Abstract
Introduction:
Swimming practice has been associated with eosinophilic inflammation, however, the underlying mechanisms are not fully understood. The eosinophil cationic protein (ECP) in induced sputum may be used as a potential biomarker to assess airway eosinophilic inflammation among elite swimmers. The objective of this study is to characterize ECP levels in sputum supernatant in elite swimmers and evaluate ECP as an eosinophilic inflammatory marker.
Material and methods:
Elite swimmers annually screened in our department (n = 27) were invited to participate in this cross-sectional study. Swimmers who agreed to participate (n = 24, 46% girls) performed lung function and skin-prick tests. Induced sputum was also collected and analyzed for differential cell counts and ECP measurements in sputum supernatant (ImmunoCAPTM 100, ECP, Thermo Fisher Scientific, Uppsala, Sweden).
Results:
The median ECP level was 15.60 μg/L (6.02–38.75 μg/L) and higher levels were found among boys (27.90 (11.20–46.30) μg/L vs 6.65 (2.82–22.80) μg/L, P = .02). In addition, ECP levels in the sputum supernatant were positively correlated with eosinophil cell counts in the induced sputum (r = 0.583, P = .08).
Conclusions:
ECP levels correlated positively with eosinophil counts in the induced sputum in elite swimmers. The measurement of ECP in sputum supernatant may be a useful marker to assess and manage eosinophilic inflammatory changes in the airways of elite swimmers.
Keywords: biomarker, elite swimmers, eosinophil cationic protein, eosinophilic inflammation, sputum
Introduction
Swimming practice has been associated with an increased risk of airway disfunction, even in athletes with no prior history of respiratory disorders.1,2 Cumulative lifetime exposure to chlorine by-products derived from swimming pool disinfectants can cause airway irritation, epithelial damage, airway inflammation, and pulmonary function decline.3 The high ventilation rate during swimming may determine the quantity of inhaled chlorine by-products; elite swimmers, who spend many hours in pools, are likely to be more affected by those products.4 Eosinophilic airway inflammation has been reported after long-term exposure to the pool environment in elite swimmers, but not in recreational swimmers.5 Previous studies reported airway vascular permeability in elite swimmers similar to individuals with asthma; thus, swimming practice may promote airway inflammation by enhancing local vascular permeability.6 Moreover, the prevalence of atopy is higher in swimmers who are repeatedly exposed to chlorine by-products. The epithelial damage caused by swimming pools environment may facilitate mucosal penetration of allergens inducing allergic sensitization.7,8,9
The induced sputum of elite swimmers has increased eosinophil and neutrophil counts compared to healthy controls.5,10,11,12 Eosinophils contain granule proteins like eosinophil cationic protein (ECP) that have proinflammatory effects and can be released when eosinophils reach the local tissue site of inflammation.13,14 Increased ECP levels and eosinophils have been associated with atopic diseases, such as asthma and atopic dermatitis.15 ECP can be found in many body fluids, including serum, sputum, and bronchoalveolar lavage fluid, and maybe a potential biomarker of airway eosinophilic inflammation.16 Sputum supernatant is a fluid obtained during sputum collection and analysis, which may be useful to study and quantify several biomarkers. Previous studies have shown its utility to assess and monitor lower airway inflammation among individuals with asthma.17,18 The use of sputum allows a direct and representative sample of the local inflammation, and therefore a more accurate measure to identify the presence, type, and severity of airway inflammation.
The combined effects of chronic endurance exercise and repeated exposure to chlorine by-products may cause structural and functional changes, including airway remodeling and an increase of inflammatory mediators.1 Despite the efforts of previous studies on elite swimmers, the mechanisms of airway disorders in this population is not fully understood. We believe that by characterizing inflammatory markers, we can better understand the mechanisms underlying airway changes reported in competitive swimmers. In this study, we aim to characterize the ECP levels in the sputum supernatant among elite swimmers. Further-more, we hypothesize that ECP levels may be used as a marker of eosinophil counts in the induced sputum of elite swimmers.
Material and methods
Study design
The participants included in this cross-sectional study have been previously described.19 Briefly, from the 27 elite swimmers screened in our department, 24 agreed to participate in this study. Swimmers eligible for this study aged ≥ 14 years old, trained at least 10 hours per week, and were free from respiratory infection in the last 3 weeks. The screening visits occurred at the beginning of the training season, during the summer. In these visits, swimmers were asked to withhold anti-asthmatic and/or anti-allergic medication and performed a specific evaluation to collect induced sputum before the training session. Written informed consent and ethical commission approval were obtained previously to any study procedure.
Study procedures
Lung function
Spirometry was performed according to the European Respiratory Society guidelines,20 and reversibility was assessed 15 min after 400 μg of inhaled salbutamol. Clinically significant reversibility was defined as a ≥12% and 200 mL increase in the forced expiratory volume in the first second of expiration (FEV1) after inhalation of salbutamol.
Skin prick tests
Skin prick tests were performed in accordance with the international guidelines21 using a standard battery of commercial extracts for common aeroallergens (Leti®, Madrid, Spain). Atopy was defined by the presence of a positive test on at least 1 of the allergens.
Collection and analysis of induced sputum
Sputum collection and analysis were performed as previously described.22 Briefly, sputum induction was performed using inhalation of hypertonic saline (4.5%) through a mouthpiece connected to an ultrasonic nebulizer (OMRON NE-U17; Omron Healthcare Europe, Netherlands).23 Sputum was then sampled and treated with dithiothreitol (Sputolysin; Calbiochem Corporation, USA). The suspension was centrifuged; the cell pellet was resuspended while the supernatant was collected and stored. Cytospins were prepared and stained using May-Grunwald/Giemsa. Differential cell counts were made by counting a minimum of 500 nonsquamous cells. The lower respiratory origin was confirmed by the presence of macrophages and bronchial epithelial cells. Normal values for sputum differential cell percentages were based on published healthy subjects counts.24,25,26,27 These measurements were done blindly to the clinical characteristics of the subjects.
Quantification of molecular markers in induced sputum supernatant
ECP in sputum supernatant was measured using ImmunoCAP™ 100, ECP, Thermo Fisher Scientific, Uppsala, Sweden. The detection limit was 0.5 μg/L.
Participants
The median age of participants was 17years old. The prevalence of asthma was 21% (n = 5) according to self-reporting medical diagnosis and 4% (n = 1) had atopy (Table 1). Differential cell counts were performed only in 10 participants (3 girls and 7 boys). Participants with asthma showed poorer lung function, specifically, lower FEV1 and FEF25–75 percentages (Table S1, http://links.lww.com/PBJ/A10). Participants with asthma practiced swimming for longer [median (25th–75th percentile): 12 (10–12) vs 9 (7–11) years, P = .05]. Although not significant, eosinophil cell counts percentages were higher in swimmers with asthma [median (25th–75th percentile): 0.5 (0.1–2.1) vs 0.2 (0.0– 0.2), P = .61] (Table S1, http://links.lww.com/PBJ/A10).
Table 1.
Demographic characteristics of swimmers.
| Characteristics | Total (n = 24) | Girls (n = 11) | Boys (n = 13) | P value |
|---|---|---|---|---|
| Age (years)∗ | 17.00 (16.00–20.50) | 17.00 (14.00–19.00) | 17.00 (16.00–21.00) | .13 |
| Years of competition∗ | 10.00 (7.00–11.50) | 9.00 (7.00–10.00) | 10.00 (7.00–12.00) | .33 |
| Training hours per week | 18.00 (16.00–20.00) | 18.00 (17.00–19.00) | 18.00 (14.00–20.00) | .87 |
| Asthma, n (%) | 5 (21) | 2 (18) | 3 (23) | .77 |
| Atopy, n (%) | 1 (4) | 1 (9) | 0 | – |
| FVC (%) ∗ | 114.50 (107.00–122.00) | 115.00 (107.00–122.00) | 114.00 (110.00–124.00) | .65 |
| FEV1 (%)∗ | 115.50 (105.00–124.00) | 111.00 (105.00–117.00) | 119.00 (104.00–126.00) | .27 |
| FEV1/FVC (%) ∗ | 85.20 (81.70–93.10) | 84.30 (82.50–93.70) | 86.15 (80.70–93.05) | .65 |
| FEF25–75 (%) ∗ | 112.00 (81.00–128.00) | 98.00 (83.50–114.00) | 117.00 (80.00–128.00) | .37 |
| FEV1 increase after BD (%) | 4.50 (1.00–7.00) | 5.00 (1.00–7.00) | 3.00 (0.50–6.00) | .41 |
BD: bronchodilation; FEV1: forced expiratory volume in the first second of expiration; FEF25–75: forced expiratory flow middle portion of FVC; FVC: forced vital capacity.
Data are presented as median (25th–75th percentile).
Statistical analysis
Categorical variables were summarized as absolute numbers and percentages, and continuous variables were expressed as median (25th–75th percentile). Given the small sample size, nonparametric tests were used to assess differences by sex and asthma diagnosis. Mann-Whitney U test was used for continuous variables and Chi-square tests for categorical variables. The correlation between ECP levels and eosinophils in induced sputum was assessed using the Spearman rank correlation. Statistical significance was set as P-values less than .05. Statistical analyses were performed using SPSS 26.0 (IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY, USA: IBM Corp).
Results
The median ECP level was 15.60 μg/L (6.02–38.75 μg/L) and higher levels were found in boys (27.90 (11.20–46.30) μ/L vs 6.65 (2.82–22.80) μg/L, P = .02). The median (25th–75th percentile) percentages of eosinophils and neutrophils in the induced sputum were 0.18% (0%–0.83%) and 1.15% (0.32%– 13.90%), respectively. Although no statistically significant differences were found, the median levels of eosinophils and neutrophils were higher among boys. A moderate correlation was observed between ECP levels in the sputum supernatant and eosinophils in induced sputum (r = 0.583, P = .08). This correlation was significant among boys (r = 0.857, P = .01) (Table 2). Additionally, there were no differences in the ECP levels between swimmers with asthma and healthy swimmers (Table S1, http://links.lww.com/PBJ/A10).
Table 2.
Measurements of inflammatory markers.
| Total (n = 24) | Girls (n = 11) | Boys (n = 13) | P value | |
|---|---|---|---|---|
| IS neutrophils (%) ∗ | 1.15 (0.32–13.90) | 0.32 (0.32–2.11) | 1.47 (0.45–19.30) | .30 |
| IS epithelial cells ∗%)∗ | 47.98 (18.10–51.40) | 50.96 (11.81–50.96) | 45.00 (18.10–57.60) | .73 |
| IS eosinophils (%) | 0.18 (0.00–0.83) | 0.00 (0.00–0.17) | 0.19 (0.16–1.30) | .08 |
| ECP∗(μg/l)† | 15.60 (6.02–38.75) | 6.65 (2.82–22.80) | 27.90 (11.20–46.30) | .02 |
| ECP∗IS eosinophils | 0.58 (P = .08) | ‡ | 0.86 (P = .01) |
Data are presented as median (25th–75th percentile).
IS: induced sputum; ECP: eosinophilic cationic protein.
IS differential cell counts were performed only in 10 participants (3 girls and 7 boys).
ECP levels were measured in sputum supernatant.
The sample size among the girl's group (n = 3) was too low to perform the correlation coefficient.
Discussion
In this study, we characterize ECP levels in sputum supernatant among elite swimmers. Our results suggest that ECP may be used as a marker of eosinophilic granulocyte activation. Moreover, we found a positive correlation between ECP levels in the sputum supernatant and eosinophil cell counts in the induced sputum of elite swimmers, being significant among boys.
Our study has some limitations. First, the cross-sectional study design did not allow for monitoring the evolution of measurements. Second, variability in the execution or in the selected method for processing sputum and measuring ECP should be considered when comparing with previous studies. Third, the number of athletes assessed was small and induced sputum was performed only in a subgroup of participants, which may be explained by the complexity of collecting induced sputum with nebulized saline in non-productive subjects.27 This technical difficulty may also explain the number of differential cell counts observed in our study. Nevertheless, sputum induction is a noninvasive, reliable, and safe method when assessing airway inflammation.17,28 Although sputum eosinophilic counts are a direct marker of airway inflammation, the analysis is time-consuming and technically difficult, limiting the number of individuals that can be examined simultaneously.18,29
Therefore, the measurement of ECP levels in sputum supernatant to assess inflammatory response occurring in the airways of athletes might be a useful marker. Clinically, this method has the potential to be used as an evaluation tool to optimize the management, screening, and prevention of airway disorders in elite swimmers. In addition, low within-subject variability in ECP measurements has been previously reported, supporting the repeatability of this method.24
Similar results have been previously reported in patients with known eosinophilic airway inflammatory diseases, such as asthma, chronic obstructive disease, and cystic fibrosis.25,30,31 ECP levels in our swimmers are in line with results reported for patients with asthma and higher than those found for healthy controls, considering previous studies using a similar method to measure ECP.26,28 Our results suggest that the mechanism behind the increment in ECP levels in elite swimmers may be independent of the diagnosis of asthma, supporting that the exposure to the swimming pool environment may play a role in these changes. In line with this finding, we also observed higher levels of ECP among boys, who had more years of competition and training hours per week, suggesting that the longer exposure to swimming pool environment, may be related to increased levels of ECP. Previous results in elite swimmers have also reported the adverse health effects from prolonged and continuous exposure to the swimming pool environment, also showing the role of irritant mechanism on airway inflammation.2,32 Accordingly, ECP characterization using sputum supernatant may be a simple and useful method for assessing and monitoring eosinophilic airway inflammation in elite athletes.
A 5-year prospective study reported worsening or development of eosinophilic airway inflammation among highly trained swimmers who continued their sports career.33 Repeated exposures to the swimming pool environment during training may partially explain this finding due to chlorine and its by-products, which may irritate the airways by oxidative stress. Skrgat et al34 have also shown the occurrence of oxidative stress in swimmers during high-intensity training period. Another study reported an enhanced migration of blood eosinophils to airway tissues during strenuous endurance exercise, leading to an increased degranulation of eosinophils and the release of ECP in healthy mountaineers.35 This finding is supported by reports of eosinophil granulocyte activation and ECP elevation during exercise that might be due to the activation of the complement system during exercise.36 Likewise, this mechanism may also explain an increment of ECP levels in competitive swimmers.
Exercise induces hyperventilation that can increase the osmolarity of the airway surface layer by dehydration of periciliary fluid. Via an osmotic challenge to the bronchial epithelium, hyperosmolality may indirectly trigger the release of ECP from eosinophils.10,35,37 Repeated bouts of exercise may lead to frequent episodes of ECP peaks increasing the risk of developing exercise-induced and allergic asthma in elite swimmers. This finding may partly explain the propensity of endurance athletes, including swimmers, for developing eosinophilic airway inflammation. Remodeling of the airway epithelium to a secretory phenotype has been previously implicated in the development of exercise-induced bronchoconstriction as a response to injury. This may be caused by the stimulation of mucin secretion during hyperpnea to counteract the drying of the protective lining of the airways and possibly increase the provision of water. Additionally, increased airway movements and pressure changes during intense breathing could stimulate epithelial cells to release cytokines.7 This evidence indicates that eosinophilic activation and ECP release may play a role in the pathogenesis of airway dysfunction in elite swimmers. To support this hypothesis, ECP has been also implicated in asthma as a stimulator of airway mucus secretion.14,16 Our results are in line with a previous study that reported an increased percentage of bronchial epithelial cells in swimmers.12 In 6 non-elite outdoor swimmers, Bonsignore et al10 observed higher baseline differential neutrophil counts in sputum when compared to controls, suggesting that chronic endurance exercise may play a role in the pathogenesis of airway neutrophilia. However, eosinophils were not elevated, suggesting that airway mast cells and eosinophilic infiltration, previously reported in swimmers,7 might be related to chronic exposure to irritants during indoor training.
However, other studies in asthma reported a weak or non-significant correlation between supernatant ECP levels and the percentage of eosinophil counts.18,38 These contradicting results may be explained by the activation degree of eosinophils. When highly activated by specific and/or increased degranulation agents, eosinophils may release large amounts of ECP. Therefore, ECP measured in supernatant reflects the number of activated eosinophils present in sputum, which does not necessarily correlate with sputum eosinophil counts.18 Other explanations may be related to the inclusion of diseased individuals, the total cell counts may be increased due to factors related to the disease leading to a non-specific eosinophil degranulation and a lower percentage of eosinophils, despite an increment in the absolute number of eosinophils. It has been demonstrated that the inclusion of subjects with asthma exacerbation may lead to an attenuation in the proportion of eosinophils because there might be eosinophil lysis with the release of free granules.39
Conclusion
In conclusion, the examination of ECP levels in induced sputum may be useful to examine and follow inflammatory and remodeling changes in the airways among elite swimmers. Further studies in larger populations are needed to confirm if ECP measurements in sputum supernatant are a reproducible and clinically valid method to assess airway dysfunction in this population.
Acknowledgments
We thank all subjects for their participation and the technical and administrative staff of FC Porto Swimming Section for logistical help.
Conflicts of interest
All authors declare no conflicts of interest.
Financial sources
Authors gratefully acknowledge the funding by the Project NORTE-01-0145-FEDER-000010—Health, Comfort and Energy in the Built Environment (HEBE), cofinanced by Programa Operacional Regional do Norte (NORTE2020), through Fundo Europeu de Desenvolvimento Regional (FEDER).
Presentation
None.
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