There is evidence of significant changes in athletes' immunity after training (2,7,15,22,32). Nevertheless, the linkage between these changes and the susceptibility to subclinical or overt infections are inconclusive (16,23). Bacterial and particularly viral infections have been frequently reported after intense and prolonged exercise (9,25). Neutrophils play an important role in innate immunity and act as a first line of defense against microorganisms (27). In addition, they are involved in the muscle tissue inflammatory response to exercise-induced injury (1). The sequence of events that occur in the neutrophil response to microbial invasion includes adherence, chemotaxis, ingestion, oxidative burst, degranulation, and microbial killing (27).
We have previously shown a transient reduction in the chemotactic activity 24 h postaerobic exercise (28,30,31) and returning to normal after 48 h (31). The reduction was not seen immediately postexercise (28,30) and was independent of the stimuli used (N-formyl-Met-Leu-Phe [fMLP], the activated complement [C5a], or interleukin 8 [IL-8]), indicating a failure of the cell to further process the stimuli (31). This finding could be related to reduced chemotactic receptors availability or to a malfunction in processing the signal transduction from the cell receptor to the intracellular skeleton. Proper intracellular skeleton rearrangement is needed for appropriate cell adhesion and cell polarization, which are required for a normal chemotactic response (20). In addition, for normal adherence, adequate expression of adhesion molecules on leukocytes and endothelial cells is required. These adhesion molecules undergo quantitative and qualitative changes in response tovarious stimuli and are involved in the leukocyte-endothelial cell interactions contributing to the migratory ability of the neutrophil. The β2-integrins constitute the main group of adhesion molecules expressed on neutrophils. They play an essential role in the adhesion process, and their signal transduction properties are crucial for regulation of neutrophil chemotaxis (10,20).
Considering that an impaired chemotaxis was consistently found 24 h after aerobic exercise (28,30,31), we investigated the possible mechanisms for this impairment and focused on the effect of exercise at that time point. The late effect of submaximal exercise on neutrophil chemotaxis, polarization, adhesion activity, and surface density of the β2-integrin CD11b/CD18 was examined. In addition, the surface density of the chemotactic receptor C5aR, a representative model of receptor availability, was assessed. The chemotactic receptors fMLP-R and C5aR share a common pathway for signal transduction and have a similar structure (10,31). Reduction in its availability 24 h postexercise might give an explanation for the reduced chemotaxis.
The effect of aerobic exercise on neutrophil chemotaxis and on other related functions was investigated at two time points: preexercise and 24 h postexercise.
The experimental group comprised 23 healthy, physically active, young adult males, aged 17-37 yr (average age = 30.5 ± 4.9 yr), who were not engaged in competitive sports. None of the participants was a smoker, used tobacco products, or used antiinflammatory drugs during the 4-wk preceding the course of the study. All participants (weight = 75.1 ± 6.3 kg, height = 180.0 ± 6.0 cm, body mass index [BMI] = 23.2 ± 1.8, body fat percentage = 15.0 ± 3.6%) trained in aerobic-type exercise (running, swimming, cycling) 3 to 10 times·wk−1 year-round (average weekly aerobic exercise = 6.0 ± 2.5 h). Ten healthy male volunteers (age = 32.4 ± 4.2, weight = 80.0 ± 13.0 kg, height = 179.6 ± 6.1 cm, body mass index [BMI] = 24.8 ± 3.4), who served as a nonexercise control group, were simultaneously tested with the experimental group on two consecutive days at resting conditions. These subjects were included to exclude daily temporal variability or experimental errors as an explanation for the results. Both the exercise and the nonexercise groups were matched for gender, age (P=0.33), weight (P = 0.35), height (P = 0.66), and BMI (P = 0.22).
All subjects volunteered to participate in the study and signed an informed consent form explaining the study's purpose, duration, and experimental procedures. The Institutional Review Board of Meir Medical Center approved the study.
In this within-subject design, the experimental study group was tested preexercise and postexercise and served as a "self-control group". These subjects underwent physical examination, anthropometric measures (height, weight, skinfold thickness), and determination of maximal oxygen consumption (V˙O2max) on a treadmill (see Exercise protocols section). On the following week, they performed a 30-min run on the treadmill at an intensity of 75% V˙O2max. Two blood samples were drawn from the median antebrachial vein of each subject, before exercise and 24 h after exercise. The subjects refrained from training during the 24h before and 24 h after the 30-min run. Two blood samples were also drawn from the median antebrachial vein of the subjects from the nonexercise control group in two consecutive days.
Ten milliliters of heparinized blood was taken from all participants for neutrophil function assessment that was completed within 4 h of blood withdrawal. Additional 5 mL of EDTA blood was taken for neutrophil surface molecules assessment.
Body composition measurements.
Anthropometrics measures including body weight and height were performed using standard procedures. Body fat percentage was assessed using measurements of skinfold thickness. The latter were measured in triplicate at four sites (subscapula, biceps, triceps, and suprailiac) by the same investigator (S. B. A.) using a digital caliper (Skyndex, Caldwell, Justice and Co., Inc., Fayetteville, AR). The sum of four skinfolds was used to calculate the body fat percentage using the equation derived by Durnin and Womersley (6).
Maximal oxygen consumption (V˙O2max) was determined by a progressive running test to exhaustion. Criteria for V˙O2max included reaching a respiratory exchange ratio >1.2 or an increase in V˙O2 < 250 mL·min−1 with an increase in speed or slope of the treadmill. Submaximal exercise consisted of a 5-min warm-up at 2 km·h−1 slower than the speed estimated to elicit 75% of V˙O2max. Thereafter, subjects ran for 30 min at the predetermined speed. V˙O2 was measured during 5-10, 15-20, and 25-30 min. The average submaximal V˙O2 was 41.4 ± 7.8 mL·kg−1·min−1. V˙O2 was measured with a computerized open flow metabolic system, where expired gas and ventilation were analyzed continuously "online." The system included an oxygen analyzer (Applied Electrochemistry S-3A, Pittsburgh, PA), a carbon dioxide analyzer (Applied Electrochemistry CD-3), and a flow transducer (Hewlett Packard 4730-1A). Analyzers were calibrated before each test with precision gas (16% O2 and 6% CO2), and the volume was calibrated with a 3-L syringe (Hans Rudolph, Inc., Kansas City, MO). Heart rate was continuously monitored using a heart rate telemetry system (Sports Tester; Polar Electro, Kemplele, Finland).
Isolation of polymorphonuclear leukocytes.
Polymorphonuclear leukocytes (PMN) were isolated (99% purity) from heparinized blood by dextran sedimentation, histopaque gradient, and erythrocyte lysis, as described byBöyum (4). PMN were resuspended for chemotaxis studies in M199 medium (Beit-HaEmek, Israel) (106 cells·mL−1) for adhesion in Hank's balanced salt solution with calcium and magnesium (HBSS+) (Sigma-Aldrich, St. Louis, MO), and for polarization in phosphate-buffered saline with 0.1% albumin and 0.2% glucose (5 × 106 cells·mL−1).
The chemotaxis was assessed in a 48-well chemotaxis chamber, through a 3-μm pore size filter (8), and induced by the chemoattractant 1 μM fMLP (Sigma Chemical Co). Random migration was conducted in the presence of medium M199. Cells were counted in nine fields using light microscopy and the average number was recorded. Data were expressed as number of migrating cells per field. To express the chemotactic activity, net chemotaxis was calculated by subtracting the random from the chemotactic migration. All procedures were performed in quadruplicates, in a blinded fashion, by two investigators (R. G., T. A. A.).
Surface expression of the membrane chemotactic receptor C5aR.
Direct immunofluorescence and flow cytometric analysis on a FACScan (Becton Dickinson, Mountain View, CA) were performed to determine the expression of the receptor C5aR from whole blood (11). FITC-labeled antiC5aR (CD88) (Serotec, Oxford, UK) or isotype for CD88 (FITC-IgG1; Dako, Glostrup, Denmark) was added to each tube for 30 min in the dark at room temperature. Hemolysis of the red blood cells and fixation of the PMN were done by the Q-prep (Beckman Coulter) technique.
Surface adhesion antigen expression.
A modified version of the direct immunofluorescence and flow cytometric analysis was performed on a FACScan to determine the antigen expression of the integrin CD11b/CD18 from whole blood (11). After addition of 0.1 μM fMLP to the whole blood, for 15 min in 37°C, placing the tubes in ice stopped the reaction. Prelabeled monoclonal mouse antihuman CD11b or isotype as negative control (PreIgG1; Dako) were added to each tube for ten min in 4°C. Hemolysis of the red blood cells and fixation of the PMN were done in the manner mentioned earlier.
Cell adhesion to gelatin surface was assessed as previously reported (19). Briefly, cells were labeled with 0.1% calcein (MW 994.87; Molecular Probes, Eugene, OR) and incubated for 30 min at 37°C, which stimulates either PMA (100 ng·mL−1) or fMLP (0.1 μM) in a 24-well plate. HTAB lysis solution (0.1% Tween 20 [Sigma], 0.1% HTAB [Sigma], 0.2% BSA, 20 mM EDTA) was added. Fluorescence of the lysates was measured by a spectroflourophotometer (Shimadzu, Tokyo, Japan) at excitation 485 nm and emission 535 nm.
The neutrophil polarization assay (29,31) was performed for assessing the cell shape changes that occur after stimulation. Given that this assay was introduced during the study, it was performed only in 14 participants. Yet, their anthropometric measures and neutrophil function were not statistically different (data not shown) from that of the remaining subjects. Isolated neutrophils were incubated in a shaking bath at 37°C, with or without 0.1 μM fMLP, added directly into the medium for 15 min. Fixation of the cells was achieved by adding 1% glutaraldehyde and phase microscope evaluation was accomplished (Olympus, Japan). Two experienced investigators (R. G., B. W.) assessed the pre- and postexercise samples in a blinded fashion. The progress of neutrophil activation was determined according to the morphological changes of the cell. Three different cell shapes were recorded: nonactivated cells (round), partially activated cells (intermediate), and fully activated cells (polar). Each cell count was established from a total count of 400 cells. The percentage of polar cells represents neutrophil polarization.
The data are expressed as mean ± SEM. When appropriate, differences between means were analyzed using the paired t-test and the general linear model for repeated measures. The Pearson's correlation coefficient was used to assess the relationship between measurements. Statistical significance was set at P = 0.05.
Impaired neutrophil chemotaxis was found 24 h postexercise (Fig. 1). The chemotactic migration decreased significantly from 96 ± 5 to 70 ± 5 cells per field (P = 0.0001), whereas the random migration did not change significantly (45 ± 3 to 39 ± 3 cells per field; P = 0.084). As expected, a parallel decrease in the net chemotaxis (from 51 ± 3 to 31 ± 3 cells per field; P = 0.0001) was observed 24 h postexercise and the overall chemotactic activity was reduced by 36%. No significant differences were found in the control group on two consecutive days, either in the chemotactic migration (100 ± 6 to 96 ± 5 cells per field; P = 0.58) or in the net chemotaxis (59 ± 4 to 57 ± 5 cells per field; P = 0.24). Further, the chemotactic migration and net chemotaxis were not significantly different between the exercise and the nonexercise groups in resting conditions (96 ± 5 vs 100 ± 6, P = 0.91; 51 ± 3 vs 59 ± 4, P = 0.37, respectively).
FIGURE 1-Neutrophil ...Image Tools
We did not find a late effect of exercise on C5aR availability or postexercise change in the expression of CD11b/CD18 molecules in basal condition or in fMLP-activated state (Table 1). Nonetheless, neutrophil adhesiveness was significantly affected by exercise. After fMLP stimulation, the neutrophil adhesiveness decreased preexercise versus postexercise from 31.5 ± 2.4% to 25.6 ± 2.3%, respectively (P = 0.002). After PMA stimulation, the neutrophil adhesiveness decreased from 43.0 ± 1.9% to 32.9 ± 3.2%, respectively (P = 0.002) (Fig. 2). The overall adhesiveness was reduced postexercise by 19% or 26% with fMLP or PMA stimulation, respectively. In the nonexercise control group, no significant changes were observed on the two consecutive days; neutrophil adhesiveness with fMLP stimulation was 29.1 ± 2.6% and 31.8 ± 5.4% (P = 0.16), whereas with PMA stimulation, the adhesiveness was 43.6 ± 4.6% and 45.4 ± 5.4% (P = 0.33), respectively.
The neutrophil polarization was significantly reduced preexercise versus postexercise from 61 ± 4% cells to 43 ± 4%, respectively (P = 0.0001) (Fig. 3). There was a 29% reduction in the polar-shaped fMLP-stimulated neutrophils 24 h postexercise, whereas in the nonexercise control group, the reduction was small and insignificant (7%, P = 0.42). The postexercise polarization (the percentage of fully activated cells) positively correlated with the postexercise neutrophil chemotactic activity (n = 14, r = 0.649, P=0.012).
FIGURE 3-Neutrophil ...Image Tools
Multifactorial elements are involved in the neutrophil behavior and in the immune responses to exercise, influencing neuroendocrine mediators (18), corticosteroid release, interleukin production (24), and oxyreduction processes associated with free radical production (1). Equally important are the methods used for neutrophil evaluation, the type of exercise, its duration and intensity, and the fitness level of the participants, which could differentially affect the cell response.
For the last 10 yr, our group has focused on neutrophil functions in response to a single bout of submaximal aerobic exercise (28,30,31). We found that although the neutrophil count is elevated immediately after exercise, neutrophil functions such as chemotaxis, oxidative burst, and bactericidal activity were unaffected. However, after a lag of 24 h postexercise, a consistent decrease of neutrophil migration in trained and untrained females with unchanged superoxide production, bactericidal activity, and F-actin skeletal polymerization was demonstrated (28,30,31). The decrease in the chemotactic ability was transient and observed regardless of the chemokine used (31). Conflicting results were reported on neutrophil functions when investigated immediately after exercise (25). Although Rodriguez et al. (21) showed no change in the chemotaxis, Ortega et al. (17) reported an increased chemotaxis of phagocytic cells immediately after exercise. Others reported increased chemotaxis in neutrophils obtained from nasal lavage immediately after a marathon run that persisted for 7 d (14). The discrepancy with our results may be related to differences in testing conditions and timing of the neutrophil function evaluation.
The present finding that complies with our previous findings on female subjects (28,30) shows a late reduction of fMLP-induced chemotaxis in physically active young adult males. Further, we looked for the mechanisms that may explain this functional decline 24 h after aerobic exercise. The surface density of the chemotactic receptor (C5aR), which serves as a representative model of receptor availability, was not affected 24 h after exercise. Moreover, the integrin CD11b/CD18, which represent one of the main receptors for neutrophil adhesiveness and crucial for normal chemotaxis, was also unaffected by exercise.
Previously, we have demonstrated that fMLP-stimulated neutrophil functions, such as F-actin polymerization and oxidative burst, were normal when tested 24 h postexercise in trained subjects (28,30,31), indicating a good receptorial availability and function for the specific fMLP receptor. All these data support the fact that neutrophil receptorial availability and function are most probably not affected by exercise and cannot explain the chemotactic impairment. Further, using different stimulating-chemokines (fMLP, C5a, and IL-8), it was shown that the chemotactic defect is present (31). Therefore, it could be assumed that the 24-h postexercise chemotactic impairment is not related to specific receptor availability.
Normal neutrophil chemotaxis requires a parallel normal neutrophil adherence. Other investigators have demonstrated an immediate effect of submaximal exercise on the reduction of the leukocyte adherence in active men (5,13,24). In the present study, we demonstrated a late effect (24 h postexercise) of submaximal exercise on neutrophil adherence reduction, which paralleled the reduction of the neutrophil chemotaxis. Reduced adherence can be related to changes in the integrin availability and function (12). Others showed that strenuous exercise changed adhesion molecule expression immediately postexercise (24,26). In our testing conditions, 24 h postexercise, the surface density of the CD11b/CD18 was not affected, nor their response to fMLP stimuli. Therefore, it is suggested that the reduced adherence 24 h postexercise is related to other cytoskeletal biophysical properties rather than to the availability of adhesion molecules.
Another possible mechanism to explain the impaired chemotaxis might be related to the cell skeleton ability to exhibit morphological changes (polarization), which is fundamental for the neutrophil movement toward stimuli. Our group already reported a reduced polarization 24 h after exercise in active females (31). In the present study, we demonstrate a similar reduction of neutrophil polarization in physically active young adult males. Consistent with these findings, the reduced polarization correlated with the reduced chemotaxis.
A 30-min bout of submaximal aerobic exercise resulted in decreased neutrophil functions such as chemotaxis, adherence, and polarization after a lag of 24 h, with no effect on membrane expression of the integrin CD11b/CD18 and the chemokine receptor C5aR. These decreased functions are probably related to a common mechanism involving cytoskeleton components and not to surface receptor availability.
The clinical implications of reduced chemotaxis should be considered. The impaired chemotaxis is often present in phagocytic disorders such as hyper-IgE syndrome, leukocyte adhesion deficiency, and Chediak-Higashi syndrome, all commonly associated with deep-seated infections (3,12). Inour study, the magnitude of exercise-induced reduction of net chemotaxis was similar to the pathological levels wefound in 13 patients with hyper-IgE syndrome (31 ± 3 vs 25 ± 4 cells per field, respectively, P = 0.21) (B. W. and R.G., unpublished data). Additional studies are needed to elucidate the effect of exercise on neutrophil function, with emphasis on prophylactic and therapeutic management.
The authors are grateful to Mrs. Aviva Zeev, The Research Department of The Zinman College of Physical Education and Sport Sciences, Wingate Institute, Netanya, for assistance in the statistical analysis and to Mrs. Rachel L. Berger, Kfar Saba, for editing assistance.
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