTE
1. Training practices during Bl, HT, and B2 Bl
% Increase Bl to HT
98.8t22.0
35&25
130.3k24.9
5,2002943
38&24
7,003-t877
Variable
Training kmlwk Training * units
RESPONSE
HT
EXERCISE
Variable VO 2 max, ml.
kg-‘. HRnax 9 beats/min TTE, s Body mass, kg
85.9+ 18.0
volume, 4,564+996
l
cessor II), an IgG or IgM peroxidase conjugate was added. Plates were incubated for another 1 h at 37”C, the washing was repeated, and there was then further incubation for 30 min in a darkened room with an o-phenylenediamine-2HCl substrate (Ortho-Diagnostic) catalyzed by 0.5% hydrogen peroxide. The reaction was halted by adding 50 ~1 of sulfuric acid to each well, and the absorbance was then determined at 492 nm. The average of the two closest optical density readings was interpreted relative to an appropriate calibration curve; the coefficient of variation between the two readings was 1.2%. Statistical Analysis Repeated-measures analysis of variance (ANOVA) in conjunction with Duncan’s post hoc multiple-range test were used to assessdifferences over time (Bl, HT, B2). Two-tailed paired t tests were used to analyze the responsesto acute exercise (before exercise compared with 5 min after exercise). The level of significance employed was P 5 0.05. Correlation analyses (Pearson correlation) were performed to assesspossible associations between lymphocyte subpopulations, helper/suppressor ratios, and mitogen-induced cellular proliferation. RESULTS
Subject Characteristics The subjects were elite distance runners, with an initial weekly baseline training volume of 98.8 t 22.0 km. Mean body mass for the group (66.1 t 4.7 kg) was low in relation to group mean height (176.8 t 4.4 cm), and there was relatively little subcutaneous fat (sum of biceps, triceps, subscapular, suprailiac, and medial calf folds, mean = 26.1 t 6.0 mm). The group mean maximal oxygen intake (65.3 t 4.9 ml. kg-’ min-‘) and the mean personal best lo-km time (31 min 43 s t 1 min 46 s) confirmed that the subjects were indeed elite runners. Despite the relative homogeneity of the sample, the personal best race times were significantly correlated with individual maximal oxygen intake values (r = -0.86, P < 0.002). l
TRAINING
2. Comparison of vo2-, at Bl and HT
B2
Values are means & SD. Bl, initial 3 wk of baseline training; HT, 3 wk of heavy training; B2, repeated 3 wk of baseline training. Training volume was approximated as a simple multiple of daily training distance and a speed-related assessment of the oxygen cost. For example, if an athlete ran a distance of 16 km at a pace of 4.47 m/s, the estimated oxygen cost would be 57.1 ml kg-’ min-’ (5), and the training volume would be 16 X 57.1 or 913.6 units; if there were five such sessions in a week, the total volume score for that individual would be 913.6 X 5, or 4,568 * units.
HEAVY
TABLE
distance,
l
AND
HR-,
and TTE
Bl
min-’
HT
65.3k4.9 186&g 650&83 66.1k4.7
65.1k4.5 181*8 664k90 65.9t4.6
Values are means + SD. VO, max, maximal 0, consumption; maximal heart rate; TTE, treadmill time to exhaustion.
Modification
HR,,
,
of Training
Each runner was individually consulted to establish a schedule of increased training that could be sustained for 3 wk without incurring gross orthopedic injuries. The average weekly distance was increased by 35%, and the estimate of training volume was increased by 38% during heavy training (Table 1). Oxygen Intake There was no significant impact of heavy training on maximal oxygen intake (Table 2). Likewise, the oxygen cost of running at a given submaximal pace was not significantly altered by 3 wk of heavy training (Table 3). The mean heart rate response to acute exercise was lower following heavy training (Table 3). Lymphocyte Subpopulations
At rest. The percentage of T lymphocytes in blood samples taken at rest were significantly lower following B2 compared with either Bl or HT percentages (Table 4). The percentage of helper cells in blood samples taken at rest tended to diminish after HT, with a further decrease evident following B2 (Table 5). The values observed at rest following B2 were significantly lower than those observed following Bl. In contrast, the percentage of suppressor cells in blood samples taken at rest tended to increase following HT, with a subsequent significant decrease observed following B2. The corresponding helper/ suppressor ratio at rest tended to be lowest following HT and significantly highest following B2 (Table 5). Acute exercise. There was a trend of a decrease in the percentages of both T and B lymphocytes 5 min after exercise, with recovery 30 min postexercise (Table 4). However, the decrease in the percentage of T lymphocytes 5 min after exercise was statistically significant only following HT. Following B2, the percentages of both T and B lymphocytes observed 30 min after exercise were higher (P < 0.05) than preexercise values. Under all three conditions, exercise tended to induce a lowering of the helper/suppressor cell ratio, a decrease that was statisti3. Comparison of submaximal responses to a 30-min treadmill run following Bl, HT, and B2 TABLE
Variable VO,,
HR,
ml kg-’ beats/min l
Bl l
min-’
51.6k3.0 163*10*
HT
B2
51.9k4.1 159+8t
Values are means t SD. Effects of training: values symbols differ significantly from each other (P < 0.05).
50.322.5 161+8* with
differing
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IMMUNE
RESPONSE
TO
ACUTE
EXERCISE
AND
HEAVY
1497
TRAINING
4. Effects of 30 min of exercise at 80% of maximal oxygen intake on lymphocyte populations following Bl, HT, and B2 TABLE
T Lymphocytes, Training Regimen
Before exercise
Bl HT B2
64.7&12.4* 62.6~13.4*” 55.2+12.9ta
%
B Lymphocytes,
5 min After exercise
30 min After exercise
57.5-tl3.6 54.3+13.gb 49.9kl2.8”
61.0+10.8 61.1k12.4” 62.2+11.1b
Before exercise
4.65k2.12 4.64k2.18 3.5lk1.23”
Values are means + SD. Data are percentages of the total lymphocyte count as seen in the peripheral Effects of acute exercise: values with differing letters differ significantly from each other (P < 0.05). Effects symbols differ significantly from each other (P < 0.05).
tally significant only following B2. Following HT, the helper/suppressor ratio was higher 30 min after exercise than observed preexercise (Table 5). Mitogen-Induced
Cell Proliferation
At rest. Following HT, the blood samples taken at rest showed a 24.6% increase of response to PHA and a 32.2% increase of response to ConA, the latter change being statistically significant (P < 0.05; Table 6). Following B2, the elevated proliferative response at rest remained significantly higher than observed before HT. Acute exercise. Following both Bl and B2, acute exercise induced no significant change in the proliferative response to mitogens (Table 6). In contrast, acute exercise induced a significant 17.9% suppression of the proliferative response to PHA following HT. Although not statistically significant, the proliferative response to ConA was suppressed 11.7%. After HT, preexercise PHA values were reestablished by 30 min after exercise. At B2, ConA values at 30 min after exercise were significantly greater than preexercise and 5 min postexercise values. Mitogen-Induced Ig Synthesis At rest. Data from resting subjects showed a trend to decreased IgG and IgM syntheses after HT, with significant increases in Ig production following B2 (Table 7). Acute exercise. Acute exercise did not alter Ig production following Bl, HT, or B2. In contrast to the data following Bl and B2, both IgG and IgM syntheses were significantly elevated 30 min postexercise after heavy training. DISCUSSION
Lymphocyte Subpopulations
The observed trend to an acute exercise-induced decrease in the percentage of T-cells, exacerbated by heavy
%
5 min After exercise
30 min After exercise
4.02k1.91 3.77zk2.21 2.89kO.99”
5.0122.58 5.28k2.53 4.68k1.48b
blood mononuclear cell suspension. of training (rest): values with differing
training, is in keeping with the larger responses previously described in less well-trained individuals (8,15,16). Nevertheless, there was no correlation between T-cell counts and the stimulation of T-cell activity by mitogens, emphasizing that simple blood counts (which fail to consider the responsiveness of the T-cells) can give very misleading information about immune function. Some previous authors have suggested that acute bouts of exercise induce an increase in the number and percentage of B-cells (8, 16, 19), whereas in the present study the trend was to a decrease in the proportion of B-cells under all three training conditions. This discrepancy may reflect differences of experimental technique. Earlier studies did not use monoclonal antibodies but rather by counting small samples (100-200 cells) identified B-cells as those that were non-T-cells (non-E-rosette-forming cells) or as those that possessed surface markers for Igs and/or Fc receptors, an approach that could lead to both counting errors and an inclusion of natural killer cells in the B-cell total (4). It is well recognized that natural killer cell populations increase with acute exercise (14). The trend to an acute exercise-induced decrease in the helper/suppressor cell ratio is in acccordance with the responses observed in less well-trained individuals (1, 2, 7, 12, 13). Although alterations in the helper/suppressor ratio have been suggested to influence alterations in mitogen-induced cellular proliferation (9, l2), no such correlation was found in the present study. Cell-Mediated Immune Function
There was extreme interindividual togen-induced proliferative response cytes, with the initial values at rest to 73,714 counts/min (cpm). Such plains the large deviations around
variation in the miof isolated lymphoranging from 18,503 large variation exthe reported means
TABLE 5. Response of helper and suppressor cells to 30 min of exercise at 80% of maximal oxygen intake following Bl, HT, and B2 Helper Training Regimen
Bl HT B2
Before exercise
38.2+ 14.0* 33.2+12.7*? 30.5+11.8t
Cells,
5 min After exercise
31.6k11.8 30.2k12.2 26.7k6.8
%
Suppressor 30 min After exercise
34.6k13.9 32.0k16.9 29.6k11.9
Cells,
%
Helper/Suppressor
Ratio
Before exercise
5 min After exercise
30 min After exercise
Before exercise
5 min After exercise
30 min After exercise
16.6+5.7*? 18.0+6.7* 12.5+7.6-f
19.2k7.0 17.6k6.8 14.5k8.1
14.0k5.2 14.2t8.8 11.6k9.8
2.91+2.25* 2.05&1.02*‘” 3.49+2.41ta
1.84kO.87 1.89kO.91” 2.53k1.62b
2.74kl.45 3.17k2.12b 3.9822.40”
Values are means k SD. Effects of acute exercise: values with differing (rest): values with differing symbols differ significantly from each other
letters differ (P < 0.05).
significantly
from
each other
(P < 0.05).
Effects
of training
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1498
IMMUNE
RESPONSE
TO
ACUTE
EXERCISE
AND
HEAVY
TRAINING
6. Effect of 30 min of exercise at 80% of maximal oxygen intake on mitogen-induced peripheral blood mononuclear cell proliferation following Bl, HT, and B2
TABLE
PHA, Training Regimen
Bl HT B2 Values significantly
Before exercise
cpm
ConA,
5 min After exercise
37,087+ 16,707* 46,218+14,04l*t” 48,680k15,202tab
30 min After exercise
34,979?20,153 38,162+13,49gb 44,610+17,750b
39,401+17,640 43,660+ 18,936ab 54,197+ 18,936”
are means k SE. PHA, phytohemagglutinin; ConA, concanavalin from each other (P < 0.05). Effects of training (rest): values with
(Table 6). Accordingly, there was also a large interindividual variation in the proliferative response (in absolute cpm) to acute exercise. At each end of the range, a similar 20% exercise-induced change would translate into a response that would range almost fourfold (3,700 and 14,743 cpm, respectively) in absolute numbers. A second source of variation around the exercise-induced changes in proliferation was a dichotomy of response, with a few subjects tending to show an exercise-induced enhancement and the remainder tending to show suppression. The dichotomy of response seemed to depend on the rested state of the individual, as discussed below. The statistical analysis employed in this study used each subject as their own control, thereby accounting for these interindividual variations. Both initially and after return to the normal regimen of training, the bout of acute exercise had no significant influence on the average proliferative response to either the cortisone-resistant (PHA) or the cortisone-suppressed (ConA) mitogen. However, when subjects performed the same bout of acute exercise immediately following heavy training, the proliferative response to PHA was significantly suppressed, with a similar trend in response to ConA. Our findings are in agreement with reported animal experimentation in which an acute exercise challenge is superimposed on the stress of longitudinal training (9). Analysis of the runners’ diaries further supports the suppressant effect of acute exercise when superimposed on heavy training. Well-rested individuals initially showed an exercise-induced enhancement of immune function, whereas those who were training harder relative to their usual habits tended to a negative response. However, even when the subjects were heavily trained, the functional impact of the acute exercise bout was very transient, with the mitogen response becoming normal within 30 min. It is thus most unlikely that the observed TABLE
Before exercise
29,060+ 38,433+ 39,609-t
16,735” 11,896t 11,268t”
cpm
5 min After exercise
30 min After exercise
28,525-t 18,335 33,810+13,595 38,876+12,756”
33,209-t 16,041 37,278+ 14,776 49,101k18,660b
A. Effects of acute exercise: values differing symbols differ significantly
with from
differing letters differ each other (P < 0.05).
change in immune function would have adverse clinical implications. Humoral Immune Function In contrast to a previous report (7), our data showed no acute exercise-induced reduction of the in vitro production of either IgG or IgM, regardless of the training regimen. The larger depression of immune response observed in the experiments of Hedfors et al. (7) may reflect their use of cell suspensions that were twice the concentration (2 X lo6 cells/ml) as those in the present study. Thus exercise-induced trends may have been magnified. Their subjects were also less well trained so that the bout of acute exercise may have been more stressful for them. Limitations One important limitation to this exercise immunology study, as in most other reports on human subjects, is that all observations of lymphocyte proportions and function were based on specimens of peripheral blood. Transient increases of peripheral lymphocyte counts could arise through an exercise-induced mobilization of sequestrated cells, and there can be no guarantee that either cell proportions or function as observed in peripheral venous blood are representative of other parts of the circulation. Moreover, measurements of function made in vitro provide only a general indication of responses in vivo. The importance of the transient changes observed in this and other exercise immunology studies has not been established. Increase of Training Volume The two methods of quantitating heavy training (distance and volume) ensured that the subjects did not increase distance at the expense of intensity. The 38% in* . 1
1
. 1
1 0.
1
7. Effect of 30 min of exercise at 80% of maximal oxygen vataRe on poReweed mttogen-vaaucea
Ig synthesis following Bl, HT, and B2 IgG, rig/ml Training Regimen
Bl HT B2
IgM,
mg/ml
Before exercise
n
5 min After exercise
n
30 min After exercise
n
Before exercise
n
5 min After exercise
n
30 min After exercise
n
644+656*t 537+412*a 884&433-j-
10 9 10
913k843 488k367” 847+555
8 10 10
7Olk851 904zk635b 828+475
9 9 10
73O-t600* 585k445*a 1,178+626”f
10 9 10
765+663 589+319” 1,052+360
8 10 10
836k581 908k617b 1,098+663
9 9 10
Values are means + SD; n, no. of men. Ig, immunoglobulin. Effects of acute exercise: values with differing letters differ other (P < 0.05). Effects of training (rest): values with differing symbols differ significantly from each other (P < 0.05).
significantly
from
each
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IMMUNE
RESPONSE
TO ACUTE
crease of average training volume was substantial, resulting in a significant increase in fatigue as assessed by psycho1 .ogical ev aluation (unpublished data). Because of fatigue, most of the runn ers were unable to repeat their initial training schedules, as reflected in the lower training volumes averaged during B2. For most of the runners, the 3 wk of heavy training was representative of a “peak” training schedule used in their ‘‘peak and taper” approach to competition. Conclusions
On the basis of this small sample and the specific in vitro measurements of immunity employed, individuals who are well trained can undertake a 30-min bout of acute exercise with at most a minimal and short-lived . suppression of cell-mediated immunity. Such changes are exacerbated by a deliberate increase of training, but they remain transient in nature and within the physiological rather than the pathological range of stress responses. I‘f the volume 0 If endu rance training is determined by the i.ndividua .l rather than an overambitious coach, there seems little danger of causi ng distu rbances of cell- media ted im munity that will have adverse 1clinical consequences. We thank Dr. Paul Corey for advice on statistical aspects of this study. We also thank the Defence and Civil Institute of Environmental Medicine and the Faculty of Medicine, University of Toronto, for the use of special laboratory facilities. This research was supported in part by a research grant from the United States Olympic Committee. Address for reprint requests: T. J. Verde, Graduate Hospital Human Performance and Sports Medicine Center, 200 W. Lancaster Ave., Wayne, PA 19087. Received 10 January 1991; accepted in final form 27 April 1992. REFERENCES 1. BERK, L. KRAMER,
S., D. NIEMAN, S. A. TAN, S. NEHLSEN-CANNARELLA, J. W. C. EBY, AND M. OWENS. Lymphocyte subset changes during acute maximal exercise (Abstract). Med. Sci. Sports Exercise
18: 706, 1986. 2. BRAHMI, Z., J.
E. THOMAS, M. PARK, AND I. R. G. DOWDESWELL. The effect of acute exercise on natural killer cell activity of trained and sedentary human subjects. J. Clin. Immunol. 5: 321-328,1985. 3. DESHAZO, R. D., M. LOPEZ, AND J. E. SALVAGGIO. Use and interpretation of diagnostic immunologic laboratory tests. J. Am. Med. Assoc. 258: 3011-3031, 1987. 4. DEUSTER, P. A., A. M. CURIALE,
M. L. COWAN,
AND
F. D. FINKEL-
EXERCISE
AND HEAVY
TRAINING
1499
Exercise-induced changes in populations of peripheral blood mononuclear cells. Med. Sci. Sports Exercise 20: 276-280, 1988. DILL, D. B. Oxygen used in horizontal and grade walking and running on the treadmill. J. Appl. Physiol. 20: 19-22, 1965. FAUCI, A. S., AND D. C. DALE. The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J. Clin. Inuest. 53: 240MAN.
246, 1974. HEDFORS,
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