Lymphocyte proliferation responses after exercise in men: fitness, intensity, and duration effects B. MAcNEIL, L. HOFFMAN-GOETZ, Departments of Kinesiology and Health Waterloo, Ontario N2L 3G1, Canada
A. KENDALL, M. HOUSTON, Studies, University of Waterloo,
AND
Y. ARUMUGAM
the administered exercise. This has not been the case with much of the previous work in this area and is a after exercise in mea: jitness, intensity, and duratioln effects. J. major reason for the incomparability of reported findAppl. Physiol. 70(l): 179-185, 1991.--This study investigated ings. In the past, studies have included such exercise as 5 the effects of intensity and duration of exercise on lymphocyte min of stair climbing (8), 10 min of moderate cycle erproliferation asa measureof immunologic function in men of gometry (13), brief exhaustive exercise (33), and mardefined fitness. Three fitness groups-low [maximal 0, uptake (9). More recent studies have incorpo(90~~~) = 44.9t 1.5 ml O2 kg-‘. mine1and sedentary], moder- athon running rated much better control over the length and intensity ate Cvo2 max = 55.2& 1.6 ml O2 kg-’ min-l and recreationally active), and high (oo,,, = 63.3 t 1.8 m1.O24kg-l min-l and of the exercise test (25,35); however, differences in exerand duration endurance trained)-and a mixed control group (vozmax = cise protocols with respect to intensity 52.4 t 2.3 ml O2 kg-l min-l) participated in the study. Sub- make comparisons across studies difficult. Moreover, jects completed four randomly ordered cycle ergometer rides: studies of short maximal exercise bouts are not set at a ride I,30 min at 65p/, 90~~~~; ride2,60 min at 30% vozmax; ride constant duration, and prolonged exercise protocols 3, 60 min at 75% Vozmsx;and ride 4, 120 min at 65% vo2max. have varied in intensity, thus exposing subjects to unBlood sampleswere obtained at various times before and after equal levels of stress within a study (6,ZO). Coupled with the exercise sessions.Lymphocyte responsesto the T cell mi- this are various levels of subject fitness across and sometogen concanavalin A were determined at each sample time through the incorporation of radiolabeled thymidine ([3H]- times within studies and differences in sampling times measures (16). The purpose of this TdR). Despite differences in resting levels of $H]TdR uptake, and immunologic a consistent depressionin mitogenesiswas present 2 h after study was to characterize the effects of acute bouts of an exercise bout in all fitness groups. The magnitude of the exercise on immune function when intensity and durareduction in T cell mitogenesiswas not affected by an increase tion were independently varied in subjects of defined in exercise duration. A trend toward greater reduction was fitness levels. A description of the time course of immupresent in the highly fit group when exercise intensity was nologic events following exercise was sought. MACNEIL, B.,L. HOFFMAN-GOETZ,A. KENDALL, M. HousTON,AND Y. ARUMUGAM.Lymphocyte proliferation responses
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increased. The reduction in lymphocyte proliferation to the concanavalin A mitogen after exercisewas a short-term phenomenon with recovery to resting (preexercise) values 24 h after cessationof the work bout. Thesedata suggestthat single sessionsof submaximal exercise transiently reduce lym-
phocyte function in men and that this effect occurs irrespective of subject fitness level. mitogenesis;immunity; T cell; concanavalin A
EXERCISE and the subsequent changes in immune function have been studied previously; however, precise conclusions have been difficult to delineate. Exercise is a form of physiological stress, and the degree of stress encountered is dependent on the intensity and duration of the exercise. Proportional to the level of stress are changes in metabolism, hormone secretion, and cardiovascular adjustments as well as other parameters (10,30). In particular, exercise-induced elevations in plasma catecholamines and corticosteroids are of interest, because these substances have been shown to be immunoregulatory (2, 4, 23, 31, 39). In light of this, meaningful research on exercise and immune function necessitates control over the intensity and duration of
PHYSICAL
0161~7567/91
$1.50 Copyright
METHODS
Subjects. Before the study was begun ethical approval was obtained from the University of Waterloo Office of Human Research and all subjects voluntarily signed an informed consent form. Thirty-two healthy male subjects were recruited on the basis of various health-related criteria and fitness levels. Subjects were placed in one of three fitness groups according to maximal 0, uptake (00 2max) capacity obtained on two exercise tests and recent history of endurance exercise activity. The procedure for the vo2max test has been previously described in detail (17). Subjects with a vog,,, of 40-50 ml. kg-l min-l and a sedentary lifestyle were placed in the low fitness group (LF; n = S), those with a ~oz,,, of 50-60 ml kg-l mine1 and a recreationally active lifestyle were classified as moderately fit (MF; n = 8), and those with a voz,,, >60 ml kg-l min-l and a recent history of endurance training were placed in the high fitness group (HF; n = 8). A group with mixed fitness levels served as control subjects (~2 = 8); this group underwent all experimental procedures of the exercising subjects with the exception of the exercise rides. Each control subject was sampled before and after quiet sit-
0 1991 the American
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179
180
LYMPHOCYTE
1. Anthropometric of subjects
TABLE
Age, Group
LF MF
HF Control
n
8
8 8 8
Yr
.26.1+1.0 25.7t_l.3 23.1t0.5 24.3t0.3
PROLIFERATION
and ji tness measures Height, cm
Weight, kg
176.5t3.3 18O.lt,2.6
71.2t4.4 77.1t2.9 71.4t3.4 78.lt2.1
176.9tl.7 176.6k3.1
ml
l
~%llMX, kg-’ mine1 l
44.9tl.5* 55.2tl.6-t 63.3+_1.8$ 52.4&2.3-t
Values are means t SE; n, no. of subjects. Groups with different symbols are significantly different (P < 0.05).
ting for each of the experimental ride times in the same fashion as the exercising subjects. Exercise tests. Each subject was required to complete four bicycle ergometer exercise tests. The tests consisted of 30 min at 65% Vozmax (ride I), 60 min at 30% 2max (ride Z), 60 min at 75% vo2,,, (ride 3), and 120 min at 65% VO 2max(ride 4). The tests were completed in random order at a rate of one test per week. The exercise tests were started between 9:00 and 9:30 A.M. At various intervals during each test, i702,,, and heart rate were monitored to ensure that the exercise intensity conformed to the prescribed level. Blood sampling. Subjects provided six blood samples for each exercise test. Approximately 50 ml of blood were collected at each sample point. Sample times consisted of 24 h preexercise; immediately preexercise; immediately postexercise; and 30 min, 120 min, and 24 h postexercise. With the exception of the immediately postexercise sample, all blood was obtained by venipuncture of the antecubital vein after the subject rested in a sitting position for 215 min. The immediately postexercise sample was collected as soon as possible after completion of the test ride (typically within 2-3 min). All subjects were to refrain from exercise 24 h before any blood sampling with the exception of the experimental rides. Mononuclear ceZEisobation. The procedure of mononuclear cell isolation has been described in detail elsewhere (1). Briefly, mononuclear cells were isolated from venous blood collected in heparin-treated tubes (Vacutainer, Becton Dickinson Canada). The blood was then mixed with phosphate-buffered saline, layered on a density gradient (Hi&opaque, Sigma Chemical, St. Louis, MO), and centrifuged at 400 g for 30 min. The mononuclear cell layer was removed, washed in phosphatebuffered saline, and resuspended at a final concentration of 5 X lo6 cells/ml in RPM1 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2.5 X 10m5 M 2-mercaptoethanol, 2 mM L-glutamine, 50 U penicillin/ml, 50 U streptomycin/ml, and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid buffer. Mitogenesis. The mononuclear cell suspension (5 X lo6 cells/ml, 100 ~1) was cultured in triplicate in 96-well culture plates (Falcon, Becton Dickinson Canada) with 20 ~1 of medium. Concanavalin A (Con A, Sigma Chemical) was added to each triplicate at optimal dose (20 ~1 at 2.5 pg/ml) on the basis of a dose-response curve determined before the study. Cells were incubated for 72 h in a hul
vo
AND EXERCISE
IN MEN
midified atmosphere of 5% CO2 at 37°C. Eight h before the termination of incubation the cells were pulsed with 0.5 &i of radiolabeled thymidine (yH]TdR, Amersham Canada, Oakville, Ontario) before they were harvested onto glass fiber filters with a semiautomated microculture harvester (Skatron, Lierbyen, Norway). [3H]TdR incorporation was determined using a liquid scintillation counter (Beckman LS 1801, Beckman Instruments, Mississauga, Ontario, Canada) and expressed as counts per minute after correction for unstimulated [3H]TdR uptake. Plasma cortisol. Blood freshly collected in EDTAtreated tubes (Vacutainer, Becton Dickinson Canada) was further treated with EGTA and centrifuged to obtain plasma samples, which were stored at -9OOC until analyzed for cortisol levels. A radioimmunoassay kit (Coat-A-Count Cortisol, Diagnostic Products, Los Angeles, CA) was used to determine plasma cortisol concentrations. Duplicate aliquots of 100 ,ul of plasma were placed in polypropylene tubes precoated with anticortisol antibody, and 1.0 ml of a buffered 1251-cortisol solution was added. The mixture was incubated in a 37OC water bath for 45 min. Tubes were thoroughly aspirated and counted in a Beckman Gamma 5500 counter (Beckman Instruments). The concentration of cortisol was determined from a standard curve by use of a Beckman DP 5500 data reduction system. Statistical anaZysis. An analysis of variance was used to determine the between-groups main effect of fitness level and within-groups effect of ride and sampling time. Post hoc analysis consisted of Student-NewmanKeuls tests and specific contrasts to determine ride differences. An alpha level of 0.05 was accepted as statistically significant. RESULTS
Subject data. Subject data are presented in Table 1. All three exercise groups were different in respect to fitness levels (P < 0.05), except the mixed control group, which was the same as the MF group. Resting (basal) function
(immediately
preexercise).
Baseline function was monitored throughout the 4 wk of the study period (Table 2). Resting baseline function was significantly lower in the LF group than in the control group, which was a mixed fitness group. The MF and HF groups were not significantly lower than the control group. Although somewhat variable, baseline
2. Resting values for [‘H]TdR uptake and cortisol ZeveZs averaged across 24-h preexercise and immediately preexercise samples and across all rides TABLE
Group
n
LF MF HF Control
64 64 64 64
[‘H]TdR
Uptake,
cpm
Cortisol,
39,937+2,418* 54,722+3,439 51,377+,3,657 60,852+_2,759
Values are means t SE; n, no. of subjects. *Significantly from
control
(P -c 0.05).
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pg/dl
18.0*0.82* 17.3t1.16 14.ot_o.71 15.lt0.57
different
LYMPHOCYTE 80
CPM
x10m3
ConA
PROLIFERATION
-T-
-
-r
--r--
181
IN MEN
uptake after 30 min of exercise (ride 1) was not increased by exercise of longer duration (120 min, ride 4; Fig. 5). There was also no differential effect by level of subject fitness. Plasma cortisoZ. A significant main group effect for cortisol was present when all data were averaged across all rides and times (F3 703= 27.9, P c 0.0001). The cortisol values for the LF, M’F, HF, and control groups were 18.1 t 0.64,16.6 t 0.65,13.8 t 0.46, and 12.1 t 0.31 pg/dl,
(Pre levels)
2.6
-r
AND EXERCISE
-
respectively.
--I
Statistically,
all means were different
from each other (P < 0.05). Cortisol data averaged across rides are presented in Fig. 6 for each group. Cortisol levels were elevated immediately postexercise and
Fitness m
1st Ride
m
2nd
Ride
Group m
3rd
Ride
4th
Ride
n * 1. Resting (basal) [SH]TdR incorporation in men of dehned fitness groups in chronological order of ride sequence. Values are means t SE. Ride 1, 30 min ?t 65% vozmax; ride 2, 60 min ?t 30% 0% nlax; ride 3, 60 niin at 75% Vozmax; ride 4,120 min at 65% Vogmsx. See text for description of subject fitness levels. FIG.
returned to or were slightly lower than resting levels by 2 h postexercise. Some variation was present among the groups in the pattern of cortisol responses, but this did
not correspond to changes in the [3H]TdR uptake values. In particular, resting cortisol levels (average of the 24-h preexercise and immediately preexercise samples) were
higher in the LF group than in the control group (Table 2). The resting cortisol levels correlated significantly with the resting levels of [3H]TdR uptake (r = -0.139,
function was relatively consistent within the groups over the study period and no progressive effect of the
P = 0.038). This correlation is not believed to be of any biological significance, given the low r value. An at-
experimental procedure was apparent (Fig. I).
tempt was made to also correlate the peak changes in
E’ect of exercise. An overall group analysis revealed a significant main effect between the groups for exercise v&28 = 3.47, P = 0.0292). All exercise groups had overall mean lymphocyte proliferation levels lower than the control group (Fig. 2); however, only the LF group
cortisol after exercise with the peak changes in [3H]TdR incorporation after exercise. No significant correlation
(P -C0.05) reached statistical significance (MF, P = 0.08;
preexercise to 2 h postexercise (r = 0.181, P = 0.057).
HF, P = 0.07). The overall effect of sampling time was also significant (& 140 = 9.08, P < 0.0001). All groups
DISCUSSION
demonstrated a ver$ consistent time pattern during the
study (Fig. 3) because there was no group X time interaction (P = 0.46). Incorporation of [3H]TdR was reduced immediately after an exercise bout and was not further
changed or recovered slightly 30 min postexercise. On all occasions the greatest drop in mitogenesis occurred 2 h after exercise (P c 0.05) with nearly complete recovery
by 24 h postexercise. The overall ride effect was not significant (& 83= 2.37, P = 0.0763). Because the ride analy-
was present for the change in cortisol values from imme-
diately preexercise to immediately change in [3H]TdR incorporation
postexercise and the from immediately
The major finding of this study is that after acute exercise the T cell mitogen response was clearly suppressed and reached the lowest level 2 h after the exer-
cise, with nearly complete recovery by 24 h postexercise. Although mitogenesis is an in vitro measure, this stimulation process is similar to the antigen-specific response
in terms
of requiring
the participation
of specialized
sis included the control group and two resting samples,
we believed that the ride effect could be better analszed with the use of specific contrasts on the change in PHITdR uptake seen with each ride. A delta score was calculated from the difference between the 2-h postexercise sample and the immediately preexercise or baseline sample to normalize for resting levels of mitogenesis and levels of mitogenesis due to the consis tent d.epression of pro lliferation 2 h postexercise. These delta scores were then used to make specific comparisons between groups for ride differences. Intensity of exercise (ride 2 vs. ride 3). At a set duration of 60 min, increasing the intensity of exercise from 30% vo 2max (ride 2) to 75% 602max (ride 3) resulted in a greater, although not statistically significant, decline in proliferation (Fig. 4). There was also a trend (NS) for subjects to be more severely affected as their fitness level increased. Duration of exercise (ride 1 vs. ride 4). At a constant intensity of 65% 7(702 max,the extent of decline in [3H]TdR
CPM x10e3 (Overall -
70
ConA
Means)
2.5
60 50 40 30 20 10 0
i LF
MF
Fitness
HF
C
Group
FIG. 2. Mean r3H]TdR incorporation in men of defined fitness groups collapsed across all rides and times. Values are means f SE. t P -C 0.05 compared with controls. *Significant a
Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
182
LYMPHOCYTE
CPM x10-’ -
80
ConA
PROLIFERATION
AND
(Low Fit)
ConA
60
40
40
20
20
-24hrs
-3min
+3min
+30min
+2hrs
0
+24hra
IN
CPM ~10~~ (Moderate -
80
2.6
60
0
EXERCISE
MEN
Fit)
2.6
-3min
-24hrs
+3min
Time CPM x10s3 (High Fit) -
80
CenA
+30min
+2hrs
+24hrs
Time 60
CPM ~10~~ (Control) T-
2.6
60
60
40
40
20
20
ConA
2.6
J
0
-24hrs
-3min
+3min
+30min
+2hrs
*24hrs
-24hrs
Time FIG.
group
3. Mean [3H]TdR for all time points.
+3Omin
+3min
Time
across all rides and displayed by incorporation in men of defined fitness grou ps averaged with immedi ately preexercise. Values are means + SE. *Significant at P < 0.05 compared
cells (32). Thus in vitro should responsiveness Exercise had
accessory cells and inducing the production of soluble mediators with growth-promoting activity (32). Lectininduced proliferation also reflects T cell growth factordependent expansion of T cell growth factor-reactive T
the T cell response to suitable mitogens reasonably portray the state of immune in vivo. a clear and distinct effect on mitogene-
A CPM X-IO-~ (2h post-pre)
FIG. 4. Reduction in TH]TdR incorporation from resting (basal) values as a function of exercise intensity in men of defined fitness groups. Values (means k SE) are calculated from 2-h postexercise sample minus immediately preexercise sample.
MF
HF
30%
V02max
75%
\jO,
max
C
Fitness Group Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
LYMPHOCYTE
PROLIFERATION
A @PM x10S3 (2h post-pre)
-30
/-
I1
B@B 2.5
-
65
%
i0,
LF
FIG.
min min
max
MF
HF
Fitness values groups. sample
30 120
ConA
-40
/ -L
Group
5. Reduction in [3H]TdR incorporation from resting (basal) as a function of exercise duration in men of defined fitness Values (means -t SE) are calculated from Z-h postexercise minus immediately preexercise sample.
sis as seen in all exercise groups There was, however, an apzparently separate effect aside from exercise at rest. Resting function in the LF group was reduced compared
ug\dL
(Low
AND
EXERCISE
183
IN MEN
with the control group. Cortisol levels were slightly, albeit significantly, higher in the L group than in the control group, and these values were significantly correlated to the resting mitogenesis levels. It is possible that anxiety on the part of the LF subjects, and to a lesser extent in the MF and HF subjects, may be responsible for the lower resting function. The increase in cortisol levels in LF subjects is one physiological measure of psychological stress and supports the argument that the LF group perceived the test situation and exercise SSsions as stressful. It is possible that this stress was reduced to some extent in the MF and HF groups likely because of more frequent exposure to exercise. The laboratory setting may still create some level of anxiety even in those well acquainted with exercise. Previous reports have shown that emotional state clearly influences immune function and that altered endocrine responses are not the only variables responsible for changes in immune function due to emotional state (18). Resti togenesis has been reported to both decrease (15, and increase (36,37) stimulation compared with tary controls after a period of training. Although it decreased immediately after exerci greatest depression in proliferative capacity repe occurred 2 h after the exercise bout. This was the case
Fit)
26
26
20
20
16
16
10
10
6
0
-3min
+Qmin
*3
in
in
+3min
26
6
0
-24hra
-3min
*3Omin
l 3min
Time FIG.
all time
+2thrs
*24hre
0
-24hro
-3min
+3min
+3Qwiin
+2!hrs
Tim
across all rides and displayed by group 6. Mean cortisol levels in men of defined fitness grou ps averaged with immedi ately preexercise. points. Values a re means 2 SE. *Significant at P< 0.05 compared
for
Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
+24hrs
184
LYMPHOCYTE
PROLIFERATION
regardless of exercise intensity or duration. Numerous studies have reported a reduction in mitogenesis immediately after exercise (9, 13, 14, 21, 25), but few have measured a similar time course into recovery. Using phytohemagglutinin as the mitogen, Tvede et al. (35) sampled at similar time points but reported a recovery by 2 h postexercise. Data are available to demonstrate that differences exist in the inherent potencies of the Con A and phytohemagglutinin mitogens to stimulate T cell proliferation (3). These differences as well as unequal doses of mitogen employed in the respective studies may account for some of the apparent discrepancy between the two studies. It is not known whether the downward trend in the mitogenesis observed in the present study continues after 2 h or begins to return to resting levels. Corresponding to the 2-h postexercise sample, an expected decline in the control group was present as a result of diurnal variation; however, this reduction (9,556 cpm) was not statistically significant. Diurnal fluctuations of this magnitude have been demonstrated previously (12, 35). Care was taken to ensure that the experimental procedures were begun at the same time each day and that control and exercise subjects were sampled at the same time to minimize the variation in lymphocyte responsiveness due to diurnal fluctuations (18). Given the significant reduction in lymphocyte proliferation after exercise, it appears that exercise has an effect greater than can be accounted for solely by diurnal variation. It is interesting to note that mitogenesis may recover somewhat 30 min postexercise before falling to its lowest level 2 h postexercise. We have previously shown in these subjects that blood T cell subset number and percentage return to basal values 2 h after exercise (17). This rapid normalization of the T cell subset response has also been observed by other investigators (6,20,22). It is, therefore, unlikely that the changes in proliferative function in T cells are directly explained by numerical shifts. The hormonal changes seen with exercise are reversed for the most part 30 min after exercise (5, 10, 17,19,29), suggesting that the main factor is not a direct effect of hormone fluctuations. In the present study cortisol values were elevated immediately postexercise and were still slightly higher by 30 min postexercise. However, cortisol levels were not different from or lower than resting values by 2 h postexercise. The hormonal changes during exercise may trigger changes in cellular processes including protein synthesis or expression of cell surface receptors for soluble mediators of cell function. The control of growth initiation is external, and it is these external signals that trigger the intracellular machinery to progress from the G1 phase to the S phase of the cell cycle as well as regulate the proliferation process (28). There are many possible factors released during exercise that may provide the external regulating signal. The time course of these events is quite variable. Messenger RNA production in response to an external signal may require 15 min to 6 h to appear after stimulation by the external signal (7, 28, 38). Cortisol has been reported to require 80-120 min before altering protein synthesis rates (24). In this study maximal reduction in mitogenesis was not seen until 2 h after exer-
AND EXERCISE
IN MEN
cise. These kinetics are not outside the above-mentioned ranges for alteration of cell proliferation. However, there was no significant correlation between exerciseinduced changes in cortisol levels immediately postexercise and [3H]TdR incorporation 2 h postexercise. The results of this study suggest that intensity rather than duration is a factor in determining the extent of mitogen impairment. Also the HF subjects appeared to be more prone to immunosuppression after high-intensity exercise. It might be argued that the LF group was less affected by high-intensity exercise because of an already low resting function; however, this is probably not the case for the longer-duration rides where the effect on the LF group was not different from that on the HF group. Oshida et al. (25) also examined trained and untrained subjects but only over one exercise bout (120 min of cycle ergometry at 60% BOB,,). No difference was noted in the extent to which the two groups were affected, which supports the findings obtained here (ride 4; 120 min of cycle ergometry at 65% VO~~.-J. Other studies examining the effects of exercise on mitogenesis offer inconsistent results. Green et al. (11) reported no change in phytohemagglutinin stimulation in marathon runners compared with normal subjects. However, the sampling times varied among the marathon runners from 1 h to 30 days after a marathon. After a heavy training period, Verde (36) found mitogenesis to be suppressed after an acute bout of exercise. Before the heavy training period, acute exercise had no effect on mitogenesis. After a single bout of exhaustive exercise, trained mice had a reduced response to Con A compared with untrained animals (14), while trained rats had less severe suppression than untrained animals (21). In both of these studies, there were large differences in durations of exercise between animal groups, the type of exercise, and the intensity used to exhaust the animals. All these factors make meaningful comparisons very difficult and likely result in the conflicting reports. In summary, cycle ergometry exercise transiently reduces T cell function, which is most severe 2 h after the exercise bout regardless of subject fitness level. This reduction in lymphocyte proliferation cannot be due to altered T cell subsets, because these have returned to normal levels by 30 min after exercise. Although the LF group had a lower baseline function, their T cell response was not as severely affected by high-intensity exercise as the MF and HF groups. Additionally, the duration of exercise had no effect on the extent of immune suppression after exercise. This study employed single isolated bouts of exercise; however, active individuals will often exercise intensely on sequential days. Further investigation on the effects of repetitive bouts of exercise on lymphocyte function is required. It is possible that overlapping bouts of activity before full recovery from a previous exercise session may induce a chronic state of reduced lymphocyte function in certain individuals who engage in successive sessions of exercise. This
study
was supported
by the Department
(Canada) and Natural Sciences and Engineering Canada Grant A7645.
of National
Defense
Research Council of
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LYMPHOCYTE
PROLIFERATION
Address for reprint requests: L. Hoffman-&e@ Dept. of Health Sciences, Faculty of Applied Health Sciences, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Received 3 April 1990; accepted in final form 8 August 1990. REFERENCES
1. BOYUM, A. Isolation of mononuclear cells and granulocytes from blood. &and. J. CZin. Lab. Invest. Suppl. 97: 77-89, 1968. 2. BURMAN, K. D., E. W. FERGUSON, Y. Y. DJUN, L. WARTOFSKY, AND K. LATHAM. Beta receptors in peripheral mononuclear cells increase acutely with exercise. Acta Endmrinoh 109: 563~568,1985. 3. COFFEY, R. G., E. M. HADDEN, AND J. W. HADDEN. Evidence for cyclic GMP and calcium mediation of lymphocyte activation by mitogens. J. ImmunoC 119: 138?-1394,19??. 4. CRARY, B., M. BORYSENKO, D. C. SUTHERLAND, I. KUTZ, J. 2. BORYSENKO, AND H. BENSON. Decrease in mitogen responsiveness of mononuclear cells from peripheral blood after epinephrine administration in humans. J. Imm;zcnoZ. 130: 694-697,1983. 5. DEUSTER, P. A., G. P. CHROUSOS, A. LUGER, J. E. DEBOLT, L. L. BERNIER, U. H. TROSTMANN, S. B. KYLE,L. C. MONTGOMERY, AND D. L. LORIAUX. Hormonal and metabolic responses of untrained, moderately trained, and highly trained men to three exercise intensities. Metabolism 38: 141-148, 1989. 6. DEUSTER, P. A., A. M. CURIALE, M. L. COWAN, AND F. D. FINKELMAN. Exercise-induced changes in populations of peripheral blood mononuclear cells. Med. Sci. Sports Exercise 20: 276-280, 1988. 7. DINARELLO, C. A. Interleukin-1 and its biologically related cytokines. In: Advances in Immunology, edited by F. J. Dixon. Toronto: Academic, 1989, p. 153-205. 8. EDWARDS, A. J., T. H. BACON, C. A. ELMS,R. VERARDI, M. FELDER, AND S. C. KNIGHT. Changes in the populations of lymphoid cells in human peripheral blood following exercise. Clin. Exp. Immunol. 58: 420-428,1984. 9. ESKOLA, J., 0. RUUSKANEN, E. SOPPI, M. K. VILJANEN, M. JARVINEN, H. TOIVONEN, AND K. KOWALAINEN. Effect of sport stress on lymphocyte transformation and antibody formation. CZin. Exp. Immunol. 32: 339-345,19?8. 10. GALBO, H., J. J. HOLST,AND N. J. CHRISTENSEN. Glucagon and plasma catecholamine responses to graded and prolonged exercise in man. J. Appl. Physiol. 38: 70-76, 1975. 11, GREEN, R. L., S. S. KAPLAN, B. S. RABIN, C. L. STANITSKI, AND U. ZDZIARSKI. Immune function in marathon runners. Ann. Allergy 47: 73-75,198l. 12. HAUS, E., D. J. LAKATUA, J. SWOYER, AND L. SACKETT-LUNDEEN. Chronobiology in hematology and immunology. Am. 9. Anat. 168: 46?-51?,1983. 13. HEDFORS,
E., G. HOLM,AND B. OHNELL. Variations of blood lymphocytes during work studied by cell surface markers, DNA synthesis and cytotoxicity. CZin. Exp. Immunol. 24: 328-335, 1976. 14. 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.
15, HOUSTON, H. The Eflect of Acute and Chronic Exercise on Immune Function in Man (MSc dissertation). Waterloo, Ontario, Canada: Univ. of Waterloo, 1987. 16. KEAST, D., K. CAMERON, AND A. R. MORTON. Exercise and the immune response. sms Med. 5: 248-267,1988. 17. KENDALL, A., L. HOFFMAN-GOETZ, M. HOUSTON, B. MACNEIL,AND Y. ARUMUGAM. Exercise and blood lymphocyte subset responses: intensity, duration and subject fitness effects. J. Appl. PhysioZ. 69: 251-260,199O. 18. KIECOLT-GLASER, J., AND R. GLASER. Methodological issues in behavioral immunology research with humans. Brain Behav. Immunity
2: 6?-78,1988.
19. KJAER, M. Epinephrine and some other hormonal responses to exercise in man: with special reference to physical training. In!. J. Sports Med. 10: 2-15,1989.
9ND EXERCISE IN MEN
185
20. LEWICKI, R., H. TCHORZEWSKI, E. MAJEWSKA, 2. NOWAK, AND Z. BAJ. Effect of maximal physical exercise on T-lymphocyte subpopulations and on interleukin-1 (11-l) and interleukin-2 (B-2) production in vitro. I&. J. Sports Med. 9: 114-117, 1988. 21. MAHON, M. P., AND M. R. YOUNG. Immune parameters of untrained or exercise-trained rats after exhaustive exercise. J. AppZ. Physiol. 66: 282-28?,1989. cm Lb. MCCARTHY, D. A., AND M. M. DALE.The leukocytosis of exercise. A review and model. Sports Med. 6: 333-363,1988. 23. MELMON, K. L., H. R. BOURNE, Y. WEINSTEIN, G. M. SHEARER, J. KRAM, AND S. BAUMINGER. Hemolytic plaque formation by leukocytes in vitro: control by vasoactive hormones. J. Clin. Invest. 53: 13-21,19?4. 24. MUNCK, A., A. N~RAY-FEJES-T~TH, AND P. M. GUYRE. Mechanisms of glucocorticoid actions on the immune system. In: Hormones and Immunity, edited by I. Berczi and K. Kovacs. Lancaster, UK: MTP, 1986, p. 20-37. 25. OSHIDA, Y., K. YAMANOUCHI, S. HAYAMIZU, AND Y. SATO. Effect of acute physical exercise on lymphocyte subpopulations in trained and untrained subjects. Int. J Sports Med. 9: 13?-140,1988. 26. PAHLAVANI, M. A., T. H. CHEUNG, J. A. CHESKY, AND A. RICHARDSON. Influence of exercise on the immune function of rats of various ages. J. Appl. Physiol. 64: 1997-2001, 1988. 27. PAPA, S., M. VITALE, G. MAZZOTTI, L. M. NERI, G. MONTI, AND F. A. MANZOLI. Impaired lymphocyte stimulation induced by long term training. ImmunoZ. Lett. 22: 29-33, 1989. 28. PARDEE, A. B. G1 events and regulation of cell proliferation. Science Wash. DC 246: 603-608,1989. 29. ROBERTSON, A. J., K. C. R. B. RAMESAR, R. C. Poms, J. H. GIBBS, M. C. K. BROWNING, R. A. BROWN, P. C. HAYES, AND J. SWANSONBECK. The effect of strenuous physical exercise on circulating blood lymphocytes and serum cortisol levels. J. CZin. Immunol. 5: 53-5?,1981. 30. ROWELL, L. B. Human Circulation Regulation During Physical Stress. New York: Oxford Univ. Press, 1986, p. 213-256. 31. SAXON, A., R. H. STEVENS, S. J. RAMER, P. J. CLEMENTS, AND D. T. Y. Yu. Glucocorticoids administered in vivo inhibit human suppressor T lymphocyte function and diminish B lymphocyte responsiveness in in vitro immunoglobulin synthesis. J. CZin. Invest. 61: 92%930,1978. 32. SEVERINSON, E., AND E.-L. LARSSON. Lymphocyte responses to polyclonal B and T cell activators. In: Handbook of Experimental Immunology, edited by D. M. Weir. Boston, MA: Blackwell, 1986, p. 63.1-63.18. 33. SOPPI, E., P. VARJO, J. ESKOLA, AND L. A. LAITINEN. Effects of strenuous physical stress on circulating lymphocyte number and function before and after training. J. Clin. Lab. ImmunoZ. 8: 43-46, 1982. 34. TAVADIA, H. B., K. A. FLEMING, P. D. HUME, AND H. W. SIMPSON. Circadian rhythmicity of human plasma cortisol and PHA-induced lymphocyte transformation. CZin. Exp. ImmunoZ. 22: 190193,19?5.
35. TVEDE, N., B. K. PEDERSEN, F. R. HANSEN, T. BENDIX, L. D. CHRISTENSEN, H. GALBO, AND J, HALKJAER-KRISTENSEN. Effect of physical exercise on blood mononuclear cell subpopulations and in vitro proliferative responses. &and. J. Immunol. 29: 383-389,1989. 36. VERDE, T. J. The Effects of Acute Exercise and Heavy Training on Immune Function in Elite Athletes (PhD dissertation). Toronto, Ontario, Canada: Univ. of Toronto, 1990. 37. WATSON, R. R., S. MORIGUCHI, J. C. JACKSON, L. WERNER, J. H. WILMORE, AND B. J. FREUND. Modification of cellular immune functions in humans by endurance training during @-adrenergic blockade with atenolol or propranolol. Med. Sci. Sports Exercise 18: 95-100,1986. 38. WEISS, A., AND J. B. IMBODEN. Cell surface molecules and early events involved in human T lymphocyte activation. In: Advances in Immunology, edited by F. J. Dixon. Toronto: Academic, 1987, p. l-38.
39. Yu, D. T. Y., P. J. CLEMENTS, AND C. M. PEARSON. Effect of corticosteroids on exercise induced lymphocytosis. CZin. Exp. Immunol. 28: 326-331,19??.
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A. KENDALL, M. HOUSTON, Studies, University of Waterloo,
AND
Y. ARUMUGAM
the administered exercise. This has not been the case with much of the previous work in this area and is a after exercise in mea: jitness, intensity, and duratioln effects. J. major reason for the incomparability of reported findAppl. Physiol. 70(l): 179-185, 1991.--This study investigated ings. In the past, studies have included such exercise as 5 the effects of intensity and duration of exercise on lymphocyte min of stair climbing (8), 10 min of moderate cycle erproliferation asa measureof immunologic function in men of gometry (13), brief exhaustive exercise (33), and mardefined fitness. Three fitness groups-low [maximal 0, uptake (9). More recent studies have incorpo(90~~~) = 44.9t 1.5 ml O2 kg-‘. mine1and sedentary], moder- athon running rated much better control over the length and intensity ate Cvo2 max = 55.2& 1.6 ml O2 kg-’ min-l and recreationally active), and high (oo,,, = 63.3 t 1.8 m1.O24kg-l min-l and of the exercise test (25,35); however, differences in exerand duration endurance trained)-and a mixed control group (vozmax = cise protocols with respect to intensity 52.4 t 2.3 ml O2 kg-l min-l) participated in the study. Sub- make comparisons across studies difficult. Moreover, jects completed four randomly ordered cycle ergometer rides: studies of short maximal exercise bouts are not set at a ride I,30 min at 65p/, 90~~~~; ride2,60 min at 30% vozmax; ride constant duration, and prolonged exercise protocols 3, 60 min at 75% Vozmsx;and ride 4, 120 min at 65% vo2max. have varied in intensity, thus exposing subjects to unBlood sampleswere obtained at various times before and after equal levels of stress within a study (6,ZO). Coupled with the exercise sessions.Lymphocyte responsesto the T cell mi- this are various levels of subject fitness across and sometogen concanavalin A were determined at each sample time through the incorporation of radiolabeled thymidine ([3H]- times within studies and differences in sampling times measures (16). The purpose of this TdR). Despite differences in resting levels of $H]TdR uptake, and immunologic a consistent depressionin mitogenesiswas present 2 h after study was to characterize the effects of acute bouts of an exercise bout in all fitness groups. The magnitude of the exercise on immune function when intensity and durareduction in T cell mitogenesiswas not affected by an increase tion were independently varied in subjects of defined in exercise duration. A trend toward greater reduction was fitness levels. A description of the time course of immupresent in the highly fit group when exercise intensity was nologic events following exercise was sought. MACNEIL, B.,L. HOFFMAN-GOETZ,A. KENDALL, M. HousTON,AND Y. ARUMUGAM.Lymphocyte proliferation responses
l
l
l
l
l
l
increased. The reduction in lymphocyte proliferation to the concanavalin A mitogen after exercisewas a short-term phenomenon with recovery to resting (preexercise) values 24 h after cessationof the work bout. Thesedata suggestthat single sessionsof submaximal exercise transiently reduce lym-
phocyte function in men and that this effect occurs irrespective of subject fitness level. mitogenesis;immunity; T cell; concanavalin A
EXERCISE and the subsequent changes in immune function have been studied previously; however, precise conclusions have been difficult to delineate. Exercise is a form of physiological stress, and the degree of stress encountered is dependent on the intensity and duration of the exercise. Proportional to the level of stress are changes in metabolism, hormone secretion, and cardiovascular adjustments as well as other parameters (10,30). In particular, exercise-induced elevations in plasma catecholamines and corticosteroids are of interest, because these substances have been shown to be immunoregulatory (2, 4, 23, 31, 39). In light of this, meaningful research on exercise and immune function necessitates control over the intensity and duration of
PHYSICAL
0161~7567/91
$1.50 Copyright
METHODS
Subjects. Before the study was begun ethical approval was obtained from the University of Waterloo Office of Human Research and all subjects voluntarily signed an informed consent form. Thirty-two healthy male subjects were recruited on the basis of various health-related criteria and fitness levels. Subjects were placed in one of three fitness groups according to maximal 0, uptake (00 2max) capacity obtained on two exercise tests and recent history of endurance exercise activity. The procedure for the vo2max test has been previously described in detail (17). Subjects with a vog,,, of 40-50 ml. kg-l min-l and a sedentary lifestyle were placed in the low fitness group (LF; n = S), those with a ~oz,,, of 50-60 ml kg-l mine1 and a recreationally active lifestyle were classified as moderately fit (MF; n = 8), and those with a voz,,, >60 ml kg-l min-l and a recent history of endurance training were placed in the high fitness group (HF; n = 8). A group with mixed fitness levels served as control subjects (~2 = 8); this group underwent all experimental procedures of the exercising subjects with the exception of the exercise rides. Each control subject was sampled before and after quiet sit-
0 1991 the American
l
l
l
l
Physiological
l
Society
Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
179
180
LYMPHOCYTE
1. Anthropometric of subjects
TABLE
Age, Group
LF MF
HF Control
n
8
8 8 8
Yr
.26.1+1.0 25.7t_l.3 23.1t0.5 24.3t0.3
PROLIFERATION
and ji tness measures Height, cm
Weight, kg
176.5t3.3 18O.lt,2.6
71.2t4.4 77.1t2.9 71.4t3.4 78.lt2.1
176.9tl.7 176.6k3.1
ml
l
~%llMX, kg-’ mine1 l
44.9tl.5* 55.2tl.6-t 63.3+_1.8$ 52.4&2.3-t
Values are means t SE; n, no. of subjects. Groups with different symbols are significantly different (P < 0.05).
ting for each of the experimental ride times in the same fashion as the exercising subjects. Exercise tests. Each subject was required to complete four bicycle ergometer exercise tests. The tests consisted of 30 min at 65% Vozmax (ride I), 60 min at 30% 2max (ride Z), 60 min at 75% vo2,,, (ride 3), and 120 min at 65% VO 2max(ride 4). The tests were completed in random order at a rate of one test per week. The exercise tests were started between 9:00 and 9:30 A.M. At various intervals during each test, i702,,, and heart rate were monitored to ensure that the exercise intensity conformed to the prescribed level. Blood sampling. Subjects provided six blood samples for each exercise test. Approximately 50 ml of blood were collected at each sample point. Sample times consisted of 24 h preexercise; immediately preexercise; immediately postexercise; and 30 min, 120 min, and 24 h postexercise. With the exception of the immediately postexercise sample, all blood was obtained by venipuncture of the antecubital vein after the subject rested in a sitting position for 215 min. The immediately postexercise sample was collected as soon as possible after completion of the test ride (typically within 2-3 min). All subjects were to refrain from exercise 24 h before any blood sampling with the exception of the experimental rides. Mononuclear ceZEisobation. The procedure of mononuclear cell isolation has been described in detail elsewhere (1). Briefly, mononuclear cells were isolated from venous blood collected in heparin-treated tubes (Vacutainer, Becton Dickinson Canada). The blood was then mixed with phosphate-buffered saline, layered on a density gradient (Hi&opaque, Sigma Chemical, St. Louis, MO), and centrifuged at 400 g for 30 min. The mononuclear cell layer was removed, washed in phosphatebuffered saline, and resuspended at a final concentration of 5 X lo6 cells/ml in RPM1 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2.5 X 10m5 M 2-mercaptoethanol, 2 mM L-glutamine, 50 U penicillin/ml, 50 U streptomycin/ml, and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid buffer. Mitogenesis. The mononuclear cell suspension (5 X lo6 cells/ml, 100 ~1) was cultured in triplicate in 96-well culture plates (Falcon, Becton Dickinson Canada) with 20 ~1 of medium. Concanavalin A (Con A, Sigma Chemical) was added to each triplicate at optimal dose (20 ~1 at 2.5 pg/ml) on the basis of a dose-response curve determined before the study. Cells were incubated for 72 h in a hul
vo
AND EXERCISE
IN MEN
midified atmosphere of 5% CO2 at 37°C. Eight h before the termination of incubation the cells were pulsed with 0.5 &i of radiolabeled thymidine (yH]TdR, Amersham Canada, Oakville, Ontario) before they were harvested onto glass fiber filters with a semiautomated microculture harvester (Skatron, Lierbyen, Norway). [3H]TdR incorporation was determined using a liquid scintillation counter (Beckman LS 1801, Beckman Instruments, Mississauga, Ontario, Canada) and expressed as counts per minute after correction for unstimulated [3H]TdR uptake. Plasma cortisol. Blood freshly collected in EDTAtreated tubes (Vacutainer, Becton Dickinson Canada) was further treated with EGTA and centrifuged to obtain plasma samples, which were stored at -9OOC until analyzed for cortisol levels. A radioimmunoassay kit (Coat-A-Count Cortisol, Diagnostic Products, Los Angeles, CA) was used to determine plasma cortisol concentrations. Duplicate aliquots of 100 ,ul of plasma were placed in polypropylene tubes precoated with anticortisol antibody, and 1.0 ml of a buffered 1251-cortisol solution was added. The mixture was incubated in a 37OC water bath for 45 min. Tubes were thoroughly aspirated and counted in a Beckman Gamma 5500 counter (Beckman Instruments). The concentration of cortisol was determined from a standard curve by use of a Beckman DP 5500 data reduction system. Statistical anaZysis. An analysis of variance was used to determine the between-groups main effect of fitness level and within-groups effect of ride and sampling time. Post hoc analysis consisted of Student-NewmanKeuls tests and specific contrasts to determine ride differences. An alpha level of 0.05 was accepted as statistically significant. RESULTS
Subject data. Subject data are presented in Table 1. All three exercise groups were different in respect to fitness levels (P < 0.05), except the mixed control group, which was the same as the MF group. Resting (basal) function
(immediately
preexercise).
Baseline function was monitored throughout the 4 wk of the study period (Table 2). Resting baseline function was significantly lower in the LF group than in the control group, which was a mixed fitness group. The MF and HF groups were not significantly lower than the control group. Although somewhat variable, baseline
2. Resting values for [‘H]TdR uptake and cortisol ZeveZs averaged across 24-h preexercise and immediately preexercise samples and across all rides TABLE
Group
n
LF MF HF Control
64 64 64 64
[‘H]TdR
Uptake,
cpm
Cortisol,
39,937+2,418* 54,722+3,439 51,377+,3,657 60,852+_2,759
Values are means t SE; n, no. of subjects. *Significantly from
control
(P -c 0.05).
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pg/dl
18.0*0.82* 17.3t1.16 14.ot_o.71 15.lt0.57
different
LYMPHOCYTE 80
CPM
x10m3
ConA
PROLIFERATION
-T-
-
-r
--r--
181
IN MEN
uptake after 30 min of exercise (ride 1) was not increased by exercise of longer duration (120 min, ride 4; Fig. 5). There was also no differential effect by level of subject fitness. Plasma cortisoZ. A significant main group effect for cortisol was present when all data were averaged across all rides and times (F3 703= 27.9, P c 0.0001). The cortisol values for the LF, M’F, HF, and control groups were 18.1 t 0.64,16.6 t 0.65,13.8 t 0.46, and 12.1 t 0.31 pg/dl,
(Pre levels)
2.6
-r
AND EXERCISE
-
respectively.
--I
Statistically,
all means were different
from each other (P < 0.05). Cortisol data averaged across rides are presented in Fig. 6 for each group. Cortisol levels were elevated immediately postexercise and
Fitness m
1st Ride
m
2nd
Ride
Group m
3rd
Ride
4th
Ride
n * 1. Resting (basal) [SH]TdR incorporation in men of dehned fitness groups in chronological order of ride sequence. Values are means t SE. Ride 1, 30 min ?t 65% vozmax; ride 2, 60 min ?t 30% 0% nlax; ride 3, 60 niin at 75% Vozmax; ride 4,120 min at 65% Vogmsx. See text for description of subject fitness levels. FIG.
returned to or were slightly lower than resting levels by 2 h postexercise. Some variation was present among the groups in the pattern of cortisol responses, but this did
not correspond to changes in the [3H]TdR uptake values. In particular, resting cortisol levels (average of the 24-h preexercise and immediately preexercise samples) were
higher in the LF group than in the control group (Table 2). The resting cortisol levels correlated significantly with the resting levels of [3H]TdR uptake (r = -0.139,
function was relatively consistent within the groups over the study period and no progressive effect of the
P = 0.038). This correlation is not believed to be of any biological significance, given the low r value. An at-
experimental procedure was apparent (Fig. I).
tempt was made to also correlate the peak changes in
E’ect of exercise. An overall group analysis revealed a significant main effect between the groups for exercise v&28 = 3.47, P = 0.0292). All exercise groups had overall mean lymphocyte proliferation levels lower than the control group (Fig. 2); however, only the LF group
cortisol after exercise with the peak changes in [3H]TdR incorporation after exercise. No significant correlation
(P -C0.05) reached statistical significance (MF, P = 0.08;
preexercise to 2 h postexercise (r = 0.181, P = 0.057).
HF, P = 0.07). The overall effect of sampling time was also significant (& 140 = 9.08, P < 0.0001). All groups
DISCUSSION
demonstrated a ver$ consistent time pattern during the
study (Fig. 3) because there was no group X time interaction (P = 0.46). Incorporation of [3H]TdR was reduced immediately after an exercise bout and was not further
changed or recovered slightly 30 min postexercise. On all occasions the greatest drop in mitogenesis occurred 2 h after exercise (P c 0.05) with nearly complete recovery
by 24 h postexercise. The overall ride effect was not significant (& 83= 2.37, P = 0.0763). Because the ride analy-
was present for the change in cortisol values from imme-
diately preexercise to immediately change in [3H]TdR incorporation
postexercise and the from immediately
The major finding of this study is that after acute exercise the T cell mitogen response was clearly suppressed and reached the lowest level 2 h after the exer-
cise, with nearly complete recovery by 24 h postexercise. Although mitogenesis is an in vitro measure, this stimulation process is similar to the antigen-specific response
in terms
of requiring
the participation
of specialized
sis included the control group and two resting samples,
we believed that the ride effect could be better analszed with the use of specific contrasts on the change in PHITdR uptake seen with each ride. A delta score was calculated from the difference between the 2-h postexercise sample and the immediately preexercise or baseline sample to normalize for resting levels of mitogenesis and levels of mitogenesis due to the consis tent d.epression of pro lliferation 2 h postexercise. These delta scores were then used to make specific comparisons between groups for ride differences. Intensity of exercise (ride 2 vs. ride 3). At a set duration of 60 min, increasing the intensity of exercise from 30% vo 2max (ride 2) to 75% 602max (ride 3) resulted in a greater, although not statistically significant, decline in proliferation (Fig. 4). There was also a trend (NS) for subjects to be more severely affected as their fitness level increased. Duration of exercise (ride 1 vs. ride 4). At a constant intensity of 65% 7(702 max,the extent of decline in [3H]TdR
CPM x10e3 (Overall -
70
ConA
Means)
2.5
60 50 40 30 20 10 0
i LF
MF
Fitness
HF
C
Group
FIG. 2. Mean r3H]TdR incorporation in men of defined fitness groups collapsed across all rides and times. Values are means f SE. t P -C 0.05 compared with controls. *Significant a
Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
182
LYMPHOCYTE
CPM x10-’ -
80
ConA
PROLIFERATION
AND
(Low Fit)
ConA
60
40
40
20
20
-24hrs
-3min
+3min
+30min
+2hrs
0
+24hra
IN
CPM ~10~~ (Moderate -
80
2.6
60
0
EXERCISE
MEN
Fit)
2.6
-3min
-24hrs
+3min
Time CPM x10s3 (High Fit) -
80
CenA
+30min
+2hrs
+24hrs
Time 60
CPM ~10~~ (Control) T-
2.6
60
60
40
40
20
20
ConA
2.6
J
0
-24hrs
-3min
+3min
+30min
+2hrs
*24hrs
-24hrs
Time FIG.
group
3. Mean [3H]TdR for all time points.
+3Omin
+3min
Time
across all rides and displayed by incorporation in men of defined fitness grou ps averaged with immedi ately preexercise. Values are means + SE. *Significant at P < 0.05 compared
cells (32). Thus in vitro should responsiveness Exercise had
accessory cells and inducing the production of soluble mediators with growth-promoting activity (32). Lectininduced proliferation also reflects T cell growth factordependent expansion of T cell growth factor-reactive T
the T cell response to suitable mitogens reasonably portray the state of immune in vivo. a clear and distinct effect on mitogene-
A CPM X-IO-~ (2h post-pre)
FIG. 4. Reduction in TH]TdR incorporation from resting (basal) values as a function of exercise intensity in men of defined fitness groups. Values (means k SE) are calculated from 2-h postexercise sample minus immediately preexercise sample.
MF
HF
30%
V02max
75%
\jO,
max
C
Fitness Group Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.
LYMPHOCYTE
PROLIFERATION
A @PM x10S3 (2h post-pre)
-30
/-
I1
B@B 2.5
-
65
%
i0,
LF
FIG.
min min
max
MF
HF
Fitness values groups. sample
30 120
ConA
-40
/ -L
Group
5. Reduction in [3H]TdR incorporation from resting (basal) as a function of exercise duration in men of defined fitness Values (means -t SE) are calculated from Z-h postexercise minus immediately preexercise sample.
sis as seen in all exercise groups There was, however, an apzparently separate effect aside from exercise at rest. Resting function in the LF group was reduced compared
ug\dL
(Low
AND
EXERCISE
183
IN MEN
with the control group. Cortisol levels were slightly, albeit significantly, higher in the L group than in the control group, and these values were significantly correlated to the resting mitogenesis levels. It is possible that anxiety on the part of the LF subjects, and to a lesser extent in the MF and HF subjects, may be responsible for the lower resting function. The increase in cortisol levels in LF subjects is one physiological measure of psychological stress and supports the argument that the LF group perceived the test situation and exercise SSsions as stressful. It is possible that this stress was reduced to some extent in the MF and HF groups likely because of more frequent exposure to exercise. The laboratory setting may still create some level of anxiety even in those well acquainted with exercise. Previous reports have shown that emotional state clearly influences immune function and that altered endocrine responses are not the only variables responsible for changes in immune function due to emotional state (18). Resti togenesis has been reported to both decrease (15, and increase (36,37) stimulation compared with tary controls after a period of training. Although it decreased immediately after exerci greatest depression in proliferative capacity repe occurred 2 h after the exercise bout. This was the case
Fit)
26
26
20
20
16
16
10
10
6
0
-3min
+Qmin
*3
in
in
+3min
26
6
0
-24hra
-3min
*3Omin
l 3min
Time FIG.
all time
+2thrs
*24hre
0
-24hro
-3min
+3min
+3Qwiin
+2!hrs
Tim
across all rides and displayed by group 6. Mean cortisol levels in men of defined fitness grou ps averaged with immedi ately preexercise. points. Values a re means 2 SE. *Significant at P< 0.05 compared
for
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regardless of exercise intensity or duration. Numerous studies have reported a reduction in mitogenesis immediately after exercise (9, 13, 14, 21, 25), but few have measured a similar time course into recovery. Using phytohemagglutinin as the mitogen, Tvede et al. (35) sampled at similar time points but reported a recovery by 2 h postexercise. Data are available to demonstrate that differences exist in the inherent potencies of the Con A and phytohemagglutinin mitogens to stimulate T cell proliferation (3). These differences as well as unequal doses of mitogen employed in the respective studies may account for some of the apparent discrepancy between the two studies. It is not known whether the downward trend in the mitogenesis observed in the present study continues after 2 h or begins to return to resting levels. Corresponding to the 2-h postexercise sample, an expected decline in the control group was present as a result of diurnal variation; however, this reduction (9,556 cpm) was not statistically significant. Diurnal fluctuations of this magnitude have been demonstrated previously (12, 35). Care was taken to ensure that the experimental procedures were begun at the same time each day and that control and exercise subjects were sampled at the same time to minimize the variation in lymphocyte responsiveness due to diurnal fluctuations (18). Given the significant reduction in lymphocyte proliferation after exercise, it appears that exercise has an effect greater than can be accounted for solely by diurnal variation. It is interesting to note that mitogenesis may recover somewhat 30 min postexercise before falling to its lowest level 2 h postexercise. We have previously shown in these subjects that blood T cell subset number and percentage return to basal values 2 h after exercise (17). This rapid normalization of the T cell subset response has also been observed by other investigators (6,20,22). It is, therefore, unlikely that the changes in proliferative function in T cells are directly explained by numerical shifts. The hormonal changes seen with exercise are reversed for the most part 30 min after exercise (5, 10, 17,19,29), suggesting that the main factor is not a direct effect of hormone fluctuations. In the present study cortisol values were elevated immediately postexercise and were still slightly higher by 30 min postexercise. However, cortisol levels were not different from or lower than resting values by 2 h postexercise. The hormonal changes during exercise may trigger changes in cellular processes including protein synthesis or expression of cell surface receptors for soluble mediators of cell function. The control of growth initiation is external, and it is these external signals that trigger the intracellular machinery to progress from the G1 phase to the S phase of the cell cycle as well as regulate the proliferation process (28). There are many possible factors released during exercise that may provide the external regulating signal. The time course of these events is quite variable. Messenger RNA production in response to an external signal may require 15 min to 6 h to appear after stimulation by the external signal (7, 28, 38). Cortisol has been reported to require 80-120 min before altering protein synthesis rates (24). In this study maximal reduction in mitogenesis was not seen until 2 h after exer-
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cise. These kinetics are not outside the above-mentioned ranges for alteration of cell proliferation. However, there was no significant correlation between exerciseinduced changes in cortisol levels immediately postexercise and [3H]TdR incorporation 2 h postexercise. The results of this study suggest that intensity rather than duration is a factor in determining the extent of mitogen impairment. Also the HF subjects appeared to be more prone to immunosuppression after high-intensity exercise. It might be argued that the LF group was less affected by high-intensity exercise because of an already low resting function; however, this is probably not the case for the longer-duration rides where the effect on the LF group was not different from that on the HF group. Oshida et al. (25) also examined trained and untrained subjects but only over one exercise bout (120 min of cycle ergometry at 60% BOB,,). No difference was noted in the extent to which the two groups were affected, which supports the findings obtained here (ride 4; 120 min of cycle ergometry at 65% VO~~.-J. Other studies examining the effects of exercise on mitogenesis offer inconsistent results. Green et al. (11) reported no change in phytohemagglutinin stimulation in marathon runners compared with normal subjects. However, the sampling times varied among the marathon runners from 1 h to 30 days after a marathon. After a heavy training period, Verde (36) found mitogenesis to be suppressed after an acute bout of exercise. Before the heavy training period, acute exercise had no effect on mitogenesis. After a single bout of exhaustive exercise, trained mice had a reduced response to Con A compared with untrained animals (14), while trained rats had less severe suppression than untrained animals (21). In both of these studies, there were large differences in durations of exercise between animal groups, the type of exercise, and the intensity used to exhaust the animals. All these factors make meaningful comparisons very difficult and likely result in the conflicting reports. In summary, cycle ergometry exercise transiently reduces T cell function, which is most severe 2 h after the exercise bout regardless of subject fitness level. This reduction in lymphocyte proliferation cannot be due to altered T cell subsets, because these have returned to normal levels by 30 min after exercise. Although the LF group had a lower baseline function, their T cell response was not as severely affected by high-intensity exercise as the MF and HF groups. Additionally, the duration of exercise had no effect on the extent of immune suppression after exercise. This study employed single isolated bouts of exercise; however, active individuals will often exercise intensely on sequential days. Further investigation on the effects of repetitive bouts of exercise on lymphocyte function is required. It is possible that overlapping bouts of activity before full recovery from a previous exercise session may induce a chronic state of reduced lymphocyte function in certain individuals who engage in successive sessions of exercise. This
study
was supported
by the Department
(Canada) and Natural Sciences and Engineering Canada Grant A7645.
of National
Defense
Research Council of
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Address for reprint requests: L. Hoffman-&e@ Dept. of Health Sciences, Faculty of Applied Health Sciences, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Received 3 April 1990; accepted in final form 8 August 1990. REFERENCES
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