Lymphocyte submaximal
subset responses to repeated exercise in men
LAURIE HOFFMAN-GOETZ, JANIS RANDALL SIMPSON, NICHOLAS YOGAJOTHI ARUMUGAM, AND MICHAEL E. HOUSTON Departments of Health Studies and Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3GI, Canada
HOFFMAN-GOETZ, LAURIE, JANIS RANDALL SIMPSON, NIE. CHOLAS CIPP, YOGAJOTHI ARUMUGAM, AND MICHAEL HOUSTON. Lymphocyte subset responses to repeated submaximal exercise in men. J. Appl. Physiol. 68(3): 1069-1074,1990.-The
effects of repeated bouts of submaximal cycle ergometry exercise on changes in the percentage of peripheral blood T-lymphocytes, the T-helper/inducer and T-cytotoxic/suppressor subsets, and natural killer (NK) cells were studied in 18 healthy young men who had no history of regular exercise training. Subjects were matched on the basis of maximal O2 uptake and assigned randomly to exercise or control groups, with controls resting quietly during the exercise sessions. The percentage of peripheral blood mononuclear leukocytes that reacted with monoclonal antibodies specific for T-lymphocytes (CD3+ cells), the helper/inducer subset (CD4+ cells) and cytotoxic/suppressor subset (CD8+ cells) of T-lymphocytes, and cells with NK activity (Leu7+ cells) were enumerated by fluorescence-activated flow cytometry for samples obtained immediately before and after exercise on days 1,3, and 5 of a b-day exercise regimen. The results of this study were mixed with decreases in the percentage of T-lymphocytes before vs. after exercise on days 1 and 3 (P c O.OOl), a decrease in the percentage of T-helper/ inducer cells before vs. after exercise on day 3 (P < 0.05), no effect of exercise on the percentage of T-cytotoxic/suppressor cells, and a marked increase in the percentage of NK cells after exercise on days 1 (P < 0.05) and 3 (P < 0.01). The total number of recovered NK cells in the mononuclear leukocyte fraction of blood also increased significantly after exercise on days 1 (P < 0.05) and 3 (P < 0.01). These findings 1) suggest that repeated exposure to submaximal exercise results in consistent increases in the percentage of NK cells, 2) demonstrate that the exercise effects on T-lymphocyte subset percents were variable over time, 3) confirm earlier reports on the impact of a single bout of submaximal exercise on percent and numerical shifts in peripheral blood T-lymphocytes and NK cells, and 4) extend the findings of earlier studies to include individuals of low fitness levels. flow cytometry; monoclonal antibodies; cycle ergometry
YEARS, considerable interest has been directed to the effects of exercise, a replicable and quantifiable stressor, on immune function (see reviews in Refs. 8,14). Understanding the clinical significance of functional changes in the immune system with exercise depends, in part, on the ability to accurately enumerate the component cell populations (e.g., T-lymphocytes) and their various subsets (e.g., T-helper/inducer cells). The introduction of monoclonal antibodies reacting with specific IN RECENT
0161-7567/90
$1.50 Copyright
CIPP,
cell surface antigens, coupled with immunofluorescent flow cytometry analysis, has greatly facilitated the evaluation of exercise-induced changes in immune parameters. For example, the conclusions drawn about the clinical relevance of a finding of no change in the capacity of natural killer (NK) cells to lyse tumor targets immediately after exercise are quite different when this information is combined with the finding of concurrent decreases in the absolute or relative number of NK cells (21) Although enumeration of lymphocyte subsets by flow cytometry is widely used clinically, there are only a limited number of reports enumerating the lymphocyte population shifts with exercise. Generally, the direction of the lymphocyte population changes with exercise is dependent on whether the work intensity is maximal or submaximal. Three recent studies describing a single maximal exercise bout found an increase (6, 16, 17) or no change (2) in the percentage of peripheral blood Tlymphocytes, an increase (2, 5) or decrease (27) in the percentage of NK cells, and an increase in the percentage of the cytotoxic/suppressor T-cell subpopulation (1, 16, 17). A single session of submaximal exercise was associated with a decrease in the percentage of blood T-lymphocytes and T-helper/inducer cells in untrained subjects of moderate fitness [maximal O2 uptake (VOW,,,) of 53.5 t 2.4 ml kg-’ min-‘1 (25) and an increase in the percentage of NK cells in athletes only. In contrast, others have reported an increase in the percentage of blood T-lymphocytes after submaximal exercise in subjects of unspecified fitness (6, 15). Variations in the direction and magnitude of lymphocyte numerical changes with exercise may be the result of the different types of exercise as well as the different fitness levels of the subjects. In the present study, we determined the effects of repeated submaximal exercise on changes in the distribution of peripheral blood Tlymphocytes, T-lymphocyte subsets (helper/inducer and cytotoxic/suppressor), and large granular lymphocytes, including NK cells, as determined by monoclonal antibodies and flow cytometry. We hypothesized that repeated exposure to an exercise stress would abrogate, or at least reduce, the magnitude of the lymphocyte phenotype changes reported after a single session of submaximal work. Additionally, we wanted to ascertain whether the types of lymphocyte numerical changes in-
0 1990 the American
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duced by exercise, which have been reported for moderate and highly fit subjects, also characterized individuals of lower fitness levels. MATERIALS
AND METHODS
Subjects. Eighteen male subjects were recruited from the University student population. These men occasionally participated in recreational activities, but they were not involved in any regular exercise training. The subjects completed an activity and lifestyle profile and signed consent forms approved by the Committee on Human Research. The subjects were considered “untrained” based on their activity profiles and Vozmax (~50 ml. kg-‘. min-‘; see Table 1). . Experimental design. Subjects performed a progressive vo &ax test on an electrically braked cycle ergometer by using a protocol described previously (13). Thereafter, subjects were paired on the basis of a best match with their previous activity and Vozmax values. The subjects were monitored in pairs over a 5-day period in which one of the subjects cycled at a load representing 65% of vozrnax each day for 1 h. This work load was selected because it represents the midpoint of the range prescribed for conditioning intensity for endurance exercise by average persons. The control subject sat beside the exercising subject on another cycle ergometer. Initially, the subject pairs were informed that the decision as to who would exercise and who would rest would be made randomly each day. Our intent was to keep the anticipation of exercise similar for the exercising and control subject pairs. However, once the initial choice was made as to who would exercise, that subject performed the exercise each day for 5 consecutive days. Blood samples were drawn from a forearm vein by venipuncture immedi .ately before and after the exercise bouts on the 1st, 3rd, and 5th consecutive days. Morning blood samples (between 8 and 1OA .M.)at rest were drawn after the subjects had relaxed in a chair for at least 15 min. Postexercise samples were drawn 3 min after the termination of exercise (exercise subjects) or sitting on the cycle ergometer (control subjects). The exercise bouts were carried out in the morning, and at the same time for each subject pair. Experimental procedures. Mononuclear leukocytes were-isolated from heparinized blood by the method of Boyum (4). Briefly, blood was diluted twofold with phosphate-buffered saline (PBS; 0.01 M, pH 7.2), layered over Histopaque (Sigma Chemical, St. Louis, MO), and centrifuged at 400 g for 30 min. The mononuclear interface was collected and washed three times and the volume adjusted to 1 ml in PBS. Mononuclear cell counts were performed manu .ally on a Nikon Optiphot phase-c sontrast microscope, and cell viability was assessed by the trypan blue exclusion technique. Cell viability, after separation on Histopaque, was always >92%. Lymphocyte subpopulations were quantitated by the technique of direct immunofluorescence by using monoclonal antibodies specific for peripheral total T-cells (CD3+), helper/inducer T-cells (CD4+), suppressor/cytotoxic T-cells (CD8+), and large granular lymphocytes, a subset that expresses NK functional activity (Leu7+).
SUBMAXIMAL
EXERCISE
Monoclonal antibodies were obtained from Becton Dickinson and used with a fluorescent-activated cell sorter (Coulter EPICS IV FACS) with an argon laser (488nm wavelength) to excite the fluorescein isothiocyanate(FITC) conjugated monoclonal antibodies. Background autofluorescence was determined for each sample, and an average of 20,000 cells/sample was counted. Labeled cell suspensions were also manually verified by fluorescence microscopy using a field of 100 mononuclear cells. Details for the flow cytometric technique are given in Ortaldo et al. (24), Renzi and Ginns (29), and Becton Dickinson technical report 23-1372-01. Aliquots of whole blood were obtained from the exercising and control subjects before and after the exercise bouts on the lst, 3rd, and 5th days. The blood was deproteinized with 0.6 M perchloric acid, neutralized with 1.0 M NaHC03, and centrifuged, and the supernatant was frozen for later analysis of lactate concentration (19). Blood samples were also analyzed for hematocrit changes. Statistical analysis. Immunological data were analyzed using an SAS repeated measures analysis of variance model (31) to determine differences across time and by group. Paired t tests were done to determine before and after differences on each test day for control and exercised groups. Statistical significance was set at the 0.05 RESULTS
Physical characteristics of the control and exercised subjects before the experiment are shown in Table 1. There were no significant differences between the two groups of subjects for age, body weight, or Tjogmax values. Table 2 shows values for hematocrit, recovered mononuclear leukocyte number, and blood lactate concentration for the two groups of subjects determined in venous blood samples obtained at rest and after exercise bouts on days 1, 3, and 5. Hematocrit percent increased -significantly immediately after exercise at 65% of voarnax, representing a percent increase of 6.5, 5.2, and 3.8% for days 1, 3, and 5, respectively (P < 0.05). Mononuclear leukocyte number increased significantly after exercise on days 1 and 3 (P < 0.05). Mononuclear leukocyte numbers were virtually unchanged from before to after sampling times in the control subjects. Blood lactate concentrations increased significantly before to after exercise on days 1, 3, and 5 for the exercised group (P < 0.001); lactate concentrations were unchanged in the control subjects as a function of time. The changes in the percent distribution of cell populations are presented in Table 3. The percentage of peripheral mononuclear leukocytes that reacted with TABLE 1. Physical characteristics and control subjects Group
n
Control Exercised
9 9
Age, Yr
22.8H.2 24.5k0.9
of the exercised
VO
2max
Weight, kg
l/min
ml kg-’ min-’
74.7k3.5 75.2k4.1
3.46kO.18 3.49k0.12
46.3k2.1 46.426.4
l
l
Values are means k SE.
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TABLE 2. Hematological and lactate concentration before and after exercise over 5 days Parameter
Hematocrit,
%
Mononuclear cells, X lo7 cells/ml Lactate, mM
Day
After
44.1t0.8
Before
After
44.5t0.6 42.5t0.5
45.1t0.5 44.7&0.6*
44.51ko.7 43.5-eo.5*
1.82t0.4 2.39kO.5 0.9tO.l
44.2t0.9 41.9t0.5 1.79kO.3 1.58kO.3
1.75kO.5
1.96k0.3
1.98t0.4
1.86t0.3
2.72t0.5*
1.701ko.4
1.01*0.1
l.OtO. 1
l.OtO.l
1.0tO.l
3.0*0.5?
1.2tO.l
different
Day 5
After
2.04kO.4
Values are means t SE. * P < 0.05 significantly
3
Before
45.420.7 45.8t0.7*
43.0t0.9
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EXERCISE
and exercised subjects
1
Before
Control Exercised Control Exercised Control Exercised
SUBMAXIMAL
values for control
Day
Group
REPEATED
2.620.37
before vs. after values; t P < 0.001 significantly
1.67k0.3
0.9tO.l
1.OkO.l 2.9+0.4-f different before vs. after values. 1.5zkO.2
TABLE 3. Percentage values for CD3+ and percentage of the subsets CD4+ and CDF, for control and exercised subjects before and after exercise over 5 consecutive days Lymphocyte Subset, %
CD3+ CD4+ CD8+
Day
Group
Control Exercised Control Exercised Control Exercised
Before
1
Day
3
Day 5
After
Before
After
Before
7O.lk3.8 72.2t3.1
65.1t4.1 60.3k5.0.f
69.4t3.8 64.0&3.1?
71.3t3.4
37.7t2.3 40.7t3.8
36.6t2.8
71.3t5.0 72.1t3.3 39.7t3.4
19.3t1.5 18.6t2.3
36.4t2.9 18.4k2.4 21.1t3.0
43.7t3.4
37.6k3.2 36.2*3.8$
16.3t2.3 17.7t2.6
15.2t2.0 19.3t2.3
Values are means t SE. * P < 0.05 significantly different before vs. after values; 7 P c 0.001 significantly < 0.01 significantly different before vs. after values.
CD3+ pan T-cell antibody decreased significantly after exercise on days 1 and 3 (P < 0.001) relative to beforeexercise values. Control subjects showed a significant decrease in the percentage of T-lymphocytes at day 5 sampling (P < 0.05). Submaximal exercise induced a small decrease in the percentage of T-helper/inducer cells across days 1, 3, and 5 relative to before-exercise values; however, this decrease in T-helper/inducer cell percent was statistically significant only after the day 3 exercise session (P < 0.01). Control subjects tended to a decrease in the percentage of CD4+ cells at the after sampling points; however, these fluctuations did not reach statistical significance. The percentage of peripheral blood lymphocytes that expressed the CD8+ antigen (i.e., cytotoxic/suppressor subset) was unaffected by exercise; paradoxically, there was a small but significant decrease in the percentage of this subset in control subjects sampled at day 5 (P < 0.05, before vs. after). The percentage of peripheral blood leukocytes that expressed the NK-associated antigen (Leu7+) was markedly affected by exercise with increases of 55.4% (P < 0.05) and 73.0% (P < 0.01) immediately after exercise on days I and 3, respectively (Fig. 1). This trend for an increase after exercise in the percent of Leu7+ cells continued, albeit not significantly, on the final sampling point (day 5 = sample 3). The percentage of Leu7+ cells in peripheral blood was unchanged before vs. after sampling times among control subjects. Table 4 shows the absolute numbers of lymphocytes recovered, by subset, in the mononuclear fraction (i.e., recovered mononuclear cells X subset percent). When the data are expressed this way, there was a significant lymphocytosis for CD3+ and CD4+ cells after exercise on day 3. Figure 2 shows the effect of repeated exercise on the absolute number of Leu7+ lymphocytes. Cells ex-
n
CNTRL-PRE
w
CNTRL-POST
q q
EX-PRE
After
63.1&3.4* 62.9k2.7 32.5t1.9 32.9k2.9 17.5t2.5 13.7zk2.3* 16.4t2.1 16.5t2.5 different before vs. after values; $ P 68.023.1 38.3t2.8 38.1t4.2
EX-POST
*
1
2
3
EXERCISE SAMPLE 1. Changes in percentage of Leu7+ lymphocytes in men before and after exercise. Exercise samples 1-3 refer to days I, 3, and 5 of a 5-day cycle ergometry protocol. Percentage of Leu7+ lymphocytes was determined by flow cytometry counting 20,000 mononuclear cells. All values are means + SE with n = 9 per group. * P < 0.05 vs. beforeexercise base line; ** P < 0.01 vs. before-exercise base line (by paired t tests). CNTRL, control subjects; EX, exercised subjects. FIG.
pressing the NK-associated antigen (Leu7) increased significantly before vs. after exercise on the first (P C 0.05) and second (P < 0.01) sampling points (i.e., days 1 and 3 of exercise). Control values remained unchanged before vs. after sampling on the consecutive days except for CD8+ cells, which decreased at the final test session (P < 0.05).
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TABLE 4. Total number of lymphocytes, by subset, recovered from mononuclear interface after density gradient centrifugation of peripheral blood for control and exercised subjects before and after exercise over 5 consecutive days Day
Lymphocyte Subset
Control Exercised Control Exercised Control Exercised
CD3’ CD4+ CD8’
Values are means k SE expressed
n
CNTRL-PRE
a
CNTRL-POST
0
EX-PRE
&j
EX-POST
n % r x
1
Day
3
Day
5
Group Before
After
Before
After
1.4320.3 1.34t0.3 0.77t0.2 0.78t0.2 0.37kO.l 0.38kO.l
1.23t0.2 1.6520.4 0.73kO.l 0.96t0.2 0.33t0.1 0.63kO.2
1.45kO.3 1.2OkO.2 0.76t0.2 0.71kO.l 0.29&O. 1 0.3220.1
1.25t0.3 1.5o,to.1* 0.70-1-0.2 0.85tO.l* 0.24kO.l 0.43-1-0.1
1.27t0.2 1.07t0.2 0.68t0.1 0.5720.1 0.30t0.1 0.25tO.l
1.06t0.2 1.05t0.3 0.5420.1 0.53t0.1 0.2oto.r-f 0.30t0.2
fraction.
* P c 0.05 significantly
different
before vs. after values.
as no. of cells x 107/ml of mononuclear
* 0.6
2
1 Exercise
3 Sample
2. Changes in absolute numbers of Leu7+ lymphocytes in men before and after exercise. Exercise samples 1-3 refer to days I, 3, and 5 of a 5-day cycle ergometry protocol. Absolute numbers of Leu7+ lymphocytes lo7 per ml of mononuclear fraction (see MATERIALS AND METHODS for details about collection of this fraction from whole blood). All values are means t SE with n = 9 per group. * P 0.05 vs. before-exercise base line; ** P < 0.01 vs. before-exercise base line (by paired t tests). CNTRL, control subjects; EX, exercised subjects. FIG.
x
c
DISCUSSION
Acute exercise of either maximal or submaximal intensity has been associated with alterations in the absolute number of leukocytes (10, 11, 23) and in the percentage of lymphoid populations (2, 6, 25). In our study, mononuclear leukocyte number and the percent distribution of lymphocyte populations of untrained young adult males, who had undergone five repeated bouts of submaximal exercise, were compared with untrained control subjects. The lymphocyte populations measured were Tcells, T-helper/inducer cells, T-cytotoxic/suppressor cells, and NK cells. Repeated physical exercise resulted in an increase in recovered mononuclear leukocyte numbers before vs. after, although the magnitude of this increase in leukocyte number was significant only after days 1 and 3 of
Before
After
exercise. From before to after exercise mononuclear leukocyte numbers increased by 46% (day I), 41% (day 3), and 11% (day 5) in agreement with the magnitude of whole blood leukocytosis (21-37%) reported by Gimenez and colleagues (11) for submaximal exercise. The results of this study show that, depending on the lymphoid cell population enumerated and the chronicity of the exercise, the effect of repeated submaximal work is a variable percent shift in lymphocyte populations. An initial bout of exercise was associated with a significant decrease in the percentage of peripheral blood lymphocytes expressing the pan-T surface antigen (CD3+), whereas repeated exercise attenuated this effect (i.e., day 1 vs. day 5). These findings on T-lymphocyte numerical changes after submaximal work also demonstrate that this parameter is inconsistently affected by exercise. Furthermore, generalizations about the effects of exercise on immune parameters drawn from a single session of work may not be entirely accurate. Although the responses at day 1 would suggest a depression in T-cell numbers, subsequent sampling (e.g., day 5) would not lead to this same conclusion. In contrast, large granular lymphocytes (including cells expressing functional NK activity) systematically increased on a percent basis after cycle ergometry work. To our knowledge, this is the first study that demonstrates the persistence of the NK cell percent response over repeated exercise trials. Paradoxically, control subjects showed significant changes in the percentage of CD3+ and in the percent and total number of CD8+ cells at the day 5 sampling point. The reasons for these percent shifts are unknown. Although we tried to control anticipatory stress across the groups, it is possible that psychological factors (anticipation, anxiety) may have been involved. Psychological stress is well known to activate the sympathetic nervous system with concomitant effects on the immune system (3,32). By the final sampling point (day 5) it was evident that subjects had been preassigned to treatment groups; whether this was a factor in the lymphocyte subset effects observed in the controls at day 5 is not clear from the data. Although percent shifts in lymphocyte numbers after acute exercise have been reported previously (see review in Ref. 8), the relationship of the percentage of subsets to the absolute numbers of leukocytes is probably more relevant clinically. We calculated the absolute number of lymphocyte subsets in the recovered mononuclear
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1073
notypes will require further investigation. Nevertheless, fraction of blood (rather than in whole blood to minimize the potential confound of differential cell loss and adhe- exercise-associated changes in immune parameters may sion during staining procedures for flow cytometry). Our prove to be important factors in susceptibility to infecresults demonstrate a T-lymphocytosis and an increase tious disease, especially of the respiratory tract (28). It in NK cell numbers after exercise at days I and 3 but is also possible that exercise-associated increases in NK not at day 5. Lewicki and colleagues (17) recently re- cells may contribute to resistance to cancer; early studies show a reduced growth of transplanted tumors in vigorported an absolute increase in the number of T-cells after a single session of maximal exercise, and Pedersen ously exercised rodents compared with controls (30), and et al. (26) described an increase in the total numbers of NK cells are known to be effector cells in the destruction NK cells immediately after 60 min of cycle ergometry at of tumor targets (18). 80% of TjoPmax.Our data (day 1 of exercise) confirm these The authors gratefully acknowledge the secretarial support of N. earlier reports and show that the exercise effect on Tand the technical assistance of R. Thorne. The use of the FACS cell and NK cell numbers occurs even at a lower work Poole unit (Dr. P. Pauls) at the University of Guelph is acknowledged. intensity (i.e., 65% of VOzmax). This investigation was made possible by a grant from the Canadian Although the mechanisms underlying the rapid and Fitness and Lifestyle Research Institute. Address reprint requests to L. Hoffman-Goetz. transient alterations in the lymphocyte phenotypes with exercise are not definitively known, there are some data Received 20 June 1989; accepted in final form 20 October 1989. in the literature to suggest hemodynamic factors. The leukocytosis of exercise is a well-documented phenomeREFERENCES non, and rapid alterations in the patterns of tissue perexercise 1. BERK, L. S., D. C. NIEMAN, AND S. A. TAN. Maximal fusion occur with high-intensity work. The alterations modifies lymphocytes and subpopulations T helper and T suppresin the percent and number of lymphoid cells may reflect sor and ratio in man. (Abstract). Med. Sci. Sports Exercise 19: S43, movement of cells into and out of the circulation and 1989. 2. BERK, L. S., D. NIEMAN, S. A. TAN, S. NEHLSEN-CANNARELLA, mobilization from tissue reservoirs (e.g., spleen, lung). J. KRAMER, W. C. EBY, AND M. OWENS. Lymphocyte subset The mechanical effect of increased cardiac output parchanges during acute maximal exercise. (Abstract). Med. Sci. allels the transient changes in lymphocyte populations. Sports Exercise 18: 706, 1986. Foster et al. (9) reported that the leukocytosis of exercise 3. BLALOCK, J. E. The immune system as a sensory organ. J. Immunol. 132: 1067-1070,1984. is predominantly the result of increased cardiac output cells and granulocytes from 4. BOYUM, M. Isolation of mononuclear (rather than of the effects of catecholamines) and subhuman blood. Stand. J. Clin. Lab. Invest. 21, Suppl. 97: 77-78, sequent demargination of leukocytes from endothelial 1968. surfaces. It is possible that there is a degree of demargin5. DEUSTER, P. A., A. M. CURIALE, M. L. COWAN, AND F. D. FINation of T-lymphocytes and NK cells in the lungs. The KELMAN. Exercise-induced changes in populations of peripheral blood mononuclear cells. Med. Sci. Sports Exercise 20: 276-280, large increase in blood flow could dislodge these lympho1988. cytes from the capillary segments in the alveolus, con6. EDWARDS, A. J., T. H. BACON, C. A. ELMS, R. VERARDI, M. tributing to the overall increase in mononuclear cell FELDER, AND S. C. KNIGHT. Changes in the population of lymphoid number. Depending on the size and particular adhesion cells in human peripheral blood following physical exercise. Clin. Exp. Immunol. 58: 420-427,1984. characteristics of the lymphocytes, this could result in 7. ESTERLING, B., AND B. S. RABIN. Stress-induced alteration of Tselective increases or decreases of the subsets in the lymphocyte subsets and humoral immunity in mice. Behav. iVeucirculation. rosci. 101: 115-119, 1987. An important finding of this study is that, irrespective 8. FITZGERALD, L. Exercise and the immune system. Immunol. Today 9: 337-339,1988. of whether the results are expressed as absolute or rela9. FOSTER, N. K., J. B. MARTYN, R. E. RANGO, J. C. HOGG, AND R. tive lymphocyte numbers, the exercise effect on T-lymL. PARDY. Leukocytosis of exercise: role of catecholamines. J. Appl. phocytes disappears at the day 5 sampling time. This Physiol. 61: 2218-2223, 1986. suggests that the changes in lymphocyte phenotypes that J. C. HUMBERT, N. DE TAL10. GIMENEZ, M., T. MONAN-KUMAR, ANCE, AND J. BUISINE. Leukocyte, lymphocyte and platelet reoccur immediately after exercise may represent a general sponse to dynamic exercise. Duration or intensity effect? Eur. J. response to acute stress rather than a specific response Appl. Physiol. Occup. Physiol. 55: 465-470, 1986. to exercise. A variety of acute physical stressors, such as 11. GIMENEZ, M., T. MONAN-KUMAR, J. C. HUMBERT, N. DE TALrotational and restraint stress, have marked effects on ANCE, M. TEBOUL, AND F. J. ARINO BELENGUER. Training and leukocyte, lymphocyte and platelet response to dynamic exercise. the populations of lymphoid cells resulting in decreases Int. J. Sports Med. 27: 172-177, 1987. in T-lymphocyte numbers (7, 33, 34). Alterations in T12. HEDFORS, E., G. HOLM, M. IVANSEN, AND J. WAHREN. Physiologlymphocyte and NK cell numbers have been documented ical variation of blood lymphocyte reactivity: T-cell subsets, imafter cortisol and epinephrine infusions in men (35), munoglobulin production, and mixed lymphocyte reactivity. Clin. Immunol. Immunopathol. 27: 9-14, 1983. hormones that increase during physical stress and exer13. HUGHSON, R. L., J. M. KOWALCHUK, W. M. PRIME, AND H. J. cise. GREEN. Open-circuit gas exchange analysis in the non-steady state. To conclude, although a single submaximal exercise Can. J. Appl. Sport Sci. 5: 15-18, 1980. session produces small but significant alterations in the 14. KEAST, D., K. CAMERON, AND A. R. MORTON. Exercise and the immune response. Sports Med. 5: 248-267, 1988. percent and total number of T-lymphocytes and NK cells in peripheral blood, repeated exercise over 5 days results 15. LANDMANN, R. M. A., F. B. MULLER, C. PERINI, M. WESP, P. ERNE, AND F. B. BUHLER. Changes of immunoregulatory cells in accommodation or normalization of the phenotype induced by psychological and physical stress: relationship to shifts. The physiological mechanisms accounting for plasma catecholamines. Clin. Exp. Immunol. 58: 127-135, 1984. these changes in the distribution of lymphoid cell phe- 16. LEWICKI, R., H. TCHORZEWSKI, A. DENYS, M. KOWALSKA, AND Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 26, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
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REPEATED
A. GOLINSKA. Effect of physical exercise on some parameters of immunity in conditioned sportsmen. Int. J. Sports Med. 8: 30% 313,1987. LEWICKI, R., H. TCHORZEWSKI, E. MAJEWSKA, 2. NOWAK, AND 2. BAJ. Effect of maximal physical exercise on T-lymphocyte subpoplations and on interleukin 1 (ILl) and interleukin 2 (IL2) production in vitro. Int. J. Sports Med. 9: 114-117, 1988. LOTZOVA, E. Effector immune mechanisms in cancer. Nat. Immun. Cell Growth Regul. 4: 293-304, 1985. LOWRY, 0. H., AND J. V. PASSONNEAU. A Flexible System of Enzymatic Analysis. New York: Academic, 1972, p. 194-195. MACKINNON, L. T. Letter to the editor-in-chief. Med. Sci. Sports Exercise 18: 596, 1986. MACKINNON, L. T., T. W. CHICK, A. VAN As, AND T. B. TOMASI. The effect of exercise on secretory and natural immunity. Adu. Exp. Med. Biol. 216A: 869-876,1987. MCCARTHY, D. A., AND M. M. DALE. The leucocytosis of exercise. Sports Med. 6: 333-363,1988.
23. MOORTHY, A. V., AND S. ZIMMERMAN. Human leukocyte response to an endurance race. Eur. J. Appl. Physiol. Occup. Physiol. 38: 271-274,1978. 24. ORTALDO, J. R., S. 0. SHARROW, T. TIMMONEN, AND R. B, HERBERMAN. Determination of surface antigens on purified human NK cells by flow cytometry with monoclonal antibodies. J. Immunol. 127: 2401-2409,1981. 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: 137-140, 1988. 26. PEDERSEN, B. K., N. TVEDE, F. R. HANSEN, V. ANDERSEN, T. BENDIX, G. BENDIXEN, K. BENDTZEN, H. GALBO, P. M. HAAHR,
SUBMAXIMAL
EXERCISE
K. KLARLUND, J. SYLVEST, B. S. THOMSEN, AND J. HALKJAERKRISTENSEN. Modulation of natural killer cell activity in peripheral blood by physical exercise. Stand. J. Immunol. 27: 673-678, 1988. 27. PEDERSEN, B. K., N. TVEDE, K. KLARLUND, L. D. CHRISTENSEN, F. R. HANSEN, H. GALBO, A. L. KHARAZMI, AND J. HALKJAERKRISTENSEN. Indomethacin in vitro and in vivo abolishes postexercise suppression of natural killer cell activity in peripheral blood. Int. J. Sports Med. In press. 28. PETERS, E. M., AND E. D. BATEMAN. Ultramarathon running and upper respiratory tract infections. S. Afr. Med. J. 64: 582-584, 1983. 29. RENZI, P., AND L. C. GINNS. Analysis of T cell subsets in normal adult. Comparison of whole blood lysis technique to Ficoll-Hypaque separation by flow cytometry. J. Immunol. Methods 98: 53-56, 1987. 30. RUSCH, H. P., AND B. E. KLINE. The effect of exercise on the growth of mouse tumor. Cancer Res. 4: 116-118,1944. 31. SAS INSTITUTE, INC. SAS User’s Guide: Statistics. Carey, NC: SAS Institute, 1982. 32. SOLOMON, G. F., A. A. AMKRAUT, AND P. KASPER. Immunity, emotions and stress. Psychother. Psychosom. 23: 209-217, 1974. 33. STEPLEWSKI, Z., AND W. H. VOGEL. Total leukocytes, T cell subpopulations and natural killer (NK) cell activity in rats exposed to restraint stress. Life Sci. 38: 2419-2427, 1986. 34. TESHIMA, H., H. SOGAWA, H. KIHARA, S. NAGATA, Y. AGO, AND T. NAKAGAWA. Changes in populations of T-cell subsets due to stress. Ann. NY Acad. Sci. 96: 459-466, 1987. 35. T~NNESEN, E., N. J. CHRISTENSEN, AND M. M. BRINKL~V. Natural killer cell activity during cortisol and adrenaline infusion in healthy volunteers. Eur. J. Clin. Invest. 17: 497-503, 1987.
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subset responses to repeated exercise in men
LAURIE HOFFMAN-GOETZ, JANIS RANDALL SIMPSON, NICHOLAS YOGAJOTHI ARUMUGAM, AND MICHAEL E. HOUSTON Departments of Health Studies and Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3GI, Canada
HOFFMAN-GOETZ, LAURIE, JANIS RANDALL SIMPSON, NIE. CHOLAS CIPP, YOGAJOTHI ARUMUGAM, AND MICHAEL HOUSTON. Lymphocyte subset responses to repeated submaximal exercise in men. J. Appl. Physiol. 68(3): 1069-1074,1990.-The
effects of repeated bouts of submaximal cycle ergometry exercise on changes in the percentage of peripheral blood T-lymphocytes, the T-helper/inducer and T-cytotoxic/suppressor subsets, and natural killer (NK) cells were studied in 18 healthy young men who had no history of regular exercise training. Subjects were matched on the basis of maximal O2 uptake and assigned randomly to exercise or control groups, with controls resting quietly during the exercise sessions. The percentage of peripheral blood mononuclear leukocytes that reacted with monoclonal antibodies specific for T-lymphocytes (CD3+ cells), the helper/inducer subset (CD4+ cells) and cytotoxic/suppressor subset (CD8+ cells) of T-lymphocytes, and cells with NK activity (Leu7+ cells) were enumerated by fluorescence-activated flow cytometry for samples obtained immediately before and after exercise on days 1,3, and 5 of a b-day exercise regimen. The results of this study were mixed with decreases in the percentage of T-lymphocytes before vs. after exercise on days 1 and 3 (P c O.OOl), a decrease in the percentage of T-helper/ inducer cells before vs. after exercise on day 3 (P < 0.05), no effect of exercise on the percentage of T-cytotoxic/suppressor cells, and a marked increase in the percentage of NK cells after exercise on days 1 (P < 0.05) and 3 (P < 0.01). The total number of recovered NK cells in the mononuclear leukocyte fraction of blood also increased significantly after exercise on days 1 (P < 0.05) and 3 (P < 0.01). These findings 1) suggest that repeated exposure to submaximal exercise results in consistent increases in the percentage of NK cells, 2) demonstrate that the exercise effects on T-lymphocyte subset percents were variable over time, 3) confirm earlier reports on the impact of a single bout of submaximal exercise on percent and numerical shifts in peripheral blood T-lymphocytes and NK cells, and 4) extend the findings of earlier studies to include individuals of low fitness levels. flow cytometry; monoclonal antibodies; cycle ergometry
YEARS, considerable interest has been directed to the effects of exercise, a replicable and quantifiable stressor, on immune function (see reviews in Refs. 8,14). Understanding the clinical significance of functional changes in the immune system with exercise depends, in part, on the ability to accurately enumerate the component cell populations (e.g., T-lymphocytes) and their various subsets (e.g., T-helper/inducer cells). The introduction of monoclonal antibodies reacting with specific IN RECENT
0161-7567/90
$1.50 Copyright
CIPP,
cell surface antigens, coupled with immunofluorescent flow cytometry analysis, has greatly facilitated the evaluation of exercise-induced changes in immune parameters. For example, the conclusions drawn about the clinical relevance of a finding of no change in the capacity of natural killer (NK) cells to lyse tumor targets immediately after exercise are quite different when this information is combined with the finding of concurrent decreases in the absolute or relative number of NK cells (21) Although enumeration of lymphocyte subsets by flow cytometry is widely used clinically, there are only a limited number of reports enumerating the lymphocyte population shifts with exercise. Generally, the direction of the lymphocyte population changes with exercise is dependent on whether the work intensity is maximal or submaximal. Three recent studies describing a single maximal exercise bout found an increase (6, 16, 17) or no change (2) in the percentage of peripheral blood Tlymphocytes, an increase (2, 5) or decrease (27) in the percentage of NK cells, and an increase in the percentage of the cytotoxic/suppressor T-cell subpopulation (1, 16, 17). A single session of submaximal exercise was associated with a decrease in the percentage of blood T-lymphocytes and T-helper/inducer cells in untrained subjects of moderate fitness [maximal O2 uptake (VOW,,,) of 53.5 t 2.4 ml kg-’ min-‘1 (25) and an increase in the percentage of NK cells in athletes only. In contrast, others have reported an increase in the percentage of blood T-lymphocytes after submaximal exercise in subjects of unspecified fitness (6, 15). Variations in the direction and magnitude of lymphocyte numerical changes with exercise may be the result of the different types of exercise as well as the different fitness levels of the subjects. In the present study, we determined the effects of repeated submaximal exercise on changes in the distribution of peripheral blood Tlymphocytes, T-lymphocyte subsets (helper/inducer and cytotoxic/suppressor), and large granular lymphocytes, including NK cells, as determined by monoclonal antibodies and flow cytometry. We hypothesized that repeated exposure to an exercise stress would abrogate, or at least reduce, the magnitude of the lymphocyte phenotype changes reported after a single session of submaximal work. Additionally, we wanted to ascertain whether the types of lymphocyte numerical changes in-
0 1990 the American
l
Physiological
Society
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1070
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duced by exercise, which have been reported for moderate and highly fit subjects, also characterized individuals of lower fitness levels. MATERIALS
AND METHODS
Subjects. Eighteen male subjects were recruited from the University student population. These men occasionally participated in recreational activities, but they were not involved in any regular exercise training. The subjects completed an activity and lifestyle profile and signed consent forms approved by the Committee on Human Research. The subjects were considered “untrained” based on their activity profiles and Vozmax (~50 ml. kg-‘. min-‘; see Table 1). . Experimental design. Subjects performed a progressive vo &ax test on an electrically braked cycle ergometer by using a protocol described previously (13). Thereafter, subjects were paired on the basis of a best match with their previous activity and Vozmax values. The subjects were monitored in pairs over a 5-day period in which one of the subjects cycled at a load representing 65% of vozrnax each day for 1 h. This work load was selected because it represents the midpoint of the range prescribed for conditioning intensity for endurance exercise by average persons. The control subject sat beside the exercising subject on another cycle ergometer. Initially, the subject pairs were informed that the decision as to who would exercise and who would rest would be made randomly each day. Our intent was to keep the anticipation of exercise similar for the exercising and control subject pairs. However, once the initial choice was made as to who would exercise, that subject performed the exercise each day for 5 consecutive days. Blood samples were drawn from a forearm vein by venipuncture immedi .ately before and after the exercise bouts on the 1st, 3rd, and 5th consecutive days. Morning blood samples (between 8 and 1OA .M.)at rest were drawn after the subjects had relaxed in a chair for at least 15 min. Postexercise samples were drawn 3 min after the termination of exercise (exercise subjects) or sitting on the cycle ergometer (control subjects). The exercise bouts were carried out in the morning, and at the same time for each subject pair. Experimental procedures. Mononuclear leukocytes were-isolated from heparinized blood by the method of Boyum (4). Briefly, blood was diluted twofold with phosphate-buffered saline (PBS; 0.01 M, pH 7.2), layered over Histopaque (Sigma Chemical, St. Louis, MO), and centrifuged at 400 g for 30 min. The mononuclear interface was collected and washed three times and the volume adjusted to 1 ml in PBS. Mononuclear cell counts were performed manu .ally on a Nikon Optiphot phase-c sontrast microscope, and cell viability was assessed by the trypan blue exclusion technique. Cell viability, after separation on Histopaque, was always >92%. Lymphocyte subpopulations were quantitated by the technique of direct immunofluorescence by using monoclonal antibodies specific for peripheral total T-cells (CD3+), helper/inducer T-cells (CD4+), suppressor/cytotoxic T-cells (CD8+), and large granular lymphocytes, a subset that expresses NK functional activity (Leu7+).
SUBMAXIMAL
EXERCISE
Monoclonal antibodies were obtained from Becton Dickinson and used with a fluorescent-activated cell sorter (Coulter EPICS IV FACS) with an argon laser (488nm wavelength) to excite the fluorescein isothiocyanate(FITC) conjugated monoclonal antibodies. Background autofluorescence was determined for each sample, and an average of 20,000 cells/sample was counted. Labeled cell suspensions were also manually verified by fluorescence microscopy using a field of 100 mononuclear cells. Details for the flow cytometric technique are given in Ortaldo et al. (24), Renzi and Ginns (29), and Becton Dickinson technical report 23-1372-01. Aliquots of whole blood were obtained from the exercising and control subjects before and after the exercise bouts on the lst, 3rd, and 5th days. The blood was deproteinized with 0.6 M perchloric acid, neutralized with 1.0 M NaHC03, and centrifuged, and the supernatant was frozen for later analysis of lactate concentration (19). Blood samples were also analyzed for hematocrit changes. Statistical analysis. Immunological data were analyzed using an SAS repeated measures analysis of variance model (31) to determine differences across time and by group. Paired t tests were done to determine before and after differences on each test day for control and exercised groups. Statistical significance was set at the 0.05 RESULTS
Physical characteristics of the control and exercised subjects before the experiment are shown in Table 1. There were no significant differences between the two groups of subjects for age, body weight, or Tjogmax values. Table 2 shows values for hematocrit, recovered mononuclear leukocyte number, and blood lactate concentration for the two groups of subjects determined in venous blood samples obtained at rest and after exercise bouts on days 1, 3, and 5. Hematocrit percent increased -significantly immediately after exercise at 65% of voarnax, representing a percent increase of 6.5, 5.2, and 3.8% for days 1, 3, and 5, respectively (P < 0.05). Mononuclear leukocyte number increased significantly after exercise on days 1 and 3 (P < 0.05). Mononuclear leukocyte numbers were virtually unchanged from before to after sampling times in the control subjects. Blood lactate concentrations increased significantly before to after exercise on days 1, 3, and 5 for the exercised group (P < 0.001); lactate concentrations were unchanged in the control subjects as a function of time. The changes in the percent distribution of cell populations are presented in Table 3. The percentage of peripheral mononuclear leukocytes that reacted with TABLE 1. Physical characteristics and control subjects Group
n
Control Exercised
9 9
Age, Yr
22.8H.2 24.5k0.9
of the exercised
VO
2max
Weight, kg
l/min
ml kg-’ min-’
74.7k3.5 75.2k4.1
3.46kO.18 3.49k0.12
46.3k2.1 46.426.4
l
l
Values are means k SE.
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LYMPHOCYTE
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AND
TABLE 2. Hematological and lactate concentration before and after exercise over 5 days Parameter
Hematocrit,
%
Mononuclear cells, X lo7 cells/ml Lactate, mM
Day
After
44.1t0.8
Before
After
44.5t0.6 42.5t0.5
45.1t0.5 44.7&0.6*
44.51ko.7 43.5-eo.5*
1.82t0.4 2.39kO.5 0.9tO.l
44.2t0.9 41.9t0.5 1.79kO.3 1.58kO.3
1.75kO.5
1.96k0.3
1.98t0.4
1.86t0.3
2.72t0.5*
1.701ko.4
1.01*0.1
l.OtO. 1
l.OtO.l
1.0tO.l
3.0*0.5?
1.2tO.l
different
Day 5
After
2.04kO.4
Values are means t SE. * P < 0.05 significantly
3
Before
45.420.7 45.8t0.7*
43.0t0.9
1071
EXERCISE
and exercised subjects
1
Before
Control Exercised Control Exercised Control Exercised
SUBMAXIMAL
values for control
Day
Group
REPEATED
2.620.37
before vs. after values; t P < 0.001 significantly
1.67k0.3
0.9tO.l
1.OkO.l 2.9+0.4-f different before vs. after values. 1.5zkO.2
TABLE 3. Percentage values for CD3+ and percentage of the subsets CD4+ and CDF, for control and exercised subjects before and after exercise over 5 consecutive days Lymphocyte Subset, %
CD3+ CD4+ CD8+
Day
Group
Control Exercised Control Exercised Control Exercised
Before
1
Day
3
Day 5
After
Before
After
Before
7O.lk3.8 72.2t3.1
65.1t4.1 60.3k5.0.f
69.4t3.8 64.0&3.1?
71.3t3.4
37.7t2.3 40.7t3.8
36.6t2.8
71.3t5.0 72.1t3.3 39.7t3.4
19.3t1.5 18.6t2.3
36.4t2.9 18.4k2.4 21.1t3.0
43.7t3.4
37.6k3.2 36.2*3.8$
16.3t2.3 17.7t2.6
15.2t2.0 19.3t2.3
Values are means t SE. * P < 0.05 significantly different before vs. after values; 7 P c 0.001 significantly < 0.01 significantly different before vs. after values.
CD3+ pan T-cell antibody decreased significantly after exercise on days 1 and 3 (P < 0.001) relative to beforeexercise values. Control subjects showed a significant decrease in the percentage of T-lymphocytes at day 5 sampling (P < 0.05). Submaximal exercise induced a small decrease in the percentage of T-helper/inducer cells across days 1, 3, and 5 relative to before-exercise values; however, this decrease in T-helper/inducer cell percent was statistically significant only after the day 3 exercise session (P < 0.01). Control subjects tended to a decrease in the percentage of CD4+ cells at the after sampling points; however, these fluctuations did not reach statistical significance. The percentage of peripheral blood lymphocytes that expressed the CD8+ antigen (i.e., cytotoxic/suppressor subset) was unaffected by exercise; paradoxically, there was a small but significant decrease in the percentage of this subset in control subjects sampled at day 5 (P < 0.05, before vs. after). The percentage of peripheral blood leukocytes that expressed the NK-associated antigen (Leu7+) was markedly affected by exercise with increases of 55.4% (P < 0.05) and 73.0% (P < 0.01) immediately after exercise on days I and 3, respectively (Fig. 1). This trend for an increase after exercise in the percent of Leu7+ cells continued, albeit not significantly, on the final sampling point (day 5 = sample 3). The percentage of Leu7+ cells in peripheral blood was unchanged before vs. after sampling times among control subjects. Table 4 shows the absolute numbers of lymphocytes recovered, by subset, in the mononuclear fraction (i.e., recovered mononuclear cells X subset percent). When the data are expressed this way, there was a significant lymphocytosis for CD3+ and CD4+ cells after exercise on day 3. Figure 2 shows the effect of repeated exercise on the absolute number of Leu7+ lymphocytes. Cells ex-
n
CNTRL-PRE
w
CNTRL-POST
q q
EX-PRE
After
63.1&3.4* 62.9k2.7 32.5t1.9 32.9k2.9 17.5t2.5 13.7zk2.3* 16.4t2.1 16.5t2.5 different before vs. after values; $ P 68.023.1 38.3t2.8 38.1t4.2
EX-POST
*
1
2
3
EXERCISE SAMPLE 1. Changes in percentage of Leu7+ lymphocytes in men before and after exercise. Exercise samples 1-3 refer to days I, 3, and 5 of a 5-day cycle ergometry protocol. Percentage of Leu7+ lymphocytes was determined by flow cytometry counting 20,000 mononuclear cells. All values are means + SE with n = 9 per group. * P < 0.05 vs. beforeexercise base line; ** P < 0.01 vs. before-exercise base line (by paired t tests). CNTRL, control subjects; EX, exercised subjects. FIG.
pressing the NK-associated antigen (Leu7) increased significantly before vs. after exercise on the first (P C 0.05) and second (P < 0.01) sampling points (i.e., days 1 and 3 of exercise). Control values remained unchanged before vs. after sampling on the consecutive days except for CD8+ cells, which decreased at the final test session (P < 0.05).
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1072
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TABLE 4. Total number of lymphocytes, by subset, recovered from mononuclear interface after density gradient centrifugation of peripheral blood for control and exercised subjects before and after exercise over 5 consecutive days Day
Lymphocyte Subset
Control Exercised Control Exercised Control Exercised
CD3’ CD4+ CD8’
Values are means k SE expressed
n
CNTRL-PRE
a
CNTRL-POST
0
EX-PRE
&j
EX-POST
n % r x
1
Day
3
Day
5
Group Before
After
Before
After
1.4320.3 1.34t0.3 0.77t0.2 0.78t0.2 0.37kO.l 0.38kO.l
1.23t0.2 1.6520.4 0.73kO.l 0.96t0.2 0.33t0.1 0.63kO.2
1.45kO.3 1.2OkO.2 0.76t0.2 0.71kO.l 0.29&O. 1 0.3220.1
1.25t0.3 1.5o,to.1* 0.70-1-0.2 0.85tO.l* 0.24kO.l 0.43-1-0.1
1.27t0.2 1.07t0.2 0.68t0.1 0.5720.1 0.30t0.1 0.25tO.l
1.06t0.2 1.05t0.3 0.5420.1 0.53t0.1 0.2oto.r-f 0.30t0.2
fraction.
* P c 0.05 significantly
different
before vs. after values.
as no. of cells x 107/ml of mononuclear
* 0.6
2
1 Exercise
3 Sample
2. Changes in absolute numbers of Leu7+ lymphocytes in men before and after exercise. Exercise samples 1-3 refer to days I, 3, and 5 of a 5-day cycle ergometry protocol. Absolute numbers of Leu7+ lymphocytes lo7 per ml of mononuclear fraction (see MATERIALS AND METHODS for details about collection of this fraction from whole blood). All values are means t SE with n = 9 per group. * P 0.05 vs. before-exercise base line; ** P < 0.01 vs. before-exercise base line (by paired t tests). CNTRL, control subjects; EX, exercised subjects. FIG.
x
c
DISCUSSION
Acute exercise of either maximal or submaximal intensity has been associated with alterations in the absolute number of leukocytes (10, 11, 23) and in the percentage of lymphoid populations (2, 6, 25). In our study, mononuclear leukocyte number and the percent distribution of lymphocyte populations of untrained young adult males, who had undergone five repeated bouts of submaximal exercise, were compared with untrained control subjects. The lymphocyte populations measured were Tcells, T-helper/inducer cells, T-cytotoxic/suppressor cells, and NK cells. Repeated physical exercise resulted in an increase in recovered mononuclear leukocyte numbers before vs. after, although the magnitude of this increase in leukocyte number was significant only after days 1 and 3 of
Before
After
exercise. From before to after exercise mononuclear leukocyte numbers increased by 46% (day I), 41% (day 3), and 11% (day 5) in agreement with the magnitude of whole blood leukocytosis (21-37%) reported by Gimenez and colleagues (11) for submaximal exercise. The results of this study show that, depending on the lymphoid cell population enumerated and the chronicity of the exercise, the effect of repeated submaximal work is a variable percent shift in lymphocyte populations. An initial bout of exercise was associated with a significant decrease in the percentage of peripheral blood lymphocytes expressing the pan-T surface antigen (CD3+), whereas repeated exercise attenuated this effect (i.e., day 1 vs. day 5). These findings on T-lymphocyte numerical changes after submaximal work also demonstrate that this parameter is inconsistently affected by exercise. Furthermore, generalizations about the effects of exercise on immune parameters drawn from a single session of work may not be entirely accurate. Although the responses at day 1 would suggest a depression in T-cell numbers, subsequent sampling (e.g., day 5) would not lead to this same conclusion. In contrast, large granular lymphocytes (including cells expressing functional NK activity) systematically increased on a percent basis after cycle ergometry work. To our knowledge, this is the first study that demonstrates the persistence of the NK cell percent response over repeated exercise trials. Paradoxically, control subjects showed significant changes in the percentage of CD3+ and in the percent and total number of CD8+ cells at the day 5 sampling point. The reasons for these percent shifts are unknown. Although we tried to control anticipatory stress across the groups, it is possible that psychological factors (anticipation, anxiety) may have been involved. Psychological stress is well known to activate the sympathetic nervous system with concomitant effects on the immune system (3,32). By the final sampling point (day 5) it was evident that subjects had been preassigned to treatment groups; whether this was a factor in the lymphocyte subset effects observed in the controls at day 5 is not clear from the data. Although percent shifts in lymphocyte numbers after acute exercise have been reported previously (see review in Ref. 8), the relationship of the percentage of subsets to the absolute numbers of leukocytes is probably more relevant clinically. We calculated the absolute number of lymphocyte subsets in the recovered mononuclear
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LYMPHOCYTE
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SUBMAXIMAL
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1073
notypes will require further investigation. Nevertheless, fraction of blood (rather than in whole blood to minimize the potential confound of differential cell loss and adhe- exercise-associated changes in immune parameters may sion during staining procedures for flow cytometry). Our prove to be important factors in susceptibility to infecresults demonstrate a T-lymphocytosis and an increase tious disease, especially of the respiratory tract (28). It in NK cell numbers after exercise at days I and 3 but is also possible that exercise-associated increases in NK not at day 5. Lewicki and colleagues (17) recently re- cells may contribute to resistance to cancer; early studies show a reduced growth of transplanted tumors in vigorported an absolute increase in the number of T-cells after a single session of maximal exercise, and Pedersen ously exercised rodents compared with controls (30), and et al. (26) described an increase in the total numbers of NK cells are known to be effector cells in the destruction NK cells immediately after 60 min of cycle ergometry at of tumor targets (18). 80% of TjoPmax.Our data (day 1 of exercise) confirm these The authors gratefully acknowledge the secretarial support of N. earlier reports and show that the exercise effect on Tand the technical assistance of R. Thorne. The use of the FACS cell and NK cell numbers occurs even at a lower work Poole unit (Dr. P. Pauls) at the University of Guelph is acknowledged. intensity (i.e., 65% of VOzmax). This investigation was made possible by a grant from the Canadian Although the mechanisms underlying the rapid and Fitness and Lifestyle Research Institute. Address reprint requests to L. Hoffman-Goetz. transient alterations in the lymphocyte phenotypes with exercise are not definitively known, there are some data Received 20 June 1989; accepted in final form 20 October 1989. in the literature to suggest hemodynamic factors. The leukocytosis of exercise is a well-documented phenomeREFERENCES non, and rapid alterations in the patterns of tissue perexercise 1. BERK, L. S., D. C. NIEMAN, AND S. A. TAN. Maximal fusion occur with high-intensity work. The alterations modifies lymphocytes and subpopulations T helper and T suppresin the percent and number of lymphoid cells may reflect sor and ratio in man. (Abstract). Med. Sci. Sports Exercise 19: S43, movement of cells into and out of the circulation and 1989. 2. BERK, L. S., D. NIEMAN, S. A. TAN, S. NEHLSEN-CANNARELLA, mobilization from tissue reservoirs (e.g., spleen, lung). J. KRAMER, W. C. EBY, AND M. OWENS. Lymphocyte subset The mechanical effect of increased cardiac output parchanges during acute maximal exercise. (Abstract). Med. Sci. allels the transient changes in lymphocyte populations. Sports Exercise 18: 706, 1986. Foster et al. (9) reported that the leukocytosis of exercise 3. BLALOCK, J. E. The immune system as a sensory organ. J. Immunol. 132: 1067-1070,1984. is predominantly the result of increased cardiac output cells and granulocytes from 4. BOYUM, M. Isolation of mononuclear (rather than of the effects of catecholamines) and subhuman blood. Stand. J. Clin. Lab. Invest. 21, Suppl. 97: 77-78, sequent demargination of leukocytes from endothelial 1968. surfaces. It is possible that there is a degree of demargin5. DEUSTER, P. A., A. M. CURIALE, M. L. COWAN, AND F. D. FINation of T-lymphocytes and NK cells in the lungs. The KELMAN. Exercise-induced changes in populations of peripheral blood mononuclear cells. Med. Sci. Sports Exercise 20: 276-280, large increase in blood flow could dislodge these lympho1988. cytes from the capillary segments in the alveolus, con6. EDWARDS, A. J., T. H. BACON, C. A. ELMS, R. VERARDI, M. tributing to the overall increase in mononuclear cell FELDER, AND S. C. KNIGHT. Changes in the population of lymphoid number. Depending on the size and particular adhesion cells in human peripheral blood following physical exercise. Clin. Exp. Immunol. 58: 420-427,1984. characteristics of the lymphocytes, this could result in 7. ESTERLING, B., AND B. S. RABIN. Stress-induced alteration of Tselective increases or decreases of the subsets in the lymphocyte subsets and humoral immunity in mice. Behav. iVeucirculation. rosci. 101: 115-119, 1987. An important finding of this study is that, irrespective 8. FITZGERALD, L. Exercise and the immune system. Immunol. Today 9: 337-339,1988. of whether the results are expressed as absolute or rela9. FOSTER, N. K., J. B. MARTYN, R. E. RANGO, J. C. HOGG, AND R. tive lymphocyte numbers, the exercise effect on T-lymL. PARDY. Leukocytosis of exercise: role of catecholamines. J. 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