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Endocrinology and Immune Function

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Acute effects of maximal exercise and heat stress on NK cell activity

DONNA B. TATE, PETRA B. SCHULER, AND STEPHEN PRUETT

Mississippi State University, Departments of Physical Education, Health, Recreation, and Human Performance and Biological Sciences, Starkville, MS

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TATE, D. B., P.B. SCHULER, & S. PRUETT JEPonline Vol. 1, No. 2, 1998. The purpose of this study was to determine if maximal exercise, a rise in core body temperature from exercise, or implemented heat, elicit physiological and/or hormonal (cortisol) changes that acutely affect natural killer (NK) cell activity. Seven well-trained males completed each of three trials: (Trial 1-max exercise in room temperature; Trial 2-max exercise in controlled heat chamber; Trial 3-whirlpool bath). NK cell activity from isolated peripheral blood mononuclear cells (PBMC) was measured by 51Cr -release assays from pre-trial resting blood samples; pre-trial values were compared to post (10 minute) trial blood samples from each trial. Only the post-exercise values in Trial 1 and 2 were significantly increased. Serum cortisol concentrations were also measured from pre and post blood samples to determine if cortisol concentrations corresponded with NK cell activity. Radioimmunoassays showed significant increases in cortisol concentrations from pre to post in exercise Trials 1, 2, but not from Trial 3. The results of this study suggest that the effects of maximal exercise seemed to elicit acute increases in NK cell activity and cortisol concentrations. Heat alone did not significantly affect either response, and heat in addition to exercise did not affect these parameters more than maximal exercise alone.

Key Words: NATURAL KILLER (NK) CELL ACTIVITY, CORTISOL CONCENTRATIONS, MAXIMAL EXERCISE, PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) 

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Introduction
Research supports the view that physical exercise can elicit changes in the immune system. A large body of literature focusing on the immunological effects of exercise has accumulated over the past few years (1,2, 3). Even early studies dating back to the beginning of the century document both desirable and undesirable influences of exercise on certain elements of the immune system response (4).

Another theory has arisen suggesting heat (rise in core body temperature) as a possible mechanism for an immune response to exercise. Exercise, as well as passive heating (i.e., saunas and whirlpool baths), and environmentally hot temperatures can cause a rise in core body temperature. Significant increases in natural killer (NK) cell activity have been seen with increases in core body temperature (5,6). In addition to heat stress, physical activity stress can elicit the release of stress hormones and catecholamines from the neuroendocrine system (7,8). As seen in several studies, there is a pronounced suppressive effect on the activity of NK cells following strenuous exercise (8,9,10) and severe increases in temperature (5,6, 11). The suppressive effects shown on NK cell activity could be due to the release of stress hormones such as cortisol, which has been shown to be immunosuppressive (3,12,13). Therefore, similarities suggest that part of the exercise-induced changes in NK cell activity may be due to the increase in core body temperature, that is induced by the exercise, or to the release of cortisol which can be caused by exercise or heat. The purpose of this study was to measure the activity of NK cells and the levels of cortisol released after maximal exercise stresses and heat stresses to determine if heat stress, maximal physical activity stress, or the body's responses to either, have acute independent effects on the activity of NK cells.

Methods
Subjects
Seven male subjects (18-26 years old) participated in this study. The subjects were well-trained (maximal volume of oxygen consumed [VO₂ max] m = 47.2 ± 8 ml/kg/min) males who completed each of three separate trials. Criteria for being "well-trained" for this study consisted of a VO₂ max measurement of > 40 ml/kg/min on a cycle ergometer. One subject was unable to complete the final trial due to physiological complications (not able to draw blood samples). All procedures followed were in accordance with and approved by the guidelines for human testing established by the Mississippi State University Institutional Review Board.

Trial 1
The study consisted of three separate trials. The trials were not randomized so that subjects were not exposed to the heated trials until it could be determined if each subject qualified for the well-trained study criterion. The first session consisted of an orientation providing an informed consent, medical history form, and a 24-hour dietary and activity history questionnaire. Subjects then had basic anthropmetric and physiologic measures taken such as height (HT), weight (WT), resting heart rate (RHR), and body composition (% BF) using the sum of seven skinfold thicknesses (14) and the Siri equation (15). Subjects were prepared for a maximal graded exercise test according to 1996 ACSM manual guidelines for exercise testing and prescription by being hooked up to a 12-lead ECG monitor and measuring blood pressure (BP) and heart rate (HR) in each of three resting positions (supine, sitting, and standing). Pre-trial blood samples from the antecubital vein (collecting at least 10 ml into heparinized tubes) were drawn prior to the exercise. Subjects were then positioned on a Sensormedics electronically-braked cycle ergometer (Ergometrics 800S, Yorba Linda, CA) and hooked up to a metabolic cart (Sensormedics Model 2900C, Anaheim, CA) using open-circuit spirometry to assess breath by breath analysis of oxygen consumption during the test to determine VO₂max . Room temperatures for this trial were recorded between 20-22 degrees C. Core body temperature changes were measured via the tympanic membrane by a Thermoscan Pro-1 Instant thermometer and recorded every minute with pre core temperature recorded at the first minute of exercise. Although tympanic membrane measures do tend to be lower than traditional core measures (rectal, bladder, esophageal), the tympanic temperature measurements have been shown to be a valid and efficient method for measuring core body temperature changes (16,17). Post blood samples were drawn 10 minutes after the cessation of the exercise test.

Exercise Protocol
A standard workrate during the test was set at 50 watts (W) and increased every three minutes until subjects reached their maximal effort. Maximal effort was determined from subjects reaching 2 of these 3 criterion: volitional exhaustion, a respiratory exchange ratio (RER) of > 1.1 (18), and/or a HR within 10 beats per minute of age-predicted maximum HR (19). A pedal speed of 75 rev/min was maintained throughout the test. Heart rate, ratings of perceived exertion (Borg scale, 1982), and a 3-lead (V1 , V5 , II) rhythm strip were recorded every minute.

Trial 2
Subjects were brought in one week later to perform a second maximal graded exercise test on the cycle ergometer. A controlled heat chamber was used with heat implemented at 29-36 degrees C and a humidity measured at 50-70%. Exercise protocol was the same as in Trial 1, as well as HR, BP, ECG monitoring, core temperature changes, and pre and post blood samples.

Trial 3
The final trial was carried out in a whirlpool bath heated to 39-41 degrees C. Pre blood samples, HR, and BP were taken prior to submersion. Subjects were then submersed to the neck in the bath until pre temperatures reached the maximum core body temperature obtained in the exercise trials (total time approximately 14 minutes to equilibrate the average time measured in the exercise trials). HR was monitored throughout the trial. Post blood samples were taken 10 minutes after the trial.

Immunological Assessment
Throughout the testing procedures, all blood samples were kept refrigerated or on ice until they could be assessed in the lab. Whole blood samples were transferred to sterile tubes with equal parts saline to isolate the natural killer (NK) cells represented in the peripheral blood mononuclear cell (PBMC) population. This was accomplished by using a Ficoll-paque gradient. Plasma samples were obtained by separation from the whole blood, and stored in -80 degrees freezer until further analysis were done. PBMC (effector cells) were then washed and adjusted to 1 x 10⁷ cells/ml. K562 (NK-susceptible cells) and P815 (NK-resistant cells) from culture were then labeled with ⁵¹Cr and incubated for 90 minutes. P815 cells were used as a control to insure that there was no abnormal non-NK activity in the PBMC population of the subjects. After incubation, the labeled cells were washed 3-4 times in supplemented saline and cell numbers adjusted to 1 x 10⁵ cells/ml. 100 ul of target cells were added to each of a 96-well round bottom culture plate. The isolated effector cells were added in triplicate to each well at effector to target ratios of 25:1, 12:1, and 6:1. Fresh medium (RPMI with serum/antibiotics) was added to provide a total of 200 ul of solution per well. Spontaneous release of chromium was determined by adding 100 ul of fresh medium to the 100 ml of labeled target cells. Maximum release of chromium was determined by adding 100 ul of 2N HCl to 100 ul of the labeled target cells. The effector and target cell preparations were incubated for 4 hours at 37 degrees C, 5% CO₂. After incubation, the plates were centrifuged for 5 minutes at 250 x g to pellet the cells. Supernatant (100 ul) was collected and measured for radioactive chromium using a gamma counter. The activity was expressed as lytic units as described by Bryant et al. (20).

Serum Cortisol Measurement
Cortisol concentration was determined by radioimmunoassay using a commercially available kit (Diagnostic Products Corp., Los Angeles, CA). Serum from whole blood samples was separated and a standard curve established using cortisol standards provided with the kit. The cortisol concentration in each sample was determined by comparison to the standard curve. Standards were assayed in duplicate, and the variation between duplicates was less than 10% of the mean. The correlation coefficient for the standard curve was greater than 0.98.

Statistical Analysis
Immunological Assessment
A three by two repeated measures analysis of variance using the SAS statistical package was employed to assess main effects between time (pre and post) and trial modes (exercise, exercise in heat, and passive heat). Tukey's post hoc analysis was used to compare the post values among the trials and pre and post measures within the trials.

Serum Cortisol Measurement
Differences between the mean of each group and every other group were determined by ANOVA followed by Bonferroni's post hoc test. Data were also analyzed using Student's t test. All analyses were performed with the Instat software package (Graphpad Software, San Diego, CA). Values of p < 0.05 were regarded as statistically significant.

Results
Immunological Assessment
Subjects were well-trained males with VO₂ max measured greater or equal to 40 ml/kg/min (Table 1). Core body temperatures and maximal heart rates were recorded in each trial (Table 1). Venous blood samples were taken pre and post and analyzed for cellular activity in lytic units (Table 1). A repeated measures analysis of variance was employed to analyze main effects between the trials. A significant difference in NK cell activity was found between the trials (p < 0.003). A significant time effect was found between pre and post measures within the trials (p < 0.001) (Fig. 1). A Tukey post hoc analysis was performed revealing NK cell activity, measured in lytic units/107 PBMC, was significantly higher post exercise compared to pre exercise for both exercise conditions but not after the whirlpool bath (passive heating).
 

Table 1. Mean±SD; N=7 (Trial 1,2) N=6 (Trial 3)

	Trial 1	Trial 2	Trial 3
VO2 Max (ml/kg/min)	47.6 ± 7.19	46.80 ± 9.70	NA
Core Temperature (degrees C)^  PRE POST	34.87 ± 0.54  36.01 ± 0.53	35.88 ± 0.48  37.00 ± 0.55	35.68 ± 0.63  37.30 ± 0.44
Lytic units/107 PBMC#  PRE  POST	81.60 ± 65.55 214.10 ± 182.39*	119.69 ± 101.18  224.73 ± 127.73*	54.40 ± 48.78  60.09 ± 35.72
Serum Cortisol (ug/dL)  PRE  POST	10.97 ± 9.43  15.45 ± 11.96*	14.30 ± 7.41  23.14 ± 6.63*	11.40 ± 5.07  10.87 ± 5.71
Max Heart Rate (bpm)	193.70 ± 14.44	192.42 ± 9.86	108.85 ± 14.04

^core temperature measured via tympanic membrane in degrees C
#a measure of cellular activity per 10⁷ peripheral blood mononuclear cell (PBMC) population
*significantly different from the PRE measure

Significant differences were found in Trial 1, t = 2.45 (p < 0.049), and Trial 2, t = 2.88 (p < 0.027) (Table 1, Fig. 1), indicating an effect on the NK cell activity from the exercise and/or the heat incurred from a rise in core body temperature from the exercise. Passive heating alone did not seem to influence a significant change in the activity of the NK cells. Tukey's post hoc analysis was again performed revealing a significant difference between post values in Trial 1(maximal exercise at room temperature) compared to Trial 3 (passive heating) (p< 0.05), and Trial 2 (maximal exercise in heat) compared to Trial 3 (passive heating) (p < 0.05). No significant interaction (trial x time) was found. Therefore, the increase in NK cell activity in the peripheral blood after the trials seemed to be dependent upon the exercise effect alone.
 

Cortisol Measurement
Pairwise comparison of each group by Student's t test indicated a significant (p < 0.05) effect of exercise alone and exercise in heat (See Table 1, Fig. 2).
 

Discussion
This study used well-trained college aged males to determine if maximal exercise with and without heat implementation would affect the acute response of NK cell activity. Three separate trials were used to determine if exercise itself, or if heat alone and in conjunction with the rise in core body temperature from the exercise, had independent effects on NK cell activity. Significant increases were found only in the exercise trials (with and without heat implementation) from pre to post exercise. The passive heat (whirlpool bath) did not cause a significant increase in NK cell activity from pre to post. However, when the two exercise trials were compared to each other, no significant differences were found between the two trials with respect to NK cell activity. Therefore, these results appear to show that maximal exercise alone showed the dominant and only significant effect on the response (increase) in activity of the NK cells.

These results coincide with most trends established by previous research. Studies have shown an increase in NK cell activity during and immediately after maximal exercise (1,2,9). However, it is not well understood why exercise recruits NK cells into the circulation. Some research has attributed the increase in activity to the increase in total cell number (21). Other studies have correlated cardiac output (22) and/or catecholamines (7) with a rise in NK cell activity. Passive heating has also been shown to increase NK cell activity when core temperature has risen greater or equal to 39 degrees C. But in this study, heat, as also seen in current research, does not seem to be an influencing factor in the acute increase in activity of the NK cells (23). However, a dilemma is encountered in studies of this type. Extreme passive heating is required to attain statistically significant increases in body temperature. However, such extremes probably occur less frequently among persons engaged in exercise than the kinds of increases noted here. Therefore, comparing the moderate increases in core body temperature accompanying exercise (1-2 degrees C) is not going to elicit the changes seen in extreme passive heating where core temperature is elevated 3-4 degrees C (6).

Physical exercise and heat stresses have been shown to produce acute hormonal responses. Physical activity results in the greater release of immunomodulatory stress hormones (cortisol, epinephrine, norepinephrine) compared to heat stress. An increase in cortisol, for example, has been shown to be immunosuppressive from 30 minutes to 2 hours after the cessation of a stressor (i.e., exercise) (3). In this study with maximal exercise, cortisol seemed to mirror the changes in NK cell activity 10 minutes post trials resulting in significantly increased cortisol concentrations from pre to post in both exercise trials. In a study by Hoffman et al.(24), high-intensity, short-termed, intermittent exercise in the heat showed a significant decrease in cortisol concentration. Differing from the current study, Hoffman et al., used 5- 15 second anaerobic power tests with 30 seconds of active recovery between each test. However, similar to the current study, as well as others (13, 25), heat alone did not seem to alter serum cortisol concentrations from pre to post trial. Although not statistically significant in this study, serum cortisol concentration decreased slightly in passive heat from pre to post trial. Therefore, according to these results exercise can cause an acute increase in NK cell activity accompanied by acute increases in serum cortisol concentrations up to 10 minutes post exercise. However, NK cell activity could be due to a rise in the total number of NK cells in the peripheral blood. Nevertheless, because of the lack of statistical significance in the temperature changes among the exercise trials of our study, the most accurate conclusion is that maximal exercise in moderately warm environmental temperatures does not affect NK cell activity or cortisol levels differently than maximal exercise at lower temperatures.

Conclusions
NK cell activity has been shown to increase after maximal exercise alone or in the presence of heat exposure. Serum cortisol concentrations have also been shown to increase after exercise and exercise in heat exposure. Neither seemed to be affected by heat alone. This may indicate that up to 10 minutes after maximal exercise, the acute immune response to exercise can be increased without a suppressive effect from an acute increase in serum cortisol concentrations. 

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Acknowledgments:Debbie Kiel , Stephanie Collier, and Wen-jun Wu from the Biological Sciences Immunology Laboratory at Mississippi State University are acknowledged for their great support and technical assistance. Dr. John Lamberth is also acknowledged for his support and assistance. The Longest Student Health Center is acknowledged for their nursing staff assistance and support. 

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Communications and reprints directed to: Donna B. Tate, Vanderbilt University Medical Center, 712 MRB II, 2220 Pierce Ave., Nashville, TN 37232; phone: (615) 936-1824; FAX: (615) 936-1667.

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