1 Institute of Respiratory Pathophysiology, Italian National Research Council, 2 Institute of Human Physiology, and 4 Institute of Pneumology, University of Palermo, 90146 Palermo; and 3 Institute of Clinical Physiology, Italian National Research Council, 56126 Pisa, Italy
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ABSTRACT |
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Elite
athletes show a high prevalence of symptoms and signs of asthma, but no
study has assessed the acute effects of endurance exercise on airway
cells in nonasthmatic athletes. We measured exhaled nitric oxide (NO)
and collected samples of induced sputum after 3% NaCl aerosol
administration for 20 min in nonasthmatic middle-aged amateur runners
after the Fourth Palermo International Marathon and 6-9 wk later
(habitual training period) at baseline. After the marathon, exhaled NO
(n = 9 subjects) was higher [27 ± 9 parts/billion (ppb)] than at baseline (12 ± 4 ppb;
P < 0.0005). Polymorphonuclear neutrophil (PMN) counts
in induced sputum were much higher in runners (91.2 ± 3.6% of
total cells postmarathon and 78.7 ± 9.1% at baseline) than in
sedentary control subjects (9.9 ± 5.9%; P < 0.001). Expression of L-selectin and CD11b/CD18 in sputum PMNs was
lower after the race than at baseline and inversely related to the
amount of exhaled NO (r = 0.66 and
0.69,
respectively; P < 0.05). Our data indicate that sputum
PMNs are increased in nonasthmatic runners both after a marathon and at
baseline and suggest that NO may modulate exercise-associated
inflammatory airway changes.
polymorphonuclear neutrophil; nitric oxide; adhesion molecules; elastase
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INTRODUCTION |
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COMPETITIVE TRAINING IN ATHLETES is often associated with symptoms and signs of airway inflammation. A questionnaire study reported a high prevalence of physician-diagnosed asthma in elite athletes compared with sedentary control subjects (8). More recently, an increased prevalence of airway hyperresponsiveness (AHR) was found in a large group of elite athletes involved in different sports (16). These changes were interpreted as secondary to repeated exercise-induced hyperventilation and/or increased airway exposure to inhaled allergens or pollutants. In elite cross-country skiers, AHR was documented at the end of an intense training period, although endobronchial biopsies showed high T-lymphocyte, macrophage, eosinophil, and PMN counts irrespective of the presence of AHR, atopy, or symptoms of asthma (11). Therefore, intense endurance training appears associated with changes in airway cells, but their relationship with asthma is unclear.
Intense and prolonged exercise also causes systemic inflammation, which appears to be tightly regulated (26, 31). Running a marathon markedly increases circulating white blood cell and PMN counts (24) and plasma levels of pro- and anti-inflammatory cytokines (26). Exercise modifies PMN adhesion molecules by decreasing the expression of L-selectin, likely in relation to mobilization of the PMN marginated pool (37) and by increasing the expression of CD11b/CD18 (34, 37). Running a marathon causes PMN activation and degranulation as indicated by an increased serum elastase level after the race (4).
In the days after a race, a high frequency of upper respiratory infections was reported by marathon runners, suggesting that intense and prolonged endurance exercise may increase the susceptibility of the respiratory tract to pathogens (25). Nasal mucociliary clearance in runners was low for several days after a marathon race (22), whereas PMN counts and chemotactic activity were increased in nasal lavage fluid (21), suggesting a role of PMNs in the pathogenesis of upper airway infections. However, no study specifically addressed the question of exercise-induced PMN involvement in lower airways or the possible relationship between the respiratory and systemic effects of exercise.
In this study, we asked whether running a marathon affects nasal or exhaled nitric oxide (NO), considered a noninvasive marker of airway inflammation (13, 14, 17). In addition, we collected samples of induced sputum to study airway cells and venous blood to explore the relationship between the effects of the race at the systemic and airway levels. To our knowledge, our study is the first on the effects of a marathon race on airway cells in healthy, nonasthmatic amateur runners.
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METHODS |
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Subjects and study design.
Nine amateur male runners were studied (age 40.1 ± 10.7 yr; body
mass index at rest 22.8 ± 1.8 kg/m2). All subjects
were nonsmokers and clinically healthy and had no history of recent
infection or other disease. No subject used nonsteroidal
anti-inflammatory agents before or during the study period. No subject
reported a clinical diagnosis of asthma, asthmalike symptoms, or
habitual use of 2-agonists. Two subjects with a history
of allergic rhinitis were asymptomatic and under no treatment at the
time of the study; induced sputum was not obtained in them. The study
was approved by the local Ethical Committee, and all subjects gave
written informed consent. One subject refused to continue the study
after the measurements on the day of the marathon; in addition, his
blood sample underwent hemolysis. Complete sets of measurements were
obtained in the rest of the subjects.
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Measurements.
In each subject and condition, a 20-ml blood sample was drawn from the
antecubital vein for complete blood cell counts and preparation of
plasma and serum aliquots. These were stored at 20°C for subsequent
determination of cortisol, elastase, albumin, IgA,
1-protease inhibitor (
1-PI), histamine,
and NO metabolite concentrations.
Induced sputum production and processing. Sputum induction and processing were according to the method of Fahy et al. (5) with slight modifications (39). Briefly, after the collection of samples of saliva, the subjects were exposed to a hypertonic saline (3%) aerosol for 20 min. The aerosol was administered with an ultrasonic nebulizer (median particle diameter 2.5 µm, output 1 ml/min; Fisoneb, Fisons Italchimici Spa, Rome, Italy). The subject regularly interrupted the procedure to cough and expectorate sputum into 50-ml sterile ampoules. The oral cavity was washed with saline before the sputum was expectorated, and salivary contamination was minimized by blowing the nose. After the procedure, the absence of bronchospasm was confirmed by spirometry.
Sputum and saliva samples were added to an equal volume of a 0.1% dithiothreitol-saline solution (Sigma, St. Louis, MO). They were gently mixed with a vortex mixer and placed in a water bath at 37°C for 15 min, with periodic removal from the water bath for further brief gentle mixing. Homogenized samples were centrifuged at 800 g for 10 min, and the supernatants were aspirated and frozen atExpression of adhesion molecules on sputum PMNs.
After cytocentrifugation, the cell pellets were resuspended in saline,
and slides were prepared, dried at 20°C for 30 min, fixed with
acetone-methanol at 4°C for 10 min, and incubated at 37°C for
1 h with monoclonal antibodies for against intercellular adhesion
molecule (ICAM)-1, L-selectin, CD11a/CD18 (lymphocyte function-associated antigen-1), and CD11b/CD-18 (DAKO) at dilutions of
1:2, 1:2, 1:100, and 1:100, respectively. Immunoreactivity was revealed
with the labeled streptavidin-biotin-alkaline phosphatase technique.
Four hundred cells per slide were counted by two investigators (M. Profita and A. M. Vignola), and the results are expressed as
percentages of positive PMNs. Interinvestigator agreement was excellent
( = 0.94).
Biochemical analysis on plasma, serum, and sputum supernatants.
Plasma cortisol and histamine concentrations were assessed by
radioimmunoassay (Immunotech, Marseille, France). The detectability threshold for histamine was 0.2 nM. Total elastase was measured with a
homogeneous enzyme immunoassay specific for human PMN elastase (Ecoline, Kit Merck, Darmstadt, Germany) (23), with a
detectability threshold of 20 µg/l. 1-PI, IgA, and
albumin were measured by a nephelometric assay (Beckman array protein
system) using specific monoclonal antibodies (Beckman Immunochemistry
Systems) (28). The assay detected levels of
1-PI, IgA, and albumin >2.8, 11.0 and 6.0 µg/ml,
respectively. Plasma NO concentrations were estimated by a colorimetric
assay based on the Griess reaction (6). Nitrates were
converted to nitrites by nitrate reductase, and then Griess reagents
were added to convert nitrites to a chromophore compound. Nitrite
concentration was estimated by spectrophotometry absorbance at 540 nm.
Statistics. Data are reported as means ± SD. Data obtained after the marathon and at baseline were compared by paired t-test (blood cell counts and biochemical measurements on plasma and serum) or unpaired t- or Mann-Whitney test as appropriate (sputum cell counts and biochemical analysis and NO measurements). A Kruskal-Wallis test was used to compare sputum cells in the runners after the race and at baseline and in sedentary control subjects. Relationships between variables were analyzed by linear regression or Spearman rank coefficient test. For all tests, a statistical package was used (StatView 4.5, Abacus Concept, Berkeley, CA). A level of P < 0.05 was chosen as indicating significance.
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RESULTS |
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Clinical data. No subject reported respiratory symptoms during immediate recovery after the marathon race or symptoms of respiratory infections in the weeks after the race when returning to the laboratory for baseline measurements.
Blood, plasma, and serum samples.
White blood cell and PMN counts were very high after the marathon, with
decreased percentages of lymphocytes and monocytes (Table
2). No significant difference was found
between postmarathon and baseline samples for red blood cell and
platelet counts, hematocrit, hemoglobin concentration, or absolute
lymphocyte or monocyte counts (data not shown). Circulating cortisol
and elastase levels were high, whereas the histamine level was low
after the marathon (Table 2). Serum albumin concentration showed a
small significant increase after the race. Plasma 1-PI
and IgA concentrations were normal and unaffected by the race (data not
shown). The mean plasma NO concentration in the runners was 69 ± 10 µM after the marathon and 72 ± 20 µM at baseline [not
significant (NS)], both values being, as expected (10),
significantly higher than the plasma NO concentration in the sedentary
control subjects (51 ± 14 µM; P < 0.05;
n = 7).
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Pulmonary function tests and NO measurements.
Spirometry was performed on average 103 ± 29 min after the
runners finished the race. Forced expiratory volume in 1 s and forced vital capacity were similar after the race and at baseline (Fig.
1). DLCO after
the marathon was in the normal range (105.4 ± 11.0% of predicted
value).
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Induced sputum. Induced sputum was obtained on average 202 ± 28 min after the end of the race. Technically adequate sputum samples were obtained only from five of seven subjects after the marathon and from five of six subjects at baseline. The samples rejected showed excess squamous contamination (both samples from the same subject) and low cell yield (postrace sample from another subject). Therefore, induced sputum samples were obtained only from four subjects on both occasions, and the data were analyzed by tests for unpaired comparisons.
The cells in the induced sputum from the runners were mostly PMNs (Fig. 3) as opposed to the prevalence of macrophages in the control subjects. However, the absolute macrophage count in the runners was in the normal range. Compared with postrace samples, a lower percentage of PMNs (P < 0.05) and a higher percentage of macrophages (P < 0.05) were found in the runners at baseline, but the absolute counts of both cell types did not differ significantly between experimental conditions. Eosinophil counts in the runners were low (0.4 ± 0.7% of total cells after the marathon and 1.0 ± 1.5% at baseline). Lymphocyte and bronchial cell counts were similar in the runners and control subjects and in the postrace and baseline samples (data not shown). Although the sample size was small, analysis of all induced sputum samples showed positive correlations between absolute cell and PMN counts versus plasma cortisol concentration in the runners (Fig. 4). Absolute cell and PMN counts in the sputum were unrelated to exhaled NO.
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DISCUSSION |
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Our study tested the hypothesis that intense and prolonged exercise may affect airway cell number and/or composition in nonasthmatic amateur runners, possibly accounting for the high susceptibility to respiratory infections reported after a marathon race (24, 25). After the race, we found 1) high exhaled and nasal NO concentrations; 2) high PMN counts in the induced sputum; 3) few PMNs positive for L-selectin and CD11b/CD18, possibly related to high exhaled NO and plasma cortisol concentrations; and 4) normal sputum elastase concentration, suggesting a lack of significant PMN activation in the induced sputum as opposed to the high elastase concentration in serum. Under baseline conditions, the percentage of PMNs in the induced sputum of the runners remained high compared with that in the sedentary control subjects, suggesting a chronic increase in PMNs in the airways, possibly related to habitual training.
The high percentage of PMNs in the induced sputum of the runners confirms and extends the findings obtained in other endurance athletes such as elite cross-country skiers (11) and elite swimmers (7). However, our study differs from the previous ones in two major respects: first, we studied amateur, middle-age runners compared with much younger elite athlete populations in the previous studies (7, 11), and second, our aim was to assess both the acute effects of prolonged endurance exercise (marathon samples) and the possible effects of chronic training (baseline samples), whereas the available studies collected samples only under baseline conditions (7, 11). Indeed, our data show a trend toward different amounts and functional features of PMNs in the induced sputum of the runners after the marathon compared with the baseline conditions. Because our subjects were not asthmatic, our data support the fact that endurance exercise may increase PMNs in the airways irrespective of concomitant asthma or AHR (11).
After the race, the elastase level was very high in the serum (threefold increase over baseline conditions) (4) and normal in the induced sputum, indicating that the high number of PMNs in the induced sputum was not associated with a significant degree of PMN activation. The pattern of adhesion molecules in the induced sputum also suggested a low grade of activation (1) in airway PMNs after the marathon. Few PMNs in the induced sputum were positive for CD11b/CD18 after the race, in contrast to the increased percentage of PMNs expressing CD11b/CD18 found in peripheral blood after incremental exercise (37) or a marathon race (9). Conversely, expression of L-selectin was low in the induced sputum after the marathon, similar to the findings in peripheral blood PMNs after incremental (34) or moderate-intensity (15) exercise. As a possible explanation, corticosteroid-induced downregulation of both L-selectin and CD11b/CD18 (2) is supported by the correlation found between plasma cortisol and expression of both adhesion molecules in induced sputum PMNs. Alternatively, the time lag between the end of the race and data collection may have allowed a progressive loss of CD11b/CD18 by PMNs, in agreement with recent findings in infarcted myocardium and in vitro (40), suggesting involvement of PMNs not expressing CD11b/CD18 in the healing processes. Further investigation is needed to define this point.
Exhaled NO was measured in our runners as a noninvasive marker of
airway inflammation (14). During exercise, the volume of
expired NO followed the changes in minute ventilation, with a rapid
fall toward baseline in early recovery (32). About 3 h after the marathon, however, exhaled NO was high, suggesting late
changes in the time course of exhaled NO after exercise. Nasal NO was
also high after the marathon in nonrhinitic runners, possibly in
relation to marathon-induced nasal alterations (21, 22).
Although exhaled NO is traditionally considered in the asthma
literature as a marker of airway inflammation, recent data suggest some
role for NO and derived products in modulating PMN chemotactic activity
(29) and downregulating nuclear factor-B activation in
the airways (35). Our data in runners suggest that NO may
indeed be involved in the control of inflammatory processes in the
airways. High exhaled NO after the marathon race was associated with a
low expression of L-selectin and CD11b/CD18 in induced sputum PMNs, the
opposite pattern being found at baseline. Because NO donors may inhibit
the upregulation of CD18 by zymosan-activated plasma without affecting
L-selectin shedding of PMNs (30), we speculate that a
similar mechanism may explain the inverse correlation between exhaled
NO and CD11b/CD18 expression in sputum PMNs of runners. Although our
sample was too small to draw definitive conclusions, the data suggest a
possible role for NO in controlling or decreasing airway inflammation
after intense and prolonged endurance exercise. However, a simple
association of independent changes in PMN adhesion molecules and
exhaled NO cannot be ruled out at present, and further studies are
needed to confirm our hypothesis.
A trend toward increased albumin concentration in induced sputum after the race suggests ongoing inflammation and increased vascular permeability in the airway compartment. The lack of significance in comparing albumin concentration between postrace and baseline measurements is likely due to the small sample size: type II error analysis showed that 14 observations/group would be necessary to show a significant difference between postrace and baseline by unpaired t-test, assuming that mean ± SD values remained the same after the sample size was increased. Histamine concentrations in induced sputum also tended to be higher after the marathon compared with baseline (sample size required to show a significant difference, 27 observations/group). However, several findings do not support the finding that histamine is a key mediator of exercise-induced airway changes: 1) histamine is known to inhibit PMN chemotaxis (3); 2) the normal pulmonary function tests (PFTs) in our runners argue against a bronchoconstrictor effect of histamine after exercise; and 3) although histamine exerts immunoregulatory functions by increasing expression of ICAM-1 and human leukocyte antigen-DR and by promoting release of fibronectin by bronchial epithelial cells (38), ICAM-1 expression in sputum was absent after the marathon. Therefore, our data do not indicate a clear-cut role for histamine in the context of exercise-induced airway inflammation.
As for the mechanism(s) of exercise-induced airway changes in runners, we favor the explanation that they may be at least partly linked to hyperventilation for prolonged periods of time. Mechanical ventilation at high tidal volumes was shown to cause pulmonary inflammation and cytokine release (27, 36). A similar mechanism may operate in runners during intense and prolonged acute exercise and chronic training. Both elite cross-country skiers (11) and swimmers (8) show increased PMNs in endobronchial biopsies or induced sputum under baseline conditions, supporting the fact that exercise may cause low-grade airway cell changes by repeated, training-associated episodes of ventilation at high tidal volumes. Again, the small sample size of our study may be responsible for the lack of significance found between the runners and sedentary control subjects in the cellularity of the induced sputum (sample size required to show a significant difference between runners postmarathon and control subjects, 18 observations/group). Another possible explanation for the high PMN counts in the induced sputum of the runners is that air pollutants were low during the marathon due to closure of the circuit to motor vehicles. In addition, our subjects usually trained in an extraurban environment, making a chronic exposure to high levels of pollutants an unlikely explanation of our findings. The occurrence of cold air-induced bronchoconstriction is not supported by normal PFTs, and there was no report of respiratory symptoms after the race; in addition, cross-country skiers with "ski asthma" show a high percentage of lymphocytes in bronchoalveolar lavage fluid at baseline (33), suggesting at least some biological differences between exercise at moderate temperature and exercise in the cold. Finally, the very high percentage of PMNs in the induced sputum of the runners was unlikely to result from the sputum induction procedure itself (20). As for a possible relationship between high PMN counts in induced sputum and exercise-induced systemic changes, a definitive conclusion is hard to draw: induced sputum total cell and PMN counts increased with plasma cortisol, but such a correlation may simply indicate that the sputum cell content reflected the stress of a marathon race in a dose-response fashion. A larger sample size is needed to extend studies on this point.
Some features of our study need comment. First, the absence of AHR or atopy was not objectively documented in our sample. However, the clinical history was negative for symptoms and/or signs of asthma, and induced sputum cell composition or baseline exhaled NO did not support that our runners were asthmatic. Second, the small sample size was imposed by technical limitations linked to sputum processing on the day of the marathon. Third, we are aware that normal spirometric and diffusion measurements ~2 h after the end of the race cannot exclude the possibility that we may have missed transient changes occurring immediately after the race. After a marathon, forced expiratory volume in 1 s was increased (19) or unchanged (18), forced vital capacity was decreased (18), and DLCO was decreased (19) or unaffected (18), all changes returning to baseline by the following morning (18, 19). Because no study ever reported PFTs during late recovery after a marathon, we felt it was important to measure PFTs together with exhaled NO and the collection of induced sputum samples. The finding of normal PFTs at a time when markers of airway inflammation were increased suggests a light impact of exercise-induced airway changes on airway function, at least in nonelite nonasthmatic runners during late recovery after a marathon. Finally, our measurements provide a "snapshot" of a relatively late phase of recovery after the race, the complex protocol not allowing data collection at the site of race arrival. Additional studies are necessary to fully describe the time course of NO and airway cells after prolonged and intense endurance exercise. Despite these limitations and difficulties, we feel that data obtained after a real competition are valuable because they take into account aspects of a marathon (i.e., weather conditions or psychological stress) that cannot be easily mimicked in the laboratory.
In summary, in nonasthmatic middle-aged amateur runners, a marathon race increased the markers of inflammation in the airways without symptoms or late changes in the PFTs. The absence of clear signs of PMN activation in induced sputum suggests a tight control of exercise-induced airway changes, extending to the respiratory compartment the view that exercise-induced changes in biological markers of inflammation appear altogether quite small (31). Further studies are needed to fully characterize the pathogenesis of exercise-induced airway inflammation in both asthmatic and nonasthmatic athletes, the possible long-term consequences of prolonged training, and the effects on athlete's performance.
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ACKNOWLEDGEMENTS |
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We are grateful to the many people who made this study possible: the athletes who underwent a complex and time-consuming protocol; Drs. Fabio Cibella, Giuseppina Cuttitta, Pietro Abate, Daniela Guerrera, Rosanna Giaramidaro, Raffaella Hopps, and Silvio D'Anna for help in data collection; Prof. Rosario Alaimo for providing the data on air quality monitoring; Drs. Carlo Palombo and Silvia Lenzi for measuring plasma nitric oxide metabolites; and Dr. Simone Albanese for plasma cortisol determination. We are also indebted to Jerome A. Dempsey, James C. Hogg, Pang N. Shek, Shawn Rhind, Claire M. Doershuk, V. Marco Ranieri, and Arthur S. Slutsky for helpful suggestions.
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FOOTNOTES |
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This study was supported by the Italian National Research Council and Valeas Italia.
Address for reprint requests and other correspondence: M. R. Bonsignore, I.F.R.-C.N.R., Via Ugo La Malfa, 153, 90146 Palermo, Italy (E-mail: marisa{at}ifr.pa.cnr.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 February 2001; accepted in final form 23 April 2001.
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