Heavy exercise has been linked to transient alterations in immune function. Prolonged, high-intensity running and cycling has been shown to induce leukocytosis and neutrophilia immediately after exercise with subjects becoming lymphocytopenic during recovery (7,17). Additionally, resistance exercise also stimulates a similar pattern of leukocytosis postexercise, followed by lymphocytopenia in the recovery, however, to a lesser extent observed in endurance exercise (3,4,15). To characterize the immune response to exercise, most studies (2,4,6,13,15) have examined circulating immune cell numbers and/or function. However, the number of immune cells in circulation only reflects ∼0.2% of total leukocyte mass (8), the balance of leukocytes existing in lymphoid, bone marrow, and other compartments, including saliva. Saliva encompasses various enzymes, immunoglobulins, hormones, peptides, and leukocytes (20). Collection of saliva samples is less invasive than blood sampling, and may present an attractive alternative method to examine exercise-induced changes in lymphocyte numbers.
For many sports, in-season extends into the winter months or begins early in the spring. Understanding the influence of cold exposure on immune responses following an acute anaerobic training session is important for assessing the risk of decreased immune function (e.g., upper respiratory tract infections, URTIs) in athletes and other exercising individuals in a cold environment. Evidence indicates that an acute bout of intensive endurance or resistance exercise induces transient changes in cells of the immune system and has possible implications in immune function (3,4,17). Although inconclusive, single bouts of these intensive types of exercise may induce immune suppression and increase the susceptibility to upper respiratory tract infections (URTIs; (6)). Conventional wisdom holds that cold exposure also suppresses immune function and increases URTI incidence; however, this belief is not consistently supported by available evidence (26). Exercise in a cold environment has been found to produce either no difference (2) or blunted (5,12,22) responses compared with warm conditions in total and differential leukocyte counts. Establishing a relationship between exercise and immune function is important as athletic performance may be negatively impacted because of URTI following strenuous exercise, compared with their more sedentary counterparts (8).
Anaerobic training is typically used in a variety of sports settings. Because of the complexity of attributes required in sports such as soccer, basketball, and hockey, they are considered randomized intermittent, dynamic, and skilled movement type sports (RIDS). To train for RIDS, an emphasis needs to be placed on skill-related fitness components including speed, agility, and quickness (SAQ; (1,10,21)). The purpose of this study was to examine the influence of a cold environment on the salivary lymphocyte response to an acute anaerobic training session, emphasizing SAQ. We hypothesize that a cooler climate will further reduce lymphocyte count following SAQ training, which may further increase the risk for developing URTI.
Experimental Approach to the Problem
Most research has examined immune responses following either endurance or resistance exercise. Our study was unique in that it compared immune responses following anaerobic exercise in 2 different environmental conditions and measured salivary lymphocyte numbers. If the immune system is suppressed during the cool exposure, then field athletes who are acutely exposed to a cool environment may be more susceptible to URTI. Determining if a cool environment makes athletes more susceptible to URTI is important to understand, as it may be better to not allow athletes to stand outside before practice/competitions or ensure proper warm-up immediately after being exposed to the cool environment.
Nine lightly clothed (∼0.3 clo) recreationally active volunteers between the ages of 19 to 22 (7/2 women/men: age, 21 ± 1 years; height, 168.7 ± 7.3 cm; weight, 66.4 ± 8.4 kg; body fat, 20.6 ± 7.6%) from the local university were recruited for this study. Subjects were excluded if they had a cold or symptoms of infection for the week before testing. This study was approved by the Institutional Review Board and all participants gave written informed consent.
Subjects participated in a familiarization session to ensure competency of the testing. To minimize the influence on the immune system, subjects adhered to instructions to not ingest anti-inflammatory medications or exercise 24 hours before testing. Subjects then participated in 2 speed, agility, and quickness (SAQ) sessions. One session took place in a thermoneutral environment (18.9° C, 41% RH, 4.5 m·s−1 wind speed) in Biddeford, Maine, and the second took place 7 days later in a cool environment (10.4° C, 62% RH, 0.6 m·s−1 wind speed) located in Thorsmork, Iceland. Subjects participated in a 5-minute warm-up, a 15-minute environmental exposure, and a ∼30-minute nonrandomized SAQ testing session. The SAQ tests included 2 trials of: 20-m sprints, 40-m sprints, two 300-yd shuttle runs, and 3 trials of the T-Test and Box Drill. All trials had a 2-minute rest period with the exception of the shuttle run, which had a 3-minute rest period. Subjects were asked to give their maximal effort.
Subjects were allowed to return to a room temperature environment postexercise. Saliva was collected through passive drool into sterile 15 ml polypropylene conical tubes at baseline, immediately postexercise, and after 2 hours of recovery for lymphocyte. Saliva was preserved by the addition of 4 ml of saliva blood cell preservation solution (AboGen Inc., Portland, ME, USA). Saliva was stored at room temperature until processed (1 week in Maine and 2 weeks for Iceland samples). The total saliva sample collected was calculated by the final volume minus the 4 ml of preservation solution added. Lymphocytes were isolated from the saliva by using proprietary methods (AboGen Inc.). Briefly, preserved saliva sample was centrifuged (800g) for 10 minutes at 4° C. The supernatant was removed and the pellet was resuspended in phosphate-buffered saline (PBS), 1% fetal bovine serum (FBS), and 0.01% sodium azide. Samples were mixed on a rotator for 10 minutes at 4° C. Samples were layered over Ficoll-Paque PLUS, and then centrifuged at 4° C for 45 minutes at 600g. The lymphocyte fraction was extracted at interface, and then the cells were washed 3× in Hanks balanced salt solution (ThermoFisher Scientific Gibco, Waltham, MA, USA) by centrifugation at 800g for 10 minutes at 4° C. Cells were stored at 4° C in PBS, 1% FBS, 0.01% sodium azide until counted. Cells were counted at least 4 times using standard hemocytometer (Sigma-Aldrich, St. Louis, MO, USA) to calculate the number of cells per milliliter of saliva donated.
All statistical analyses used SPSS Version 21 (IBM, Chicago, IL, USA) and an alpha level of p ≤ 0.05 was used to detect any significant differences in immune responses. Data were tested for normal distribution using Kolmogorov-Smirnov tests and were found to be normally distributed (p = 0.799–0.910). Mauchly's test for sphericity indicated that the assumption of sphericity was accepted (p = 0.790). Changes in lymphocytes were analyzed using a 2 × 3 (condition by time) repeated-measures analysis of variance (ANOVA). Mean lactate levels from the cool environment were also compared with those in a thermoneutral environment using a 2 × 2 repeated-measures ANOVA.
Plasma lactate significantly increased (p < 0.001) from rest to immediately postexercise in both warm (1.06 ± 0.31 to 10.57 ± 2.29) and cool (1.01 ± 0.39 to 11.11 ± 2.65) conditions, with no difference between conditions. Salivary lymphocytes (s-LYMPH) increased (p < 0.001) immediately postexercise, followed by a decrease (p < 0.001) below baseline values after 2 hours of recovery in both conditions (Table 1). The s-LYMPH counts were lower (p < 0.001) in the cool environment than in the warm environment at the postexercise time point (Figure 1). The coefficient of variability (CV) scores for the s-LYMPH for the warm condition were 6.4% (baseline), 6.0% (postexercise), and 12.7% (2-hour postexercise), and the CV scores for the cool condition were 4.9% (baseline), 9.3% (postexercise), and 13.1% (2-hour postexercise), respectively.
The purpose of this study was to examine the influence of environmental exposure on salivary lymphocytes following an acute anaerobic training session. In the present study, engaging in anaerobic exercise resulted in significant alterations of s-LYMPH. In addition s-LYMPH were significantly elevated postanaerobic exercise in a warm environment compared with a cool environment. Research has consistently reported that acute bouts of endurance and resistance exercise have influenced the migration of immune cells in the peripheral blood (3,18,19,25). Although we examined saliva, similar to other studies (12,18,25), we also observed lymphocytosis immediately postexercise and a lymphopenia into the recovery period. This shows that acute anaerobic exercise can also produce a similar biphasic response as reported with endurance and resistance exercise (11).
As previous research has shown lymphocytes respond to increases in catecholamines and cortisol, which are typically elevated during vigorous physical activity. Our data are similar to previous findings that found lesser fluctuations in circulating lymphocytes following exercise in cold vs. warm conditions (5,14,22). In the cool environment, subjects displayed a less severe fluctuation in lymphocytes (at both postexercise and 2-hour recovery time points) than after exercise in the temperate environment. Neuroendocrine mechanisms might be responsible for this difference. The lymphocytosis during and after exercise is accomplished by flushing lymphocytes out of marginal pools. The mechanisms responsible for mediating this process are not fully understood, but available evidence implicates sympathetic nervous activity (16), catecholamines (9), and cortisol (24) as mediators of the process. The effect of temperature on the neuroendocrine response to exercise is somewhat controversial, with opposing findings available in the literature. Specifically, LaVoy et al. (13) stated that exercise in colder temperatures increases the levels of lactate, cortisol, and norepinephrine, and potentially evokes greater fluctuations in immunity. In contrast, Walsh and Whitham (26) stated the opposite: that exercise in colder conditions reduces plasma catecholamines and cortisol, and thus blunts exercise-induced changes in immune parameters. Although we did not measure cortisol or catecholamines in the present study, our findings may be viewed as supporting the conclusions of Walsh and Whitham, where cold exposure gave rise to a lesser response in lymphocytes. Furthermore, lactate, which has been proposed as a proxy measure for epinephrine (23), was measured in the present study and was found to be similar between environmental conditions.
The clinical significance of exercise-induced changes in lymphocyte numbers (either circulating or in saliva) is unclear. It is not known whether an increase in lymphocytes cells is a positive response, increasing the availability of cells to become involved in immune reactions, or if it is a negative response, meaning that cells have been diverted from areas in which they were previously involved in immune reactions. Furthermore, a simple accounting of circulating leukocyte number does not address the function of those cells. However, a conservative interpretation of the present findings suggests that SAQ exercises in cool environment resulted in a lesser disturbance in salivary lymphocyte homeostasis both immediately after exercise (cool = +13% increase; warm = +77% increase) and at 2-hour recovery (cool = −14% decrease; warm = −27% decrease). Thus, there is no indication that exercise in the cool environment presented a greater challenge to the subjects' immunity.
In summary this was the first study to report salivary immune responses (a) with anaerobic exercise and (b) in 2 different environmental conditions. The results of our study suggest that acute anaerobic exercise can elicit enough of a stress to change lymphocyte homeostasis. Furthermore, participating in a warm environmental condition may result in a more robust response of the lymphocytes immediately postexercise with a lymphocytopenia 2 hours into the recovery period.
The main finding of this study was that salivary lymphocytes are responsive to sprinting exercise, and this response was blunted following cool exposure. Although interpreting the meaning of a lesser change in salivary lymphocytes on host defense is premature, it seems conservative to interpret our data as indicating that there was no indication that exercise in the cool environment had a harmful effect on host immunity, as indicated by a lesser disturbance in salivary lymphocytes. Thus, there is no reason to believe that coaches and athletes who must practice and compete in cool environments are exposing their immune systems to a greater challenge than when exercising vigorously in a warmer environment.
This study was supported by AboGen Inc., and the New England Chapter of the American College of Sports Medicine Undergraduate Research Experience Grant. The results of the present study do not constitute endorsement of the product by the authors or the National Strength and Conditioning Association.
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