There is substantial evidence that exercise causes significant changes to various immune cell parameters (5-8,18,20,23), especially those of the innate immune system, including macrophages (5-8,23), natural killer (NK) cells (5,29), and neutrophils (3,15,16,20,22). Our laboratory has developed a mouse model of exercise and infection in which we have shown that macrophages play an important role on both the positive and negative effects of moderate and fatiguing exercise on susceptibility to infection (5-7); however, the role of neutrophils in this model is not clear. Human as well as animal studies have provided evidence for an alteration in neutrophil function after exercise (3,15,16,20,22). It has been reported that moderate exercise can increase certain activities of neutrophils, whereas vigorous exercise can suppress the activities of neutrophils. For example, the activity of hydrogen peroxide (H2O2) production by blood neutrophils is increased after moderate exercise (22), as are blood neutrophil respiratory burst activity (20) and phagocytic activity (15). Exhaustive exercise, on the other hand, has been reported to suppress the phagocytosis of blood neutrophils (3) and neutrophil oxidative burst activity (3,16). Exercise also has been shown to cause an increase in the number of circulating neutrophils (3,19,20) from endothelial cells and the bone marrow, which is thought to be mediated by stress hormones.
Various nutritional strategies, including glutamine, antioxidants, and carbohydrates, have been investigated as possible countermeasures for the immunosuppression of neutrophils during periods of stressful exercise training and competition (11). However, data are often contradictory, and firm conclusions cannot be made as to whether any of these strategies is an effective countermeasure for exercise-induced immunosuppression of neutrophil function. On the contrary, there is very little evidence on nutritional interventions in conjunction with moderate exercise as an additional stimulus to further enhance the activity of neutrophils.
β-glucans, polysaccharides derived from the cell wall of yeast, fungi, algae, and oats, have been documented to enhance the activities of both the nonspecific and the specific immune system. However, they have received little attention in the field of exercise immunology. β-glucan exerts its effects through direct stimulation of macrophage, neutrophil, and NK cells (4,26). Activation of these cells by β-glucan has been shown to protect the organism from various viral (5,6), bacterial (9), and fungal (1) challenges. Recent data from our laboratory have indicated a beneficial effect of oat β-glucan consumption on susceptibility to infection in mice exercised to fatigue. This effect was associated with attenuation of the decrease in intrinsic macrophage antiviral resistance; however, it is likely that other immune cells also play a role.
The purpose of this study was to determine the effect of the soluble oat fiber β-glucan on peritoneal neutrophil respiratory burst activity after repeated days of moderate and fatiguing exercise. The moderate exercise protocol consisted of six consecutive days of treadmill running (1 h·d−1) designed to mimic a period of moderate exercise training. The fatiguing exercise protocol consisted of three consecutive days of prolonged treadmill running to fatigue (approximately 2.5 h). A β-glucan enriched oat bran concentrate was used because of its solubility, natural occurrence in the diet, "generally recognized as safe" designation by the FDA, and documented health benefits in various pathological conditions, including diabetes and cardiovascular disease (14,30).
Male CD-1 mice, 4 wk of age, were purchased from Harlan Sprague Dawley Labs and were acclimated to our facility for at least 3 d before any experimentation. Mice were purchased as pathogen-free stock, and periodic screening of sentinel mice yielded negative results for common murine viral or bacterial pathogens. Mice were housed four per cage and were cared for in the animal facility located at the University of South Carolina School of Medicine. Mice were maintained on a 12:12-h light:dark cycle in a low-stress environment (22°C, 50% humidity, low noise) and were given food (Purina Chow) and water (or oat β-glucan dissolved in water) ad libitum. All experiments were performed at the end of the active dark cycle.
Mice were randomly assigned to one of the following six groups: exercise to fatigue with water (Ftg-H2O; N = 10), exercise to fatigue with oat β-glucan (Ftg-OβG; N = 10), moderate exercise with water (Mod-H2O; N = 10), moderate exercise with oat β-glucan (Mod-OβG; N = 10), control with water (Con-H2O; N = 20), or control with oat β-glucan (Con-OβG N = 20). The fatiguing exercise experiment (experiment 1) and the moderate exercise experiment (experiment 2) were carried out on different days; each experiment had its own control groups. Ftg-H2O, Mod-H2O, and Con-H2O mice received tap water for the 10 d before sacrifice, whereas Ftg-OβG, Mod-OβG, and Con-OβG mice were fed a solution of oat β-glucan for the 10 d before sacrifice. The oat β-glucan solution was made from an oat bran concentrate enriched to 68% soluble β-glucan (manufactured by Nurture, Inc., Devon, PA and supplied by the Quaker Oats Co., Barrington, IL), which was dissolved in the drinking water at a concentration of 0.6 mg·mL−1 and was made fresh daily. Daily consumption of fluid was measured to ensure that there were no differences in fluid ingestion between the water and the oat β-glucan solution. The body weight of each animal was monitored throughout the supplementation and exercise period to ensure that no weight loss was experienced by any group.
Treadmill acclimation and exercise protocol.
The university's institutional animal care and use committee approved the protocols described.
The effect of β-glucan on neutrophil number and function after fatiguing exercise was examined. Exercise mice (Ftg-H2O and Ftg-OβG) were acclimated to the treadmill for a period of 20 min·d−1 for the 3 d before the experimental exercise bouts. The exercise stress protocol consisted of a fatiguing exercise bout of treadmill running (performed in the morning, 7 a.m.) for three consecutive days. Mice in the exercise groups ran on the treadmill (two per lane) at a speed of 36 m·min−1 and a grade of 8% until they reached volitional fatigue. This is estimated to elicit approximately 75-90% maximal O2 uptake (10,25), assuming a maximal O2 uptake of 173-206 mL·kg−1·min−1 for mice. Fatigue was defined as the inability of a mouse to maintain the appropriate pace despite continuous hand prodding for 1 min, at which time the mouse was removed from the treadmill. Electric shock was never used in these experiments, because mice readily respond to a gentle tap of the tail or hindquarters, encouraging them to maintain pace with the treadmill. Mice rarely required this type of continual prodding until they approached the point of fatigue.
The effect of oat β-glucan on neutrophil number and function after short-term, moderate exercise was examined. The moderate exercise protocol consisted of a 1-h bout of treadmill running (performed in the morning, 7 a.m.) for six consecutive days. Mice in the exercise groups ran on the treadmill (two per lane) at a speed of 36 m·min−1 and a grade of 8%. Mice in the control groups (Con-H2O and Con-OβG) remained in their cages in the treadmill room throughout the exercise bouts. These mice were exposed to similar handling and noise in an attempt to control for extraneous stresses that may be associated with treadmill running. Control mice were deprived of food and water during the exercise sessions.
After the last bout of exercise or rest, mice were given a 1-mL i.p. injection of thioglycollate. Thioglycollate is a nonspecific inflammatory stimulus that has been used for eliciting neutrophils into the peritoneal cavity. In an initial experiment, additional mice were used to determine the time of sacrifice after thioglycollate injection. A 3-h time point was determined to have an appropriate influx of neutrophil cells and was used in subsequent experiments. Because the majority of the cells elicited at this time point were neutrophils, PECs will be referred to as neutrophils. Mice were sacrificed 3 h after injection, and neutrophils were harvested via lavage and counted. Briefly, the lavage procedure consisted of a 5-mL i.p. injection of RPMI 1640 supplemented with 10% fetal bovine serum and 2% penicillin, streptomycin, and L-glutamine (PSG); the media was recovered after gentle agitation of the mouse. The volume of media recovered was variable; therefore, the results are expressed here as total cells per milliliter of media recovered. The number of neutrophils elicited into the peritoneal cavity after thioglycollate injection is representative of a response that could be observed during infection or inflammation.
Neutrophil respiratory burst.
Neutrophils were adjusted to a concentration of 1 × 106 million cells per milliliter in RPMI 1640 supplemented with PSG. Cells were then incubated with 1 μL of 2 '7'dichlorofluorescein diacetate (DCFH/DA) (0.5 mM) per milliliter of cell suspension, for a final concentration of 250 ng·mL−1. DCFH/DA is a viable dye that is taken up by the cells and that causes a green fluorescence during the presence of a respiratory burst. Cell suspensions were incubated at 37°C for 15 min. Five 1-mL aliquoits were made for each sample as follows: 0 time: DCFH/DA only; 15 min: DCFH/DA and phorbol myristate acetate (PMA); 30 min: DCFH/DA and PMA; 45 min: DCFH/DA and PMA; 60 min: DCFH/DA and PMA. Two microliters of PMA (1 μg·mL−1) were added to the appropriate tubes. The use of PMA is a well-characterized method used to stimulate respiratory burst activity in neutrophil cells. Tubes containing DCFH/DA only are representative of endogenous respiratory burst activity, and tubes containing PMA represent stimulated respiratory burst activity. The tubes were placed in the incubator at 37°C for the indicated times. After the incubation period, 1 mL of 1% paraformaldehyde was added to the tubes. The tubes were allowed to sit at room temperature before flow cytometric analysis. A Beckman Coulter Epics XL-MCL flow cytometer equipped with an argon ion laser operating at 488 nm and Research software were used to assess activity. Samples were analyzed for green fluorescence gated on PMN using forward and side scatter. A total of 5000 cells were counted in each reaction tube.
Statistical analyses were performed using commercially available statistical packages from the SAS system SigmaStat (version 2.03, SigmaStat, SPSS, Chicago, IL). Differences in treadmill run times in experiment 1 were analyzed using the Student's t-test in SigmaStat (P < 0.05). Differences in neutrophil respiratory burst, cell number, weight gain, and fluid consumption were compared using a two-way analysis of variance in SigmaStat with Student-Neumann-Keuls post hoc analysis (P < 0.05).
Run time to fatigue was not significantly different between the fatiguing exercise groups. Average run time to fatigue during the three exercise days was 119 ± 9 min for Ftg-H2O and 118 ± 8 min for Ftg-OβG. In addition, run times were not different on days 1-3 of exercise, indicating that the protocol was relatively well tolerated by the mice and that no apparent training effect occurred during this time period.
Nutrient consumption and weight gain.
There were no differences in the average amount of fluid consumed by either the oat β-glucan- or exercise-treated groups. During the course of the 10-d ingestion period, mice consumed approximately 6.2 mL·d−1 of fluid, resulting in a daily dose of approximately 3.7 mg of oat β-glucan per mouse. There was also no difference in body weight across the groups.
Neutrophils were elicited from mice after thioglycollate injection. The number of cells mobilized into the peritoneal cavity were counted and are expressed in million cells per milliliter lavaged. Fatiguing exercise (experiment 1) (P < 0.05) and oat β-glucan (P < 0.05) significantly increased neutrophil mobilization into the peritoneal cavity 3 h after thioglycollate injection (Fig. 1A). There was no effect of moderate exercise (experiment 2) on neutrophil mobilization (Fig. 1B).
Neutrophil respiratory burst activity.
Neutrophils were elicited from mice after both oat β-glucan treatment and fatiguing exercise (experiment 1), and endogenous as well as PMA-stimulated respiratory burst activity were measured. Three days of fatiguing exercise did not result in any significant differences in endogenous or PMA-stimulated respiratory burst activity in neutrophils. Oat β-glucan consumption for 10 consecutive days (Ftg-OβG and Con-OβG) did, however, result in a significant increase in endogenous neutrophil respiratory burst activity (P < 0.05) (Fig. 2A). There were no differences between the groups after PMA activation at 15, 30, 45, or 60 min. Neutrophils were elicited from mice after oat β-glucan feedings and moderate exercise (experiment 2), and their endogenous and PMA-stimulated respiratory burst activity was examined (Fig. 2B). Six days of short-term moderate exercise (Mod-H2O and Mod-OβG) and oat β-glucan (Con- OβG) resulted in a significant increase in endogenous neutrophil burst activity in this model (P < 0.05); there were no differences between groups after activation by PMA.
There is substantial evidence that exhaustive exercise can decrease certain functions of neutrophils, whereas moderate exercise can result in an increase. This has led to many nutritional interventions to counteract the immunosuppression of exhaustive exercise, but little evidence exists regarding nutritional interventions in conjunction with moderate exercise as an additional stimulus to further enhance immune function. This study has examined the effect of oral consumption of the soluble oat fiber β-glucan on neutrophil number and function after both moderate and fatiguing exercise in mice. The data from this study suggest that both exercise and oat β-glucan feedings can increase neutrophil burst activity (number and/or function), but only oat β-glucan increased both number and function of neutrophils, and there were no additive effects.
In recent years, there has been considerable interest in how exercise may affect the immune system. Neutrophils represent one of the key nonspecific-host defense cell populations responsible for the phagocytosis of many microbial, bacterial, and viral pathogens. Additionally, neutrophils are known to be involved in the synthesis and release of immunomodulatory cytokines that influence both T cell and B cell activities. The overall activity of neutrophils is a reflection of both number and function on a per-cell basis; only by considering both of these variables is it possible to determine overall activity of neutrophils. Data from animal as well as human studies indicate that exercise can alter the number and function of neutrophils (3,15,16,20,22). Moderate exercise has been shown to increase various activities of neutrophils, whereas exhaustive exercise can suppress the activities of neutrophil cells. For example, the activity of H2O2 production (22), respiratory burst activity (20), and phagocytic activity (15) of neutrophils is increased following moderate exercise. Exhaustive exercise, on the other hand has been reported to suppress the phagocytosis 3 and oxidate burst activity (3,16) of neutrophils. Alterations in neutrophil cell numbers also have been reported after exercise. It is thought that exercise induces mobilization of neutrophil cells into the circulation (19,20); the release of stress hormones and cytokines are thought to play a role.
In this study, using thioglycollate elicited neutrophils, we found an increase in endogenous respiratory burst activity after short-term moderate exercise, consistent with the literature; however, there were no changes after short-term exhaustive exercise in this model. This may be attributable to the time point of sacrifice or, more likely, to the fact that thioglycollate may have masked any potential changes that we may have seen after exhaustive exercise. These changes were not observed after stimulation with PMA. It is likely that any changes that were observed without PMA administration were diminished after its activation. Fatiguing exercise resulted in an increase in the number of neutrophils mobilized into the peritoneal cavity after thioglycollate injection; however, there was no effect of moderate exercise on cell number. Others have reported an increase in neutrophil mobilization after exercise (19,20); the results of our study suggest that the effects of exercise on neutrophil mobilization may be related to duration, because we observed an increase with fatiguing exercise and not with moderate exercise.
Nutritional strategies such as the use of carbohydrates, glutamine, and antioxidants have had limited success in preventing suppression of neutrophils after exercise stress (11), and there is no evidence of an effect of these or of any other nutritional strategy in conjunction with moderate exercise on neutrophil number or function. Previous work with primarily insoluble yeast or fungi-derived β-glucan suggests that it can stimulate a wide range of immunological activities, including increased neutrophil function (26), and can enhance host resistance to fungal (1) and viral (5,6) diseases and cancer (17). Less is known about the immunostimulant properties of soluble oat β-glucan, which, incidentally, is an important fiber component of the "heart-healthy diet" as defined by the FDA (14). Recently in our laboratory, we have found a beneficial effect of oat β-glucan on increasing macrophage antiviral resistance in mice (6). However, these are the first data to show a benefit of oat β-glucan on neutrophil respiratory burst.
Macrophages, NK cells, and neutrophils contain specific β-glucan receptor sites on their cell membrane, such as complement receptor 3 (CR3) and dectin-1 (2) that, when bound, results in increased functional activity (4,26). Neutrophils also have been reported to possess a β-glucan receptor known as glycosphingolipid lactosylceramide, which is thought to be exclusively present on neutrophils (31). The range of biological responses elicited supports the concept of multiple β-glucan receptors. The mechanisms of stimulation can be dependent on the route of administration (e.g., intravenous, intraperitoneal, or oral) and specific characteristics of the β-glucan, including the source (e.g., oats, yeast, fungi, etc.), solubility, molecular mass, degree of branching, and conformation (ratio of 1→3 to 1→4 and 1→6 glucopyranolsyl linkages) (28). After oral administration of soluble β-glucan, pinocytic M-cells located in Peyer's patches of the small intestine can ingest the β-glucan via phagocytosis, resulting in the release of cytokines that are responsible for initiating an extensive cascade of systemic immune responses (13,21,24). It is also possible for very small β-glucan particles to be absorbed directly into the lymphatic and vascular systems, where they can interact directly with circulating immune cells via their β-glucan receptors (14,30).
Others have reported enhancement of neutrophil functions, including an enhanced blood neutrophil oxidative burst response, increased microbial killing, and activation of nuclear transcription factors (27) after treatment with β-glucan derived from yeast. Harler et al. (12) have reported a role of β-glucan on neutrophil migration. In this study, oat β-glucan significantly increased the number of thyioglycollate-elicited neutrophils mobilized into the peritoneal cavity as well as the respiratory burst activity of neutrophils.
This is the first study that has examined a possible interaction between exercise and oat β-glucan on neutrophil respiratory burst activity. We hypothesized that consumption of oat β-glucan would 1) offset the typical reduction in neutrophil function associated with fatiguing exercise, and 2) further enhance the immunostimulatory effect of moderate exercise. Unfortunately, the first hypothesis could not be tested in this study because there was no decrease in neutrophil function in response to the fatiguing exercise protocol. The second hypothesis of an additive benefit of oat β-glucan and moderate exercise was not supported. It is possible that this is attributable to a ceiling effect by which the benefits of each individual treatment on neutrophil function have been maximized.
The data from this study suggest that although not additive in their effects, both exercise and oat β-glucan had beneficial effects on either neutrophil number or function on a per-cell basis, but only oat β-glucan was effective in increasing both number and function. In general, we interpret these data to suggest that the optimal neutrophil response occurs with oat β-glucan feedings. These alterations in overall neutrophil activity may at least partially explain the effects of oat β-glucan and exercise on susceptibility to infection.
This work was funded in part by a grant from Quaker Oats Company and the Gatorade Sports Science Institute.
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