Exhaustive endurance exercise has been shown to cause activation of circulating granulocytes as part of a generalized acute inflammatory response (4,5,12,31). Activation of granulocytes in vitro and in vivo is associated with increased surface expression of the β2-integrin adhesive receptor MAC-1 (CD11b/CD18) (2,3,9,33) and the release of proteases and reactive oxygen species (32). The effects of endurance exercise on granulocyte release of reactive oxygen species and proteases have been extensively studied (8,11,18,22). However, the regulation of MAC-1 (CD11b/CD18) expression in endurance exercise is still imperfectly understood (8). Increased surface expression of the integrin adhesive receptor MAC-1 (CD11b/CD18) facilitates binding of granulocytes to IgG-like molecules (cell adhesion molecules) on endothelial cells (1,24,28,29). Adhesion to endothelial cells is one step in a cascade of events that leads to tissue infiltration of granulocytes (1,21). Increased tissue infiltration of granulocytes occurs after endurance exercise (16,19). Considering the importance of MAC-1 (CD11b/CD18) in tissue infiltration (28), regulation of this integrin in endurance exercise might be an important factor for the targeting of reactive oxygen species and proteases and thereby tissue damage and/or repair. We tested the hypothesis that the degranulation of granulocytes and upregulation of the granulocyte integrin MAC-1 (CD11b/CD18) are related to exercise duration and/or intensity. We also investigated whether or not the expression of MAC-1 (CD11b/CD18) would be influenced by body temperature or dehydration. Moreover, we tested the hypothesis that changes in leukocyte counts and changes in MAC-1 (CD11b/CD18) expression with endurance exercise are independently regulated.
Study population. Eight male amateur runners were recruited from a runner's meeting (36 ± 11 yr, 179 ± 7.4 cm, 73 ± 5.4 kg). The number of years that each runner trained endurance running and the number of marathon races before the study are given in Table 1. The study participants were queried about their medical history and given a physical examination, which included a 12-lead electrocardiogram. Individuals who had medical conditions that could be compromised by endurance exercise were excluded from the study. Written informed consent was obtained from all participants before study entry, and the protocol was approved by the University's committee on the protection of human subjects.
Each subject performed a maximum incremental load test on a treadmill, an extensive training run of approximately 3 h, and a competitive marathon race. All exercise tests were performed within 7 wk, with an interim of at least 3 wk between the 3-h run and the marathon race.
Incremental load test. The incremental load test was performed on a electronically driven treadmill (Woodway, Ergo XELG2, Weil am Rhein, Germany) in a climatized room. Treadmill inclination was 1%. The test began with a 3-min reference phase. Depending upon the sports history (e.g., running time in previous marathon races), the initial running velocity was set 2.6 or 3.0 m·s−1. The speed was increased every 3 min by 0.4 m·s−1 until exhaustion (inability to sustain running speed). Ventilatory gases were measured continuously during the entire test (Oxycon Gamma, Mijnhard, Bunnik, The Netherlands). The metabolic cart was calibrated using gases of known concentration and a syringe before each test. Heart rate (HR) was continuously monitored (PE 3000, Polar Electro, Kempele, Finland).
Training run. The training run was performed under field conditions with running on flat roads and lasted approximately 3 h. The intended running velocity for the training run was the running velocity that corresponded to 50-60% of peak O2 during the incremental load test, representing a moderate intensity long duration training for marathon runners.
Marathon race. The subjects participated in a competitive marathon race. To remain well hydrated during the marathon, subjects were advised to drink 0.1-0.2 L water or carbohydrate drinks every 5 km.
For the training run and the marathon race, mean O2 corresponding to the mean velocity was estimated by interpolating the relationship between running velocity and O2 obtained during the incremental load test. The mean running velocity was calculated as distance/running time. To calculate relative workload intensity, the O2 during training run or marathon race was expressed as percentage of peak O2 (determined during the incremental load test) (13,15).
Blood samples for immunofluorescence analysis (4 mL) and a blood cell count (2 mL) were collected from an antecubital vein into vacuum tubes containing EDTA (Vacutainer, Becton Dickinson, Rutherford, NJ) immediately before and after the incremental load test, training run, and marathon race. The leukocyte count was corrected by plasma volume changes as determined from hematocrit (Hct) and hemoglobin (Hb) values. All blood samples were obtained by the same investigator with a large bore needle with minimal manual compression. The tympanic temperature (Core Check 2090, IVAC, San Diego, CA) was measured immediately before and after every exercise. The body weight was measured immediately before and after exercise always with the same digital scale.
Immunofluorescence analysis and determination of granularity. We utilized the following monoclonal antibodies and chemicals purchased from Becton and Dickinson (San Jose, CA): CD18 (mouse IgG1), IgG1/IgG1 Simultest Control, mouse IgG1 isotype control, Cellwash®, CaliBrite®, and FACS Lysing Solution®. The following monoclonal antibodies were purchased from Dianova (Hamburg, Germany): CD11B (mouse IgG1), CD15 (IgGM), mouse IgG1 isotype control.
Fluorescent antibody cell sorting (FACS) analysis was carried out as described previously (9). Whole blood was stained with monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) within 6 h after collection. Thereafter, the blood samples were treated with FACS Lysing Solution, realizing the lysis of erythrocytes and partial fixation of leukocytes. The samples were then washed two times with optimized phosphate buffer solution (Cellwash), which resulted in a cell suspension for flow cytometry.
The cells were analyzed on a standard flow cytometer (FACScan, Becton and Dickinson, San Jose, CA). Flow cytometric standardization was achieved by running beads (CaliBrite). For each sample, 10,000 cells were analyzed on the log fluorescence scale of the flow cytometer. Granulocytes were recognized in the forward versus sideward scatter diagram and verified with CD15 as a specific marker. The granularity of granulocytes was determined from the side-ward scatter characteristics. A decrease in the granularity in the sideward scatter has been shown to indicate degranulation of granulocytes in vitro. The surface antigen expression, the mean log FITC, and SSC (sideward scatter) channel of the positively stained cells were determined from a single parameter histogram. Because this study was performed under field conditions and blood samples had to be stored, we tested whether storage of blood samples at 4°C for up to 12 h would change surface expression of CD11b or CD18 on granulocytes.
All data are expressed as mean ± standard deviation (SD). Intra-individual mean differences were compared by simple ANOVA according to Friedman and Wilcoxon tests. Relative differences were compared with the corresponding pretest values.
Multiple ANOVA. Changes in leukocyte counts and changes in CD11b expression with exercise were analyzed by multiple ANOVA. Because there were no significant changes in CD18 expression with a simple ANOVA, CD18 was not analyzed by multiple ANOVA. To explain the changes in leukocyte counts during exercise, a model that included running time, work intensity, and body-weight changes as covariates was chosen. A model to explain changes of CD11b expression during exercise including running time, work intensity, body-weight changes, and changes of leukocyte counts as covariates was constructed. A value for P < 0.05 was considered to be statistically significant.
Physiological parameters. The mean maximal running speed attained during the incremental treadmill test was 4.68 ± 0.46 m·s−1. This maximal speed reached during the incremental treadmill test corresponded to a peak O2 of 58.3 ± 4.9 mL·kg−1·min−1. The duration of the incremental treadmill test was 17.6 ± 2.5 min. The distances covered during the 3-h training run are given in Table 1. The workload intensity attained during the 3-h training run as estimated from the mean velocity and the incremental treadmill test was 50 ± 4% of peak O2. All subjects completed the full marathon distance in a mean time of 198 ± 25 min (Table 1). The slowest subject ran the marathon in 247 min and the fastest subject in 169 min. The workload intensity attained during the marathon was 74 ± 5% of peak O2 as estimated from the mean marathon velocities and velocity corresponding to peak O2.
Body weights and temperatures before and after exercise are given in Table 2. Changes in body temperature during exercise were statistically significant after the incremental treadmill test (P < 0.05), but not significant after 3-h training run and marathon. The incremental treadmill test was performed in a climatized room (26°C, 1026 mbar, 64% humidity). There was a very small but statistically significant decrease of the body weight after the incremental treadmill test (0.83 ± 0.32 kg; P < 0.05). The training run was performed during a warm summer day (27-33°C, 1026 mbar, 50-60% humidity). During the training run, the subjects lost 3.9 ± 0.57% of their body weight, indicating insufficient fluid repletion (P < 0.05). The climatic conditions for the marathon run were 20-24°C, 1030 mbar, and 66-72% humidity. There was a decrease of the body weight after the marathon by 2.8 ± 0.92% (P < 0.05).
Leukocyte counts. The absolute values of leukocyte counts before and after the maximal incremental load test, the 3-h training run, and the marathon are given in Table 3. The relative changes of leukocyte counts before and after the maximal incremental load test, the 3-h training run, and the marathon are illustrated in Figure 1. There was a significant increase (P < 0.001) in the leukocyte counts after the maximal incremental load test, the 3-h training run, and the marathon, respectively. On multiple ANOVA, 72% (P overall < 0.0001) of the variance of the leukocyte count changes could be explained by the covariates exercise duration (P < 0.0001) and intensity (P < 0.0001). The covariate body weight change during exercise was borderline significant to explain changes in leukocyte counts (P = 0.053).
Integrin expression and granulocyte granularity. The relative changes of CD11b and CD18 expression on circulating granulocytes as assessed by flow cytometry are given in Figures 2 and 3, respectively. There was no significant change in the CD18 expression after exercise. The surface expression of CD11b on circulating granulocytes was increased by 10.2 ± 9.6% (P < 0.05) after the maximal treadmill test and by 84 ± 76% (P < 0.01) after the marathon. These increases in CD11b surface expression could not be attributed to a distinct subpopulation of cells. There was no significant change in the CD11b expression on granulocytes after the 3 h training run. On multiple variance analysis, a model including the covariates weight loss during exercise (P = 0.008), leukocyte count changes (P = 0.42), exercise duration (P = 0.001), and exercise intensity (P = 0.21) explained 66% (P overall = 0.002) of the variance in CD11b changes during exercise. There was no significant change in CD11b or CD18 surface expression with storage of blood samples at 4°C for up to 12 h.
The granularities of granulocytes, as assessed by the sideward scatter characteristics before and after the maximal incremental load test, the 3-h training run, and the marathon are given in Table 4. There was no significant change in the granularity of granulocytes before and after exercise.
The main finding in this study was that the increase in CD11b expression on circulating granulocytes is related to exercise duration and dehydration (as indicated by a loss in body weight). The expression of CD11b on granulocytes was unchanged after a training run of moderate intensity. There was no significant change in CD18 expression or granulocyte granularity in response to exercise of different durations and intensities. Increased duration and intensity of endurance running were associated with increased leukocyte counts.
Upregulation of integrin adhesive receptors on granulocytes is accomplished either by de novo synthesis or by release of stored molecules from peroxidase negative granules (3,28). Release of integrins from peroxidase negative granules is accomplished within minutes (28,33). Therefore, degranulation is the most likely mechanism to explain upregulation of CD11b after intense exercise of short duration (8). According to the current understanding (8), granulocyte degranulation is likely to occur with the exercise durations and intensities achieved in our study. However, granulocyte degranulation after intense exercise as measured by sideward scatter characteristics was not reproduced in our study. Changes in sideward scatter may be less sensitive to detect granulocyte activation in vivo than immunofluorescence methods. Furthermore, sideward scatter characteristics of granulocytes might be more affected by storage than surface expression of integrin adhesive receptors. We did not observe major changes in integrin expression with storage at 4°C for up to 12 h. The effects of storage on sideward scatter characteristics of granulocytes was not systematically assessed.
De novo synthesis of integrin receptors takes at least several hours to occur (28) and would probably not be detected after short-term endurance exercise. Synthesis of integrin adhesive receptors might contribute to changes in CD11b surface expression on granulocytes after prolonged exercise such as a marathon.
In this study, the number of study subjects may have been too small to detect a change in CD18 expression with exercise. Another possible explanation for disparate effects of exercise on CD11b and CD18 expression is that the surface expression of these receptors in part may be independently regulated (10).
In our study, there was no relationship between surface expression of CD11b and changes in body temperature. We suspect that the body temperatures measured did not reflect the actual body temperatures during exercise. In particular after the marathon race, there was some delay between the completion of the race and the time that temperature measurements were taken. The changes in CD11b surface expression on granulocytes was in part explained by decreases in body weight. We suggest that the body weight decrease after endurance exercise in this study was mainly caused by dehydration. CD11b expression on leukocytes has been shown to be influenced by changes in temperature (in vitro, animal model) and osmolarity (in vitro) (30,34). Dehydration during endurance exercise may influence the regulation of CD11b expression on circulating granulocytes. To study a possible relation between body temperature and expression of CD11b on granulocytes, temperature measurements should be taken immediately after or during exercise.
As shown by multiple ANOVA, increased exercise duration partially explained the expression of CD11b on granulocytes. Furthermore, intense exercise of short duration was shown to increase CD11b expression on granulocytes. One explanation for the relationship between exercise duration and/or intensity and CD11b expression on circulating granulocytes could be related to mechanical stress imposed on blood cells during endurance exercise (7,20). Mechanical stress has been shown to upregulate CD11b surface expression on granulocytes in vitro(2).
Another speculative explanation that could relate the expression of CD11b on granulocytes to exercise duration and intensity is the release of inflammatory mediators. Inflammatory mediators that have been shown to be released in intense endurance exercise include tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), complement, and arachidonic acid metabolites (5,6,23). TNF-α and complement upregulate CD11b expression on granulocytes (2,28). In contrast to the observations with intense exercise, there was no increase in TNF-α, IL-1, or IL-6 after moderate endurance exercise (cycling at 60% of peak O2) (27). This state-of-affairs could explain the lack of a training run effect (50% of peak O2) on CD11b expression on granulocytes. It has also been shown that the redox state of cells may influence expression of surface receptors (26).
Increased exercise duration, increased exercise intensity, and weight loss were associated not only with an increase in CD11b expression on granulocytes but also, as has been shown previously (17), with an increase in leukocyte counts. There was, however, no significant relation between changes in leukocyte counts and changes in CD11b expression on granulocytes. This observation may suggest that changes in leukocyte counts and changes in MAC1 (CD11b/CD18) expression with endurance exercise are regulated relatively independently.
Our results and others' (8) suggest that CD11b is upregulated after short-term intense endurance exercise by about 10-20%. The two-fold increase in integrin expression on circulating granulocytes after the marathon race approaches the range observed in pathological conditions, such as some forms of systemic vasculitis (3-to 4-fold increase of integrin expression in active disease) (9). Whether the increase in CD11b surface expression with exercise augments tissue infiltration is not known and requires further studies.
Chronic endurance training may have effects on granulocyte integrin expression different from acute exercise. Recently, we have observed that the surface expression of several integrin adhesive receptors on granulocytes is smaller in trained than in untrained individuals (14). Similarly, leukocyte counts and activation markers at rest are decreased and the leukocytosis in exercise is blunted in trained as compared with untrained individuals (17,22).
The main weakness of this and many other studies dealing with activation markers of granulocytes is that the relatively small pool of circulating cells may not represent the entire cell population. Thus, measures of granulocyte activation after endurance exercise could be an underestimation, because activated granulocytes may be trapped in the vessel wall. Furthermore, leukocyte activation may under some circumstances be a more localized process (28). We cannot exclude the possibility that some of the changes in granulocyte surface markers after endurance exercise are due to the release of different granulocyte subpopulations from the vessel wall and, with more prolonged exercise, from bone marrow reserves (17). Another methodological problem is related to the fact that differential blood counts were not obtained. Therefore, the changes in leukocyte counts may not exactly represent the quantitative changes in circulating granulocytes. The qualitative changes in granulocytes rather than granulocyte counts were the main focus of this study.
We conclude that increases in CD11b expression on circulating granulocytes were evident after intense, short-duration, endurance exercise and after a competitive marathon race, but not after a training run of moderate intensity. Thus, exhaustive exercise may be one mechanism for the upregulation of integrin adhesive receptors on granulocytes. This phenomenon might be in part responsible for increased granulocyte tissue infiltration. Further research should clarify the question of whether increases in granulocyte integrin expression in endurance exercise contribute to tissue damage and/or repair.
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