ages of 17 and 3 1 were chosen to participate in the study. Most of the subjects ... At 071 5, subjects started their 45-min exercise session, with blood samples ...
Effect of High- Versus Moderate-Intensity Exercise on Lymphocyte Subpopulations and Proliferative Response D. C. Nieman, A. R. Miller, D. A. Henson, B. 1 Warren, G. Glnewitch, R. L. Johnson, J. M. Davis, D. E. Birttemorth, 1 L. Herring, S. L. Nehlsen-Cannarella Departments of Health, Leisure, and Exercise Science, Biology. Appalachian State University; Immunology Center, Loma Linda University Medical Center; Department of Exercise Science, University of South Carolina
Introduction Abstract
D. C. Nieman, A. R. Miller, D. A. Henson, B. 1 Warren, G. Gusewitch, R. L. Johnson, J. M. Davis, D. E. Blrttemlorth, 1 1.Herring and S. L. Nehlsen-Cannarella, Effect of High- Versus Moderate-Intensity Exercise on Lymphocyte Subpopulations and Proliferative Responses. Int. J. Sports Med., Vol. 15, No. 4, pp. 199-206, 1994. Accepted after revision: November 6, 1993. The effect of 45 min of high- (80% O02max) versus moderate- (50 % O02max) intensity treadmill exercise on circulating leukocyte and lymphocyte subpopulations, catecholamine and cortisol concentrations, and the mitogen-stimulated lymphocyte proliferative response was investigated in 10 well-conditioned (mean VOzmax 66.0 1.9 ml/kg/min), young males (mean age 22.1 k 1.3 yrs). Blood samples were taken before and immediately after exercise, with three more samples taken during 3.5 h of recovery. Treatment order on the treadmill (graded walking at 7.3 k 0.1 km/h, 6.5 0.6 % grade, versus level running at 16.1 k0.3 km/h) was counterbalanced with subjects acting as their own controls and results analyzed using a 2 x 5 repeated measures ANOVA. The concanavalin A- (Con A) stimulated lymphocyte proliferative response was decreased at 1 h and 2 h post-exercise relative to baseline levels following both exercise-intensity conditions. However, when adjusted on a per-T cell (CD3+) basis to account for the change in number of T cells in the in vitra assay, only the high-intensity exercise condition was associated with a 1-h post-exercise decrease (21 %, p = 0.05) in the proliferative response relative to baseline. Exercise at 80 % versus 50 % VOzmax resulted in significantly greater increases in cortisol and epinephrine concentrations, providing a physiological rationale for the immediate-post-exercise lymphocytosis, 1- to 3.5-h lymphocytopenia, and the decrease in Con A-stimulated lymphocyte proliferative response (per CD3+ cell) that occurred in greater measure following high-intensity exercise.
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Key words:
Immune system, lymphocytes, lymphocyte transformation, Concanavalin A, epinephrine, cortisol, exertion
Int. J. Sports Med. 15 (1994) 199-206 O Georg Thieme Verlag Stuttgart . New York
Numerous studies have now established that vigorous cardiorespiratory exercise (70 %- 80 % aerobic capacity) of 5 to 60 min duration is associated with a unique biphasic perturbation of the circulating lymphocyte count (1 0,12,14,35). Immediately post-exercise, circulating numbers of lymphocytes increase by 70 %- 130 %, followed by a 30 %-SO % lymphocytopenia that begins approximately 30min post-exercise and continues for close to 4 hours. Moderate-intensity exercise (40%-60% aerobic capacity) has been reported to induce a much smaller lymphocytosis and lymphocytopenia (22,24,35). The literature is also rather consistent that mitogen-stimulated lymphocyte proliferation is decreased 30 %60 % during recovery from acute bouts of cardiorespiratory exercise, with values approaching pre-exercise levels within 2-4 hours (8,10,11,13,18,35). The mechanism, however, underlying this decrease is disputed. with several researchers advancing the idea that it is due to a true down-regulation of T lymphocyte function because of elevated hormones and other soluble factors (13,18) while others hold that numerical shifts in lymphocyte subpopulations are responsible (10,11,35). There is also disagreement in the Literature regarding the contrast between moderate-versus high-intensity exercise, with some reporting a decrease in mitogen-stimulated lymphocyte proliferation regardless of the intensity of exercise (IS), while others report little change following moderate-intensity exercise (22,35). Interpretation of many lymphocyte proliferation studies is made difficult because lymphocyte subpopulation data are often not reported. Additionally, assay methods vary widely, highlighted by differences in mitogen doses, use of isolated mononuclear cell suspensions versus whole-blood samples, and the length of incubation with both the mitogen and radiolabeled thymidine. The purpose of this study was to compare the mitogen-stimulated lymphocyte proliferative response of highversus moderate-intensity exercise. To aid in the interpretation of the results, measurements of changes in lymphocyte subpopulations and hormone concentrations were conducted. Adjustments in post-exercise proliferative responses were made to account for alterations in the number of T cells to determine whether a true per-T lymphocyte functional change occurred in response to the acute exercise sessions.
200 Int. J. Sports Med. 15 (1994)
Material and Methods
Subjects Ten well-conditioned male runners between the ages of 17 and 3 1 were chosen to participate in the study. Most of the subjects were members of the University cross-country running team. This type of subject was chosen to ensure successful completion of a 45-min treadmill run at 80% V02max. Subjects voluntarily signed an informed consent statement approved by the University Institutional Review Board for Human Studies.
Research design One week prior to the scheduled start of the study, subjects came to the Human Performance Laboratory (HPL) for orientation, and measurement of body composition and aerobic capacity. Body composition was assessed from a seven-site skinfold test (26) and VOzmax was determined utilizing the protocol by Bruce et al. (2). Oxygen uptake and ventilation were measured using a Sensormedics MMC Horizon System metabolic cart (Sensor Medics, Yorba Linda, CA). Maximal heart rate was measured using the Polar Pacer heart rate monitor (Polar USA, lnc.). Training history was assessed through a questionnaire. Subjects were paired, and reported to the HPL at 0700 on two separate days, two weeks apart, having abstained from vigorous exercise, food, or drink other than water for 12 h. One subject of each pair was randomly assigned to run for 45 min on a level treadmill at 8 0 % V02max (high intensity) while the other engaged in 45 min of graded treadmill walking at 50 % V02max (moderate intensity). During the second visit, roles were reversed. Members of each pair exercised at the same time within the laboratory, with heart rate and metabolic measurements recorded every 5 rnin with the same equipment used during assessment of aerobic capacity to ensure that the proper intensity was being maintained throughout the 45 min exercise session. After reporting to the HPL at 0700, subjects rested quietly for 5 10 rnin. The first blood sample was taken at 07 10. At 071 5, subjects started their 45-min exercise session, with blood samples taken immediately after (0800), and then again at 1, 2, and 3.5 h of recovery (0900, 1000, and 1130). A meal of whole grain breakfast cereal, raisins, and low-fat milk adjusted to 10 kilocalories per kilogram of body weight, 70 % calories from carbohydrate, 15 % calories from fat, and 15% calories from protein, was fed to the subjects at 0915.
Blood analysis All 5 blood samples were drawn from an antecubital vein with subjects in the seated position. A clinical hematology laboratory performed routine complete blood counts (CBC) using a Coulter STKS instrument (Coulter Electronics, Inc., Hialeah, Florida). Heparinized whole blood was used for immune cell phenotyping for analysis of lymphocyte subset profiles. Lymphocyte phenotyping was accomplished by direct immunofluorescence labelling of cell surface antigens with mouse anti-human monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) from
D. C. Nieman, A. R. Millel: D. A. Henson, B. J. Warren et al. AMAC, Inc. (Westbrook ME). The immunophenotyping was performed on heparinized whole blood a n 4 after lysis of the red cells, was analyzed on a Becton Dickinson FACScan flow cytometer. Gated lymphocyte populations were verified by labelling with antibody to CD45 while anti-CD14 was used to enumerate contaminating monocytes in the lymphocyte gate. A total of 5,000 lymphocytes were counted per sample. Lymphocyte subsets were analyzed to determine percent total T (CD3+), total B (CD19+), T helperlinducer (CD3+CD4+), T cytotoxicl suppressor (CD3+CD8+), and natural killer (CD3CD16+CD56+) (NK) cell subsets. Absolute numbers were calculated using CBC data. Dual-labelled samples were run and analyzed using appropriate negative, isotype and lymphocyte controls The mitogenic response of lymphocytes was determined in whole blood culture using suboptimal mitogen doses (1.27,37). Heparinized venous blood was diluted 1: 10 with RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 IU/ml):streptomycin (100 pglml), 200 mM L-glutamine, and 5 x 1O4 M 2-mercaptoethanol. Concanavalin A (Con A) was prepared in RPMI 1640 media, at a concentration of 1 mglml, and then further diluted with RPMI 1640-FCS media to the suboptimal concentration of 20pglml for Con A. One hundred microliters diluted blood were dispensed into each of triplicate wells of a 96-well roundbottom microtiter plate. To each of these were added 100 microliters of the suboptimal concentration of Con A. Control wells received FCS-supplemented media instead of mitogen. After 72 h incubation at 37 "C, the cells were pulsed with 1 pCi of thymidine (methyl)-3H (New England Nuclear, Boston, MA) prepared with RPMI 1640-FCS. After pulsing, the plate was incubated for an additional 4 h. The cells were harvested and the radionucleotide incorporation was assessed by liquid scintillation counting using Beckman Ready Safe (Beckman Instruments Inc., Fullerton, CA) as the scintillation cocktail. Con A results are presented as both unadjusted (counts per min, cpm x 1&< with the control count subtracted out) and adjusted (cpm per T [CD3+] cell, calculated by determining the number of T cells in each assay well based on the lymphocyte count and T cell percentage). Plasma cortisol was assayed using a competitive solid-phase 1251radioimmunoassay technique (Diagnostic Products Corporation, Los Angeles, CA). For plasma catecholamines, blood samples were drawn into chilled tubes containing EGTA and glutathione (Amersham, #RPN532 Vacutainer tubes), centrifuged, and the plasma stored at - 80 "C until analyses. Plasma concentrations of norepinephrine and epinephrine were determined by high pressure liquid chromatography (HPLC) with electrochemical detection (21). Specifically, 1.0 ml aliquots of thawed plasma were added to 1.5 ml microcentrifuge tubes, each containing 5 mg Affi-Gel 601 (Bio-Rad Laboratories), which had been previously washed with 1 ml aliquots of 1 M HCI, 0.1 M NaOH and H20. An internal standard (10 p1 of 1 pM dihydroxybenzylamine) was added to each tube, and the tubes were mixed for 10 rnin at room temperature. After centrifugation, the catecholamine-free plasma was removed by aspiration and the gel washed twice with H20. Catecholamines were eluted from the gel by the addition of 200p1 of 0.75 M acetic acid and filtered through microcentrifuge filter tubes containing 0.45pm nylon membranes (Costar, TNC., Cambridge, MA). The extract containing the catecholamines was stored at -80 "C until HPLC. HPLC with electrochemical
Effect of High- Versus Moderate-Intensity Exercise on Lymphocyte Subpopulations
k t . J Sports Med. 15 (1994) 201
Table 1 Subject characteristics (mean f SE). N
Age (~rs)
Ht (cm)
Wt (kg)
% Fat
VOzrnax' (rnl/kg/rnin)
10
22.1 f1.3
181 f2.0
69.9 f2.2
7.7 f 0.7
66.0 f 1.9
HRrnaxt (b~m)
VErnaxS (I/rnin)
194 f3
173.0 f 6.3
krn/wk#
64.7 f 8.7
VOpmax = maximal 0 2 uptake; tHRmax = maximal heart rate; SVEmax = maximal ventilation rate; #km/wk = distance run per week during the past year
detection was performed on an ESA Coulochem I1 system with an ESA HR-80 catecholamine column. The mobile phase (ESA Cat-A-Phase containing 3 % methanol) was recirculated at 1.25 mllmin. Norepinephrine and epinephrine were quantified by comparison with standard solutions using the ESA dual electrode electrochemical detector (ESA model 5021 conditioning cell electrode set at 350mV, ESA model 501 1 Analytical cell electrode 1 set at 100 mV and electrode 2 set at - 300 mV). The values were corrected for recovery of the internal standard. Plasma volume changes were determined using the method of Dill and Costill (6). Lactate concentrations were analyzed in duplicate by injecting 25 yl whole blood into a lactate analyzer (YSI Inc., Yellow Springs, OH).
Statistical analysis All post-test blood values except for lactate and the three hormones were adjusted for changes in plasma volume prior to statistical analysis. For the adjusted Con-A values, the actual number of T cells present in the assay well were used when making calculations. Leukocyte and lymphocyte subsets, Con A-stimulated lymphocyte proliferative responses, and hormone values were analyzed using 2 (high- vs moderate-intensity conditions) x 5 (five times of measurement) repeated measures ANOVA with the BMDP 2v statistical software module (7). This statistical module allows a repeated measures analysis when there are two "within subjects" comparisons (in this case, within and between conditions, with subjects acting as their own controls). After determining that none of the baseline values for any of the immune system tests differed between conditions, the immediate-post--exercise, I-, 2-, and 3.5-h recovery values were compared between conditions using paired t-tests with the Statistical Analysis System software program (30). For these 4 multiple comparisons, a Bonferroni adjustment was made, with statistical significance set at P = 0.0125, with values between this and 0.05 treated as trends. Within each condition, each level of the factor (4 time measurements) was compared with baseline values using the same approach as just described for between condition comparisons. To avoid confusion, these data (within condition multiple comparisons) are not represented in most of the tables, but are mentioned in the test where appropriate.
Results The results of the baseline body composition and graded maximal exercise tests are given in Table 1. Subjects can be described as lean and highly fit, having averaged approximately 65 kmlwk in running during the past year. During the high-intensity 45-min exercise session, subjects averaged 16.1 f 0.34 kmlh, with a V 0 2 of 52.5 f
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1.0 mllkglmin (80 % V02max), ventilation of 116 5 llmin, heart rate of 176+_4beatslmin (91 % maximum heart rate), respiratory exchange ratio of 0.95*0.01, and rating of perceived exertion of 14.3k0.3 (6 to 19 scale). During the moderate-intensity 45-min exercise session, subjects averaged 7.3 0.1 kmlh on a grade of 6.5+0.6%, with a V 0 2 of 33.2*0.9 mllkglmin (50% V02max), ventilation of 5 8 f 2 llmin, heart rate of 131 f 4 beatslmin (68 % maximum heart rate), respiratory exchange ratio of 0.85 f 0.01, and rating of perceived exertion of 9.8 +_ 0.7. Pre-test lactate concentrations were 1.1 f 0.1 mmolll for both conditions, and increased to 4.8 k 0.5 and 1.6 f 0 . 2 mmolll in the high- and moderate-intensity conditions, respectively.
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All post-exercise values except for cortisol and the catecholamines have been adjusted for plasma volume shifts which measured 10.3 k 1.4 % and 8.3 k 1.1 % immediately postexercise in the high- and moderate-intensity conditions, respectively, with all other recovery values approaching baseline. Significant interaction effects occurred for total leukocytes and each of the subsets except for basophils and monocytes (Table 2). High- versus moderate-intensity exercise resulted in a greater immediate post-exercise leukocytosis and lymphocytosis. All leukocyte subset values at 1-hour recovery tended to be similar between intensity conditions, but by 2-h recovery, a leukocytosis and neutrophilia were experienced within the highintensity condition which was sustained for the remainder of the 3.5 h of recovery. Circulating numbers of lymphocytes were significantly decreased (p < 0.001) relative to baseline values by 1-h recovery, and were sustained throughout 3.5-h of recovery, reaching their lowest point at 2-h recovery (58% and 41 % decrease in the high- and moderate-intensity conditions, respectively). Significant interaction effects occurred for each of the lymphocyte subsets (Table 3). Immediately post-exercise, circulating numbers of each of the lymphocyte subsets were greater in the high- versus moderate-intensity condition. In the high-intensity condition, the immediate post-exercise lymphocytosis was primarily comprised of T and NK cells. Although 1- and 3.5-h post-exercise values were similar between conditions, circulating numbers of cells from each lymphocyte subset except for NK cells were lower at 2-h post-exercise in the high-intensity condition. For both conditions, the 2-h lymphopenia was represented largely by T cells (72 %, represented by both T helper and T suppressor cells) with NK (22 %) and B (6 YO)cells comprising the remainder. Fig. 1 depicts the unadjusted Con A-stimulated lymphocyte proliferative response while Fig. 2 expresses this on a per T lymphocyte (CD3+) basis. For the unadjusted values (Fig. I), no significant interaction effect (intensity condition over 5 time measurements) occurred (p = 0.606). However, a
202 Znt. J. Sports Med. 15 (1994) -
D. C. Nieman, A. R. Millec D. A. Henson, B. J. Warren et al.
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Table 2 Leukocyte subsets before and during recovery7 from high- versus moderate-intensity exercise (mean SE). Variable
Pre-exercise
Post-Exercise
1 h-Recovery
2 h-Recovery
3.5 h-Recovery
(109/ L)
Effect (condition x time) p value
Total Leukocytes High5 Moderate
5.98f 0.48 6.03f 0.56
Neutrophils High Moderate
2.71 f 0.30 2.75f 0.36
Lymphocytes High Moderate
2.37k0.18 2.33+0.16
Basophils High Moderate
0.05f 0.01 0.05f 0.01
Eosinophils High Moderate
0.26f0.04 0.23+ 0.02
Monocytes High Moderate
0.60f 0.04 0.67+ 0.07
*p < 0.0125 for contrast between conditions; trecovery values have been adjusted for shifts in plasma volumes; §high and moderate represent high- (80 % VOnmax) and moderate- (50 % VOnmax) intensity exercise, respectively
Table 3 Lymphocyte subsets before and during recoveryt from high- versus moderate-intensity exercise (mean f SE). Variable
Pre-Exercise
Post-Exercise
1 h-Recovery
2 h-Recovery
3.5h-Recovery
1.70f0.18 1.72f0.14
2.60i0.27' 1.78f0.19
0.95f 0.10 1.16f0.08
0.73f 0.07' 1.03f 0.09
0.93f 0.08 1.16+0.10
< 0.001
1.38f0.18' 0.97f 0.11
0.59f 0.08 0.70f 0.05
0.47It 0.06' 0.64f0.06
0.55f 0.06 0.70f 0.07
< 0.001
+
1.06f0.16' 0.65f 0.10
0.32f0.03 0.38f 0.05
0.23f 0.02* 0.34f0.04
0.31f 0.03 0.39f 0.04
0.40f 0.08 0.32f 0.05
1.01f 0.12' 0.48f 0.08
0.10f0.02 0.15f0.04
0.09f 0.01 0.11 f 0.02
0.16 f0.03 0.19 + 0.02
< 0.001
0.25f0.04 0.25f 0.03
0.41f 0.07' 0.25f 0.04
0.1750.03 0.19f0.02
0.15f 0.02' 0.20f 0.03
0.19 f 0.03 0.20f 0.03
< 0.001
(1091~) T lymphocytes (CD3+) High5 Moderate
T helper/ inducer (CD3+CD4+) High 0.99f 0.15 Moderate 1 .OOf 0.09
Effect (condition x time) p value
T suppressor/ cytotoxic (CD3+CD8+) High Moderate
0.61f 0.08 0.61 0.08
Natural Killer (CD3-CD16+CD56+) High Moderate
B lymphocytes (CD19+) High Moderate
'p < 0.0125 for contrast between conditions; trecovery values have been adjusted for shifts in plasma volumes: §high and moderate represent high- (80 % VOnmax) and moderate- (50 % VOnmax) intensity exercise, respectively
Effect of High- Versus Moderate-Intensity Exercise on Lymphocyte Subpopulations cpm x
Int. 1 Sports Med. 15 (1994)
cpmlCD3+ cell
T
22 -
Moderate lntensity 1.4
\
17-
Moderate Intensity
12High Intensity
Pre-exercise
Post-exercise
,
1-h recovery
1
. 2-h recovery
* High lntensity 0.8
3.5-h recovery
Fig. 1 Whole blood, unadjusted Con A-stimulated lymphocyte
proliferative response to high- versus moderate-intensity exercise in 10 well-conditioned young males. The pattern of change was not significantly different between intensity conditions (p = 0.606), but a strong time effect (p < 0.001) was observed for both conditions. p < 0.05, within condition versus baseline " p < 0.01 25, within condition versus baseline
Pre-exercise
Post-exercise
1-h recovery
2-h recovery
3.5-h recovery
Fig. 2 Whole blood, Con A-stimulated lymphocyte proliferative response adjusted per T cell (CD3+)to high- versus moderate-intensity exercise in 10 well-conditioned young males. The pattern of change was not significantly different between intensity conditions (p = 0.276). 'p < 0.05, within condition versus baseline
Table 4 Cortisol and catecholamine responses before and during recovery from high- versus moderate-intensity exercise (means? SE).
Variable (nmolll)
Pre-Exercise
Post-Exercise
1 h-Recovery
Cortisol
High§ Moderate
633 f 53 671 k 76
926 f 91 742 f 78
804 f 82 596 f 50
2 h-Recovery
*
694 73 535 f 66
3.5 h-Recovery
Effect (condition x time) p value
456 f 34 431 f 45
Epinephrine
High Moderate
0.394* 0.098 0.198+ 0.034
1.290k 0.177' 0.568 + 0.120
0.293f 0.093 0.171 k 0.018
Norepinephrine
High Moderate
1.59 f 0.28 2.07 ? 0.46
16.30 f 2.87' 5.56 ? 1.51
+
2.05 0.36 2.00 k 0.29
'p < 0.0125 for contrast between conditions; §high and moderate represent high- (80 % VOnmax) and moderate- (50 % VOnmax) intensity exercise, respectively
significant time effect was measured (p
