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Leptin and urinary leukotriene E4 and 9α,11β-prostaglandin F2 release after exercise challenge
Ann Allergy Asthma Immunol 111 (2013) 112e117
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Leptin and urinary leukotriene E4 and 9a,11b-prostaglandin F2 release after exercise challenge Hey-Sung Baek, MD, PhD *; Jae-Hyung Choi, MD y; Jae-Won Oh, MD, PhD y; and Ha-Baik Lee, MD, PhD y * Department y
of Pediatrics, Hallym University Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea Department of Pediatrics, Hanyang University Hospital, Hanyang University College of Medicine, Seoul, Korea
A R T I C L E
I N F O
Article history: Received for publication March 26, 2013. Received in revised form May 16, 2013. Accepted for publication May 21, 2013.
A B S T R A C T
Background: Leptin-related effects on inflammation and bronchial hyperresponsiveness (BHR) in the human airway have not been demonstrated. Objectives: To investigate the relationship between the levels of serum leptin and BHR and urinary leukotriene E4 (LTE4) and 9a,11b-prostaglandin F2 (9a,11b-PGF2) release after exercise challenge in asthmatic children. Methods: Eighty-six prepubertal children between 6 and 10 years old were enrolled and divided into 4 groups: 19 obese asthmatic children, 25 normal-weight asthmatic children, 21 obese nonasthmatic children, and 21 healthy controls. We measured serum leptin levels and urinary LTE4 and 9a,11b-PGF2 levels in children before and 30 minutes after the exercise challenge. Results: Serum leptin levels were significantly higher in obese asthmatic children compared with normalweight asthmatic children. Significant increases in urinary levels of LTE4 and 9a,11b-PGF2 were observed in obese asthmatic children after the exercise challenge. Although smaller than in obese asthmatic children, significant increases in the urinary levels of LTE4 and 9a,11b-PGF2 were also observed in the normal-weight. Asthmatic children Logarithmic serum leptin values were significantly associated with the logarithmic maximum percentage change in forced expiratory volume in 1 second, the logarithmic urinary LTE4 change, and the logarithmic urinary 9a,11b-PGF2 change from baseline to after exercise in both obese and normalweight asthmatic children. Conclusion: The serum levels of leptin were significantly associated with BHR and urinary LTE4 and 9a,11b-PGF2 release induced by exercise challenge in asthmatic children. Ó 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Introduction Exercise-induced bronchoconstriction (EIB) reflects the degree of indirect bronchial hyperresponsiveness (BHR).1 Hyperosmolar triggering of mast cells and possibly other inflammatory cells results in release of bronchoconstricting mediators (eg, cysteinylleukotrienes, histamine, and prostaglandins) during exercise challenge.2e6 Direct evidence for mediator release after exercise in asthmatic patients includes an increase in urinary levels of the mast cell marker 9a,11b-prostaglandin F2 (9a,11b-PGF2) and leukotriene E4 (LTE4) in association with EIB.4e6 Adipose tissue releases a variety of proinflammatory adipokines, including leptin and adiponectin.7,8 Leptin may play roles in the development of asthma and allergies. Studies in mouse models have demonstrated that adipokines enhance BHR, airway inflammation, and allergic responses.9e11 Another study found in vitro
Reprints: Ha-Baik Lee, MD, PhD, Department of Pediatrics, Hanyang University Hospital, Hanyang University College of Medicine, 17 Haengdang-Dong, SeongdongGu, Seoul 133-792, Korea; E-mail: [email protected]. Disclosures: Authors have nothing to disclose.
that exogenous leptin enhances leukotriene production.12 This may be clinically important because obese asthmatic patients are relatively resistant to inhaled corticosteroids13,14 but respond to antileukotrienes as do lean asthmatic patients.14 However, leptin-induced effects on inflammation and BHR in the human airway have not been demonstrated. The aim of this study was to investigate the association between the levels of serum leptin and BHR and urinary release of LTE4 and 9a,11b-PGF2 after exercise challenge in asthmatic children. Methods Study Patients Study patients were randomly recruited from outpatient clinics in Hanyang University Hospital, Seoul, Korea. Participants were prepubertal children 6 to 10 years old divided into 4 groups: 19 obese asthmatic children, 25 normal-weight asthmatic children, 21 obese nonasthmatic children, and 21 healthy controls. Age- and sexspecific body mass indices (BMIs) were calculated according to the US Centers for Disease Control and Prevention guidelines.15 Participants with a BMI less than the 85th percentile were
1081-1206/13/$36.00 - see front matter Ó 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anai.2013.05.019
H.-S. Baek et al. / Ann Allergy Asthma Immunol 111 (2013) 112e117
considered normal weight, and those with a BMI in the 95th percentile or higher were considered obese. Underweight individuals in the less than fifth percentile were excluded. Asthma was defined as the presence of symptoms either with an increase in forced expiratory volume in 1 second (FEV1) of 12% or higher after bronchodilator treatment or if less than 8.0 mg/mL of inhaled methacholine induced a 20% decrease in FEV1 (PC20).16 An exercise bronchoprovocation test was used for EIB diagnosis, the results of which were considered positive with a 15% or greater decrease in FEV1 after exercise.16 Classification of asthma was based on Global Initiative for Asthma guidelines.17 Controls were age- and sexmatched healthy children with no history of wheezing or infection during the preceding 2 weeks. Exclusion criteria included acute exacerbation of asthma within the previous 6 months and parenchymal lung disease within the previous 4 weeks. Participants were excluded if they had used inhaled corticosteroids, leukotriene modifiers, long-acting antihistamines, or long-acting b2-agonists during the previous 30 days. In the healthy group, those with any likelihood of atopy or a positive methacholine challenge result for BHR were excluded. Study Protocol Participants had a total of 3 visits. All participants initially underwent anthropometric measurements of height and weight. They were also examined for any sign of puberty during the visit. At first visit, blood tests were performed, and fractional exhaled nitric oxide levels were measured. Each patient was evaluated by skin prick tests (SPTs) and before and after bronchodilator spirometry. Skin rick testing included house dust mite, cat dander, dog dander, birch, and short ragweed (Allergopharma, Reinbek, Germany) plus negative (saline) and positive (histamine) controls. Positive responses were defined as a mean wheal diameter of 3 mm over negative with a positive response on positive control. During the second and third visits (at least 1-week interval), methacholine and exercise tests were performed. Fasting blood samples were stored at 70 C. Baseline urine samples were collected 60 minutes before exercise. Patients emptied their bladder 5 minutes before exercise, and urine was collected again 30 minutes after the end of the challenge. Patients were encouraged to drink a glass of water every hour. All urine samples were stored at 70 C. Procedures were approved by the Medical Ethics Committee of Hanyang University Hospital (approval HYUH IRB 2010-R-72), and all patients gave written consent. Exercise Challenge Exercise challenges were conducted in accordance with American Thoracic Society standards16 and performed by running on a treadmill with the nose clipped (LE 200 CE; Jaeger Co, Freiburg, Germany) using a standardized protocol. Heart rate was continuously monitored by a radiographic device (electronic ECG monitor, BCI Autocorr; Smiths Medical Inc, Waukesha, Wisconsin). The temperature in the laboratory was maintained at 22 C and humidity at 40% to 50%. Inspired air temperature and humidity were measured. The treadmill speed was increased until the heart rate was approximately 85% of the predicted maximum ([220 e age] 0.9) and maintained for 6 minutes. Spirometry was conducted 20 and 5 minutes before each exercise challenge and repeated 0, 3, 6, 10, 15, and 20 minutes afterwards.
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defined as at least one positive allergen specific IgE test result (IgE 0.35 kU/L) or SPT finding. Serum leptin and adiponectin were measured according to the manufacturer’s protocols (R&D Systems Inc, Minneapolis, Minnesota). Urinary LTE4 and 9a,11b-PGF2 were assayed in nonextracted diluted urine samples using a specific enzyme immunoassay kit (Cayman Chemical Co, Ann Arbor, Michigan).18 To correct for variations in diuresis, creatinine concentrations were measured, and urinary LTE4 and 9a,11b-PGF2 levels were expressed as picograms per milligram of creatinine (pg/ mg creatinine). Urine creatinine measurements were performed using a colorimetric assay (Sigma, St Louis, Missouri). Statistical Analysis Data analysis was conducted using SPSS statistical software, version 16.0 (SPSS Inc, Chicago, Illinois). Depending on statistical distribution, continuous data are expressed as means (SDs) or medians with interquartile ranges. Differences between baseline and postexercise urinary 9a,11b-PGF2 and LTE4 levels were assessed for statistical significance using the Wilcoxon signed-rank test. Groups were compared by Kruskal-Wallis tests for continuous variables or c2 tests for categorical variables. Post hoc pairwise comparisons were performed by Tamhane tests. Numerical parameters with nonnormal distributions (serum leptin and adiponectin levels, maximum percentage change in FEV1 from baseline to after exercise, and urinary 9a,11b-PGF2 and LTE4 changes from baseline to after exercise) were log transformed. The effects of log-transformed leptin and adiponectin on log-transformed maximum percentage change in FEV1 and log-transformed 9a,11b-PGF2 or log-transformed LTE4 changes from baseline to after exercise were analyzed using a linear regression model to adjust for BMI, age, sex, and atopy. Estimates represent regression slopes for log-transformed leptin and adiponectin levels with respect to changes from baseline to after exercise (log-transformed maximum percentage change in FEV1, log-transformed 9a,11b-PGF2 change, and log-transformed LTE4 change). Results Patient Characteristics Demographic data and peripheral blood biomarker levels are given in Table 1. Of the 44 patients with asthma, 10 had mild intermittent asthma (5 obese and 5 normal-weight patients), 13 had mild persistent asthma (6 obese and 7 normal-weight patients), and 21 had moderate asthma (8 obese and 13 normalweight patients). No significant differences were seen in asthma severity between the obese and normal-weight asthmatic patients. Mean age, height, and sex distribution values did not differ significantly among the groups, and no significant differences were seen in the rates of atopy or prior use of inhaled corticosteroids between obese and normal-weight asthmatic patients. Asthmatic patients differed significantly from nonasthmatic patients in serum total IgE, peripheral blood eosinophil, and exhaled nitric oxide levels. No significant differences in these variables were found between obese asthmatic patients and normal-weight asthmatic patients or between obese nonasthmatic patients and healthy controls. Serum leptin levels were significantly higher in both obese asthmatic patients and obese nonasthmatic patients compared with their normal-weight counterparts. No statistically significant differences were seen in serum leptin and adiponectin levels between obese and obese nonasthmatic patients.
Blood Eosinophils, Serum Total IgE, Eosinophil Cationic Protein, Leptin, Adiponectin, and Urinary LTE4 and 9a,11b-PGF2
Pulmonary Function and BHR to Methacholine and Exercise
Serum total IgE, eosinophil cationic protein, and specific IgE against the allergens used for SPTs were measured using the ImmunoCAP system (Phadia AB, Uppsala, Sweden). Atopy was
Baseline FEV1 and FEV1/forced vital capacity values in obese asthmatic patients, normal-weight asthmatic patients, and obese nonasthmatic patients were lower than in healthy controls
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Table 1 Characteristics of the study patientsa Characteristic
Age, y BMI Sex, % male Prior ICS use, % Atopy, % Total IgE, IU/mL PB eosinophils, /mL ECP, ng/mL FeNO, ppb Leptin, ng/mL Adiponectin, mg/mL Lung function FEV1, % FVC, % FEV1/FVC ratio Postbronchodilatory DFEV1, % EIB, % Maximum decrease in FEV1 after exercise, % PC20, mg/mL
Asthmatic patients
Nonasthmatic patients
P valueb
Obese (n ¼ 19)
Normal weight (n ¼ 25)
Obese (n ¼ 21)
Normal weight (n ¼ 21)
8.5 (1.4) 22.2 (2.7)c,d 68 52.6 84 290.7 (163.8-979.0)d,g 721.7 (461.9)d,g 37.8 (19.9-78.5)d,g 23.0 (15.0-36.5)d,g 11.5 (9.7-19.6)c,d 6.8 (5.0-8.3)c,d
8.3 (1.6) 16.6 (1.3) 68 48.0 80 230.6 (81.9-514.0)d,g 523.3 (349.8)d,g 32.6 (10.9-214.5)d,g 22.0 (11.3-37.5)d,g 3.3 (2.3-6.3) 11.1 (8.9-16.5)
8.9 (1.7) 22.0 (3.7)d 57 NA NA 68.0 (12.8-96.7) 143.3 (89.8) 13.8 (7.6-21.3) 12.5 (10.3-15.5) 11.7 (7.5-23.2)d 8.2 (5.3-10.5)d
7.8 (1.8) 16.3 (1.1) 43 NA NA 36.5 (17.6-64.3) 133.3 (70.2) 9.8 (4.5-15.0) 9.0 (7.3-17.8) 4.0 (1.9-5.7) 15.5 (8.1-20.8)
.20 <.001 .78e .75f .44f <.001 <.001 <.001 <.001 <.001 <.001
88.9 (11.6)d,g 93.3 (7.7) 81.5 (6.9)d 11.4 (5.3)d,g 79c,d,g 26.8 (13.3)c,d,g 3.6 (2.9)
90.3 (10.0)d 93.1 (8.6) 83.9 (3.3)d 10.3 (7.2)d 44d,g 16.4 (11.2)d,g 3.4 (2.3)
94.3 (11.5)d 98.2 (11.5) 86.4 (4.1)d 0.2 (3.9) 14 6.7 (4.7) NA
104.1 (9.9) 98.4 (8.5) 90.7 (2.2) 1.5 (3.8) 10 3.8 (6.1) NA
.02 .64 <.001 <.001 <.001 <.001 .77h
Abbreviations: BMI, body mass index; ECP, eosinophil cationic protein; EIB, exercise-induced bronchoconstriction; FEV1, forced expiratory volume in 1 second; ICS, inhaled corticosteroid; NA, not applicable; PB, peripheral blood; PC20, provocative concentration of methacholine inducing a 20% decrease in FEV1. a Data are presented as absolute numbers, means (SDs), or medians (interquartile ranges). b Kruskal-Wallis test. c P < .05 vs normal-weight asthmatic patients (post hoc comparisons with the Tamhane test). d P < .05 vs healthy patients (post hoc comparisons with the Tamhane test). e Chi-square test. f Fisher’s exact test. g P < .05 vs obese nonasthmatic patients (post hoc comparisons with the Tamhane test). h Mann-Whitney test.
(Table 1). Baseline FEV1 values were lower in obese asthmatic patients compared with obese nonasthmatic patients. The bronchodilator response of asthmatic patients was significantly greater than that of nonasthmatic patients, as were EIB frequencies and maximum percentage change in FEV1 after exercise. Further significant differences in EIB frequencies and maximum percentage change in FEV1 after exercise were found between obese and normal-weight asthmatic patients. However, no significant differences were found in PC20 between the 2 asthmatic groups. Effects of Exercise on Urinary LTE4 and 9a,11b-PGF2 Urinary LTE4 levels significantly increased after exercise in both obese (P < .001) and normal-weight (P ¼ .006) asthmatic patients. However, no significant increase was found in urinary LTE4 excretion after exercise in either obese nonasthmatic patients or healthy controls (Table 2 and Fig 1A). Urinary logarithmic D (the difference between baseline and after exercise) LTE4 values were significantly higher in obese asthmatic patients compared with normal-weight
asthmatic patients (1.67 [0.37] vs 1.27 [0.63] pg/mg creatinine; P ¼ .02). Urinary logarithmic D LTE4 values were significantly higher in normal-weight asthmatic patients compared with obese nonasthmatic patients (1.27 [0.63] vs 0.84 [0.83] pg/mg creatinine; P ¼ .04) and healthy controls (1.27 [0.63] vs 0.71 [0.95] pg/mg creatinine; P ¼ .04). No significant differences were found in urinary logarithmic D LTE4 between obese nonasthmatic patients and healthy controls (Fig 2A). Urinary 9a,11b-PGF2 levels increased significantly after exercise in both obese asthmatic patients (P < .001) and normal-weight (P ¼ .002) asthmatic patients, whereas urinary 9a,11b-PGF2 levels did not change significantly in either obese nonasthmatic patients or healthy controls (Table 2 and Fig 1B). Urinary logarithmic D 9a,11b-PGF2 was significantly higher in obese asthmatic patients compared with normal-weight asthmatic patients (1.66 [0.52] vs 1.28 [0.41] pg/mg creatinine; P ¼ .02). Urinary logarithmic D 9a,11bPGF2 was significantly higher in normal-weight asthmatic patients compared with obese nonasthmatic patients (1.28 [0.41] vs 0.80
Table 2 Baseline and postexercise urinary LTE4 and 9a,11b-PGF2 Levelsa Variable
Asthmatic patients Obese (n ¼ 19)
Urinary LTE4, pg/mg creatinine Baseline After exercise Urinary 9a,11b- PGF2, pg/mg creatinine Baseline After exercise
P valueb
Nonasthmatic patients Normal weight (n ¼ 25)
Obese (n ¼ 23)
Normal weight (n ¼ 20)
87.3 (98.7-114.8) 138.1 (41.8-175.4)c
72.1 (33.8-97.3) 90.9 (50.9-140.8)d
67.3 (35.9-106.2) 84.7 (58.8-126.8)
60.4 (42.6-82.4) 60.5 (41.5-97.3)
.18 .002
97.9 (75.0-166.0) 129.6 (81.9-263.0)c
84.1 (72.1-110.0) 100.5 (78.3-132.2)d
95.4 (77.3-137.6) 95.3 (87.4-133.9)
89.8 (73.6-132.9) 87.0 (65.0-108.8)
.44 .001
Abbreviations: 9a,11b-PGF2, 9a,11b-prostaglandin F2; LTE4, leukotriene E4; and pg/mg creatinine, picograms per milligram of creatinine. a Data are presented as medians (interquartile ranges). b Kruskal-Wallis test. c P < .001 compared with baseline (Wilcoxon signed-rank test). d P < .01 compared with baseline (Wilcoxon signed-rank test).
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Associations Between Serum Leptin Levels and Maximum Percentage Change in FEV1 and Change in Urinary LTE4 and 9a,11b-PGF2 After Exercise Logarithmic serum leptin values were significantly associated with the logarithmic maximum percentage change in FEV1 from baseline to after exercise in both obese (0.416% increase for each doubling of biomarker levels from baseline, P ¼ .01) and normalweight (0.566% increase, P ¼ .02) asthmatic patients (Table 3). Logarithmic serum leptin values were significantly associated with logarithmic urinary LTE4 change from baseline to after exercise in both obese (0.459% increase, P ¼ .04) and normal-weight (0.992% increase, P ¼ .04) asthmatic patients. Logarithmic serum leptin values were also significantly associated with logarithmic urinary 9a,11b-PGF2 change from baseline to after exercise in both obese (0.675% increase, P ¼ .03) and normalweight (0.704% increase, P ¼ .04) asthmatic patients. Logarithmic serum adiponectin values were inversely associated with the logarithmic maximum percentage change in FEV1 from baseline to after exercise in both obese (1.115% decrease, P ¼ .03) and normal-weight (0.497% decrease, P ¼ .03) asthmatic patients. In contrast, logarithmic serum adiponectin values were not significantly associated with the logarithmic urinary LTE4 change or logarithmic urinary 9a,11b-PGF2 change in asthmatic children. Discussion
Figure 1. Leukotriene E4 (LTE4) (A) and 9a,11b-prostaglandin F2 (9a,11b-PGF2) (B) levels in urine measured at baseline and then 30 minutes after exercise in obese and normal-weight asthmatic patients, obese nonasthmatic patients, and healthy controls. NS indicates nonsignificant. *P < .05; **P < .01.
[0.75] pg/mg creatinine; P ¼ .02) and healthy controls (1.28 [0.41] vs 0.70 [0.79] pg/mg creatinine; P ¼ .03). No significant differences were found in urinary logarithmic D 9a,11b-PGF2 between obese nonasthmatic patients and healthy controls (Fig 2B).
Recent studies have advanced our understanding of EIB as a distinct syndrome in asthma that is related to indirect airway hyperresponsiveness and is notable for increased production of bronchoconstrictive eicosanoids, such as cysteinyl-leukotrienes and prostaglandin D2, and shedding of epithelial cells into the airway lumen.2,19e21 Increased urinary cysteinyl-leukotrienes levels may also be detected after exercise in patients with EIB6 but not always.4 In this study, there was a significant increase in the urinary LTE4 levels from baseline to after exercise in both obese and normal-weight asthmatic patients. In addition, logarithmic urinary D LTE4 values were significantly higher in obese asthmatic patients compared with normal-weight asthmatic patients. O’Sullivan et al4 reported an increase in urinary levels of the mast cell marker 9a,11b-PGF2 in association with EIB. We documented that urinary 9a,11b-PGF2 levels increased significantly after exercise compared with baseline in both obese and normal-weight asthmatic patients and urinary logarithmic D 9a,11b-PGF2 was significantly higher in obese compared with normal-weight asthmatic patients. We reported previously that serum leptin and adiponectin levels were significantly correlated with BHR induced by exercise in both obese and normal-weight asthmatic patients22; our findings are in agreement. Shore et al9,10 observed the effects of leptin and adiponectin on mouse airway responsiveness. They found that
Figure 2. Differences in urinary leukotriene E4 (LTE4) (A) and 9a,11b-prostaglandin F2 (9a,11b-PGF2) (B) from baseline to after exercise challenge in obese and normal-weight asthmatic patients, obese nonasthmatic patients, and healthy controls. NS indicates nonsignificant. *P < .05.
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Table 3 Multiple linear regression models for change in FEV1 and Urinary LTE4 and 9a,11b-PGF2 after exercise challenge in asthmatic children Variable
Age Estimate 95% CI P valuea Sex Estimate 95% CI P valuea Atopy Estimate 95% CI P valuea BMI Estimate 95% CI P valuea Log Leptin Estimate 95% CI P valuea Log Adiponectin Estimate 95% CI P valuea
Log D FEV1
Log D LTE4
Log D 9a,11b-PGF2
Obese asthmatic patients (n ¼ 19)
Normal-weight asthmatic patients (n ¼ 25)
Obese asthmatic patients (n ¼ 19)
Normal-weight asthmatic patients (n ¼ 25)
Obese asthmatic patients (n ¼ 21)
Normal-weight asthmatic patients (n ¼ 21)
0.070 0.176 to 0.036 .17
0.038 0.130 to 0.054 .40
0.053 0.075 to 0.055 .39
0.041 0.230 to 0.148 .65
0.064 0.36 to 0.241 .64
0.100 0.301 to 0.101 .30
0.097 0.176 to 0.370 .46
0.141 0.412 to 0.130 .29
0.100 0.360 to 0.559 .64
0.227 0.362 to 0.815 .42
0.142 0.835 to 0.550 .65
0.527 1.055 to 0.001 .06
0.109 0.424 to 0.206 .47
0.337 0.56 to 0.109 .11
0.094 0.588 to 0.400 .68
0.214 0.844 to 0.417 .48
0.229 1.085 to 0.627 .56
0.030 0.330 to 0.391 .56
0.026 0.077 to 0.025 .28
0.035 0.139 to 0.070 .498
0.054 0.134 to 0.026 .17
0.149 0.402 to 0.130 .22
0.028 0.137 to 0.082 .53
0.023 0.228 to 0.182 .72
0.416 0.122 to 0.711 .01
.57 0.081 to 1.051 .02
.46 0.024 to 0.894 .04
.99 0.012 to 1.971 .04
.68 0.022 to 1.327 .03
.70 0.017 to 1.392 .04
1.115 2.063 to 0.167 .03
0.497 0.943 to 0.051 .03
0.752 1.962 to 0.458 .20
0.462 1.832 to 0.968 .495
0.832 3.581 to 1.917 .52
0.516 1.777 to 0.746 .39
Abbreviations: 9a,11b-PGF2, 9a,11b-prostaglandin F2; CI, confidence interval; FEV1, forced expiratory volume in 1 second; LTE4, leukotriene E4. P values were computed using a regression model to evaluate the difference of the estimate (slope) from zero.
a
leptin treatment augmented allergen-induced BHR but did not affect eosinophil influx or TH2 cytokine expression, suggesting that leptin augments BHR through a mechanism independent of TH2 inflammation.9 They also found in mice that adiponectin administration resulted in almost complete suppression of allergeninduced BHR and airway inflammation in the lung.10 Nair et al23 found that leptin did not stimulate proinflammatory cytokine release from airway smooth muscle cells and concluded that the proinflammatory effects of obesity in asthma were unlikely to be due to a direct effect of leptin on airway smooth muscle. Mancuso et al12 reported that leptin augmented alveolar macrophage leukotriene synthesis by increasing phospholipase activity. Hallstrand et al24 reported that group X secretory phospholipase A2 was overexpressed in airway epithelial cells and bronchial macrophages of asthmatic patients and was released in response to exercise. They suggested that group X secretory phospholipase A2 functions as a key regulator of eicosanoid formation in the airways; furthermore, this enzyme was strongly implicated in features of airway hyperresponsiveness, such as EIB. Misso et al25 demonstrated that plasma secretory phospholipase A2 activity in patients with acute asthma was increased and associated with increased BMI. We postulate that high leptin levels in asthmatic patients lead to the synthesis and release of leukotrienes and/or prostaglandins from activated mast cells, epithelial cells, or eosinophils through increasing secretory phospholipase A2 activity during exercise and ultimately enhance airway responsiveness; however, further work is necessary to demonstrate a causative relationship. Leptin levels were significantly associated with urinary LTE4 release induced by exercise in both obese and normal-weight asthmatic patients. Our results are similar to those of an earlier study, which reported that leptin and adiponectin had positive and negative associations, respectively, with urinary LTE4⁄creatinine in a selected cohort of asthmatic patients.26 We found that leptin levels were significantly associated with urinary levels of the mast cell marker 9a,11b-PGF2 induced by exercise in both obese and normal-weight asthmatic patients. Taildeman et al27 reported expression of leptin receptors on human
lung mast cells, which suggests that leptin has paracrine or autocrine immunomodulatory effects on mast cells. In contrast, adiponectin levels were not significantly associated with urinary LTE4 release or 9a,11b-PGF2 release induced by exercise in our study. In asthmatic patients, EIB is related to the presence of eosinophils. Those with EIB tend to have a greater concentration of eosinophils in sputum than those without EIB.19,28 An association between peripheral blood eosinophil counts and EIB severity has been reported.29 Fractional exhaled nitric oxide is correlated to total eosinophil count in asthmatic patients.30 In this study, no significant differences were seen in peripheral blood eosinophil count and fractional exhaled nitric oxide levels between obese and normal-weight asthmatic patients. Postexercise FEV1 decreased markedly in both the obese patients with asthma and normal-weight patients with asthma, although the obese patients without asthma, who had higher leptin and lower adiponectin levels, did not have significant exerciseinduced BHR. Because BHR to exogenous stimuli is a characteristic feature of asthma and we excluded individuals with positive methacholine challenge results for BHR from the groups without asthma, the obese patients without asthma might not have EIB. These findings suggest that leptin levels are associated with exercise-induced BHR in a synergistic manner in asthmatic patients but not in patients who are only obese. Leptin levels might play a role in augmenting exercise-induced BHR in patients with asthma, as demonstrated in the mouse study by Shore et al.9 Many unresolved questions regarding the mechanisms involved in leptin-related effects on inflammation and BHR remain. One limitation of our study was the small sample size. Because of the lack of a group of asthmatic patients with frequent exacerbations or asthma symptoms, we cannot state how our findings relate to poor asthma control. Nevertheless, our study design had strengths. To our knowledge, this is the first controlled observational study of the relationship between serum leptin levels and urinary LTE4 and 9a,11b- PGF2 release after exercise in asthmatic children. The finding of the mouse study of Shore et al9 that leptin treatment augmented allergen-induced BHR raises the possibility that the leptin levels are
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causative of EIB and/or BHR. Our study suggests that leptin might augment BHR through the mechanisms involved in LTE4 and 9a,11bPGF2 release during exercise-induced bronchoconstriction in asthmatic children, although we did not obtain any direct evidence for a cause-and-effect relationship. In summary, serum leptin levels were significantly associated with BHR and urinary LTE4 and 9a,11bPGF2 release after exercise in asthmatic children.
[15]
Acknowledgments
[16]
We thank our pediatric patients and their parents for generous donation of study samples, Mr Soon-Ho Koo for technical assistance, and Professor Young-Gwan Im and Mr Han-Sang Baek for data management. We also thank Aerocrine for their kind support of the NIOX MINO for exhaled nitric oxide measurement. References [1] Joos GF, O’Connor B, Anderson SD, et al. Indirect airway challenges. Eur Respir J. 2003;21:1050e1068. [2] Hallstrand TS, Moody MW, Wurfel MM, Schwartz LB, Henderson WR Jr, Aitken ML. Inflammatory basis of exercise-induced bronchoconstriction. Am J Respir Crit Care Med. 2005;172:679e686. [3] Carroll NG, Mutavdzic S, James AL. Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur Respir J. 2002;19:879e885. [4] O’Sullivan S, Roquet A, Dahlen B, et al. Evidence for mast cell activation during exercise-induced bronchoconstriction. Eur Respir J. 1998;12:345e350. [5] Nagakura T, Obata T, Shichijo K, et al. GC/MS analysis of urinary excretion of 9alpha,11beta-PGF2 in acute and exercise-induced asthma in children. Clin Exp Allergy. 1998;28:181e196. [6] Reiss TF, Hill JB, Harman E, et al. Increased urinary excretion of LTE4 after exercise and attenuation of exercise-induced bronchospasm by montelukast, a cysteinyl leukotriene receptor antagonist. Thorax. 1997;52:1030e1035. [7] Sin DD, Sutherland ER. Obesity and the lung, 4: obesity and asthma. Thorax. 2008;63:1018e1023. [8] Jartti T, Saarikoski L, Jartti L, et al. Obesity, adipokines and asthma. Allergy. 2009;64:770e777. [9] Shore SA, Schwartzman IN, Mellema MS, Flynt L, Imrich A, Johnston RA. Effect of leptin on allergic airway responses in mice. J Allergy Clin Immunol. 2005; 115:103e109. [10] Shore SA, Terry RD, Flynt L, Xu A, Hug C. Adiponectin attenuates allergeninduced airway inflammation and hyperresponsiveness in mice. J Allergy Clin Immunol. 2006;118:389e395. [11] Johnston RA, Zhu M, Rivera-Sanchez YM, et al. Allergic airway responses in obese mice. Am J Respir Crit Care Med. 2007;176:650e658. [12] Mancuso P, Canetti C, Gottschalk A, Tithof PK, Peters-Golden M. Leptin augments alveolar macrophage leukotriene synthesis by increasing phospholipase activity
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and enhancing group IVC iPLA2 (cPLA2gamma) protein expression. Am J Physiol Lung Cell Mol Physiol. 2004;287:L497eL502. Forno E, Lescher R, Strunk R, Weiss S, Fuhlbrigge A, Celedon JC. Decreased response to inhaled steroids in overweight and obese asthmatic children. J Allergy Clin Immunol. 2011;127:741e749. Peters-Golden M, Swern A, Bird SS, Hustad CM, Grant E, Edelman JM. Influence of body mass index on the response to asthma controller agents. Eur Respir J. 2006;27:495e503. Ogden CL, Flegal KM, Carroll MD, Johnson CL. Prevalence and trends in overweight among US children and adolescents, 1999-2000. JAMA. 2002;288: 1728e1732. Crapo RO, Casaburi R, Coates AL, et al. Guidelines for methacholine and exercise challenge testing-1999. Am J Respir Crit Care Med. 2000;161: 309e329. Bateman ED, Hurd SS, Barnes PJ, et al. Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J. 2008;31:143e178. Misso NL, Aggarwal S, Thompson PJ, Vally H. Increases in urinary 9alpha,11beta-prostaglandin F2 indicate mast cell activation in wine-induced asthma. Int Arch Allergy Immunol. 2009;149:127e132. Hallstrand TS, Moody MW, Aitken ML, Henderson WR Jr. Airway immunopathology of asthma with exercise-induced bronchoconstriction. J Allergy Clin Immunol. 2005;116:586e593. Hallstrand TS, Henderson WR Jr. Role of leukotrienes in exercise-induced bronchoconstriction. Curr Allergy Asthma Rep. 2009;9:18e25. Hallstrand TS. New insights into pathogenesis of exercise-induced bronchoconstriction. Curr Opin Allergy Clin Immunol. 2012;12:42e48. Baek HS, Kim YD, Shin JH, Kim JH, Oh JW, Lee HB. Serum leptin and adiponectin levels correlate with exercise-induced bronchoconstriction in children with asthma. Ann Allergy Asthma Immunol. 2011;107:14e21. Nair P, Radford K, Fanat A, Janssen LJ, Peters-Golden M, Cox PG. The effects of leptin on airway smooth muscle responses. Am J Respir Cell Mol Biol. 2008;39: 475e481. Hallstrand TS, Chi EY, Singer AG, Gelb MH, Henderson WR Jr. Secreted phospholipase A2 group X overexpression in asthma and bronchial hyperresponsiveness. Am J Respir Crit Care Med. 2007;176:1072e1078. Misso NL, Petrovic N, Grove C, Celenza A, Brooks-Wildhaber J, Thompson PJ. Plasma phospholipase A2 activity in patients with asthma: association with body mass index and cholesterol concentration. Thorax. 2008;63:21e26. Giouleka P, Papatheodorou G, Lyberopoulos P, et al. Body mass index is associated with leukotriene inflammation in asthmatics. Eur J Clin Invest. 2011;41:30e38. Taildeman J, Perez-Novo CA, Rottiers I, et al. Human mast cells express leptin and leptin receptors. Histochem Cell Biol. 2009;131:703e711. Yoshikawa T, Shoji S, Fujii T, et al. Severity of exercise-induced bronchoconstriction is related to airway eosinophilic inflammation in patients with asthma. Eur Respir J. 1998;12:879e884. Lee SY, Kim HB, Kim JH, et al. Eosinophils play a major role in the severity of exercise-induced bronchoconstriction in children with asthma. Pediatr Pulmonol. 2006;41:1161e1166. Covar RA, Szefler SJ, Martin RJ, et al. Relations between exhaled nitric oxide and measures of disease activity among children with mild-to-moderate asthma. J Pediatr. 2003;142:469e475.
Contents lists available at SciVerse ScienceDirect
Leptin and urinary leukotriene E4 and 9a,11b-prostaglandin F2 release after exercise challenge Hey-Sung Baek, MD, PhD *; Jae-Hyung Choi, MD y; Jae-Won Oh, MD, PhD y; and Ha-Baik Lee, MD, PhD y * Department y
of Pediatrics, Hallym University Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea Department of Pediatrics, Hanyang University Hospital, Hanyang University College of Medicine, Seoul, Korea
A R T I C L E
I N F O
Article history: Received for publication March 26, 2013. Received in revised form May 16, 2013. Accepted for publication May 21, 2013.
A B S T R A C T
Background: Leptin-related effects on inflammation and bronchial hyperresponsiveness (BHR) in the human airway have not been demonstrated. Objectives: To investigate the relationship between the levels of serum leptin and BHR and urinary leukotriene E4 (LTE4) and 9a,11b-prostaglandin F2 (9a,11b-PGF2) release after exercise challenge in asthmatic children. Methods: Eighty-six prepubertal children between 6 and 10 years old were enrolled and divided into 4 groups: 19 obese asthmatic children, 25 normal-weight asthmatic children, 21 obese nonasthmatic children, and 21 healthy controls. We measured serum leptin levels and urinary LTE4 and 9a,11b-PGF2 levels in children before and 30 minutes after the exercise challenge. Results: Serum leptin levels were significantly higher in obese asthmatic children compared with normalweight asthmatic children. Significant increases in urinary levels of LTE4 and 9a,11b-PGF2 were observed in obese asthmatic children after the exercise challenge. Although smaller than in obese asthmatic children, significant increases in the urinary levels of LTE4 and 9a,11b-PGF2 were also observed in the normal-weight. Asthmatic children Logarithmic serum leptin values were significantly associated with the logarithmic maximum percentage change in forced expiratory volume in 1 second, the logarithmic urinary LTE4 change, and the logarithmic urinary 9a,11b-PGF2 change from baseline to after exercise in both obese and normalweight asthmatic children. Conclusion: The serum levels of leptin were significantly associated with BHR and urinary LTE4 and 9a,11b-PGF2 release induced by exercise challenge in asthmatic children. Ó 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Introduction Exercise-induced bronchoconstriction (EIB) reflects the degree of indirect bronchial hyperresponsiveness (BHR).1 Hyperosmolar triggering of mast cells and possibly other inflammatory cells results in release of bronchoconstricting mediators (eg, cysteinylleukotrienes, histamine, and prostaglandins) during exercise challenge.2e6 Direct evidence for mediator release after exercise in asthmatic patients includes an increase in urinary levels of the mast cell marker 9a,11b-prostaglandin F2 (9a,11b-PGF2) and leukotriene E4 (LTE4) in association with EIB.4e6 Adipose tissue releases a variety of proinflammatory adipokines, including leptin and adiponectin.7,8 Leptin may play roles in the development of asthma and allergies. Studies in mouse models have demonstrated that adipokines enhance BHR, airway inflammation, and allergic responses.9e11 Another study found in vitro
Reprints: Ha-Baik Lee, MD, PhD, Department of Pediatrics, Hanyang University Hospital, Hanyang University College of Medicine, 17 Haengdang-Dong, SeongdongGu, Seoul 133-792, Korea; E-mail: [email protected]. Disclosures: Authors have nothing to disclose.
that exogenous leptin enhances leukotriene production.12 This may be clinically important because obese asthmatic patients are relatively resistant to inhaled corticosteroids13,14 but respond to antileukotrienes as do lean asthmatic patients.14 However, leptin-induced effects on inflammation and BHR in the human airway have not been demonstrated. The aim of this study was to investigate the association between the levels of serum leptin and BHR and urinary release of LTE4 and 9a,11b-PGF2 after exercise challenge in asthmatic children. Methods Study Patients Study patients were randomly recruited from outpatient clinics in Hanyang University Hospital, Seoul, Korea. Participants were prepubertal children 6 to 10 years old divided into 4 groups: 19 obese asthmatic children, 25 normal-weight asthmatic children, 21 obese nonasthmatic children, and 21 healthy controls. Age- and sexspecific body mass indices (BMIs) were calculated according to the US Centers for Disease Control and Prevention guidelines.15 Participants with a BMI less than the 85th percentile were
1081-1206/13/$36.00 - see front matter Ó 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anai.2013.05.019
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considered normal weight, and those with a BMI in the 95th percentile or higher were considered obese. Underweight individuals in the less than fifth percentile were excluded. Asthma was defined as the presence of symptoms either with an increase in forced expiratory volume in 1 second (FEV1) of 12% or higher after bronchodilator treatment or if less than 8.0 mg/mL of inhaled methacholine induced a 20% decrease in FEV1 (PC20).16 An exercise bronchoprovocation test was used for EIB diagnosis, the results of which were considered positive with a 15% or greater decrease in FEV1 after exercise.16 Classification of asthma was based on Global Initiative for Asthma guidelines.17 Controls were age- and sexmatched healthy children with no history of wheezing or infection during the preceding 2 weeks. Exclusion criteria included acute exacerbation of asthma within the previous 6 months and parenchymal lung disease within the previous 4 weeks. Participants were excluded if they had used inhaled corticosteroids, leukotriene modifiers, long-acting antihistamines, or long-acting b2-agonists during the previous 30 days. In the healthy group, those with any likelihood of atopy or a positive methacholine challenge result for BHR were excluded. Study Protocol Participants had a total of 3 visits. All participants initially underwent anthropometric measurements of height and weight. They were also examined for any sign of puberty during the visit. At first visit, blood tests were performed, and fractional exhaled nitric oxide levels were measured. Each patient was evaluated by skin prick tests (SPTs) and before and after bronchodilator spirometry. Skin rick testing included house dust mite, cat dander, dog dander, birch, and short ragweed (Allergopharma, Reinbek, Germany) plus negative (saline) and positive (histamine) controls. Positive responses were defined as a mean wheal diameter of 3 mm over negative with a positive response on positive control. During the second and third visits (at least 1-week interval), methacholine and exercise tests were performed. Fasting blood samples were stored at 70 C. Baseline urine samples were collected 60 minutes before exercise. Patients emptied their bladder 5 minutes before exercise, and urine was collected again 30 minutes after the end of the challenge. Patients were encouraged to drink a glass of water every hour. All urine samples were stored at 70 C. Procedures were approved by the Medical Ethics Committee of Hanyang University Hospital (approval HYUH IRB 2010-R-72), and all patients gave written consent. Exercise Challenge Exercise challenges were conducted in accordance with American Thoracic Society standards16 and performed by running on a treadmill with the nose clipped (LE 200 CE; Jaeger Co, Freiburg, Germany) using a standardized protocol. Heart rate was continuously monitored by a radiographic device (electronic ECG monitor, BCI Autocorr; Smiths Medical Inc, Waukesha, Wisconsin). The temperature in the laboratory was maintained at 22 C and humidity at 40% to 50%. Inspired air temperature and humidity were measured. The treadmill speed was increased until the heart rate was approximately 85% of the predicted maximum ([220 e age] 0.9) and maintained for 6 minutes. Spirometry was conducted 20 and 5 minutes before each exercise challenge and repeated 0, 3, 6, 10, 15, and 20 minutes afterwards.
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defined as at least one positive allergen specific IgE test result (IgE 0.35 kU/L) or SPT finding. Serum leptin and adiponectin were measured according to the manufacturer’s protocols (R&D Systems Inc, Minneapolis, Minnesota). Urinary LTE4 and 9a,11b-PGF2 were assayed in nonextracted diluted urine samples using a specific enzyme immunoassay kit (Cayman Chemical Co, Ann Arbor, Michigan).18 To correct for variations in diuresis, creatinine concentrations were measured, and urinary LTE4 and 9a,11b-PGF2 levels were expressed as picograms per milligram of creatinine (pg/ mg creatinine). Urine creatinine measurements were performed using a colorimetric assay (Sigma, St Louis, Missouri). Statistical Analysis Data analysis was conducted using SPSS statistical software, version 16.0 (SPSS Inc, Chicago, Illinois). Depending on statistical distribution, continuous data are expressed as means (SDs) or medians with interquartile ranges. Differences between baseline and postexercise urinary 9a,11b-PGF2 and LTE4 levels were assessed for statistical significance using the Wilcoxon signed-rank test. Groups were compared by Kruskal-Wallis tests for continuous variables or c2 tests for categorical variables. Post hoc pairwise comparisons were performed by Tamhane tests. Numerical parameters with nonnormal distributions (serum leptin and adiponectin levels, maximum percentage change in FEV1 from baseline to after exercise, and urinary 9a,11b-PGF2 and LTE4 changes from baseline to after exercise) were log transformed. The effects of log-transformed leptin and adiponectin on log-transformed maximum percentage change in FEV1 and log-transformed 9a,11b-PGF2 or log-transformed LTE4 changes from baseline to after exercise were analyzed using a linear regression model to adjust for BMI, age, sex, and atopy. Estimates represent regression slopes for log-transformed leptin and adiponectin levels with respect to changes from baseline to after exercise (log-transformed maximum percentage change in FEV1, log-transformed 9a,11b-PGF2 change, and log-transformed LTE4 change). Results Patient Characteristics Demographic data and peripheral blood biomarker levels are given in Table 1. Of the 44 patients with asthma, 10 had mild intermittent asthma (5 obese and 5 normal-weight patients), 13 had mild persistent asthma (6 obese and 7 normal-weight patients), and 21 had moderate asthma (8 obese and 13 normalweight patients). No significant differences were seen in asthma severity between the obese and normal-weight asthmatic patients. Mean age, height, and sex distribution values did not differ significantly among the groups, and no significant differences were seen in the rates of atopy or prior use of inhaled corticosteroids between obese and normal-weight asthmatic patients. Asthmatic patients differed significantly from nonasthmatic patients in serum total IgE, peripheral blood eosinophil, and exhaled nitric oxide levels. No significant differences in these variables were found between obese asthmatic patients and normal-weight asthmatic patients or between obese nonasthmatic patients and healthy controls. Serum leptin levels were significantly higher in both obese asthmatic patients and obese nonasthmatic patients compared with their normal-weight counterparts. No statistically significant differences were seen in serum leptin and adiponectin levels between obese and obese nonasthmatic patients.
Blood Eosinophils, Serum Total IgE, Eosinophil Cationic Protein, Leptin, Adiponectin, and Urinary LTE4 and 9a,11b-PGF2
Pulmonary Function and BHR to Methacholine and Exercise
Serum total IgE, eosinophil cationic protein, and specific IgE against the allergens used for SPTs were measured using the ImmunoCAP system (Phadia AB, Uppsala, Sweden). Atopy was
Baseline FEV1 and FEV1/forced vital capacity values in obese asthmatic patients, normal-weight asthmatic patients, and obese nonasthmatic patients were lower than in healthy controls
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Table 1 Characteristics of the study patientsa Characteristic
Age, y BMI Sex, % male Prior ICS use, % Atopy, % Total IgE, IU/mL PB eosinophils, /mL ECP, ng/mL FeNO, ppb Leptin, ng/mL Adiponectin, mg/mL Lung function FEV1, % FVC, % FEV1/FVC ratio Postbronchodilatory DFEV1, % EIB, % Maximum decrease in FEV1 after exercise, % PC20, mg/mL
Asthmatic patients
Nonasthmatic patients
P valueb
Obese (n ¼ 19)
Normal weight (n ¼ 25)
Obese (n ¼ 21)
Normal weight (n ¼ 21)
8.5 (1.4) 22.2 (2.7)c,d 68 52.6 84 290.7 (163.8-979.0)d,g 721.7 (461.9)d,g 37.8 (19.9-78.5)d,g 23.0 (15.0-36.5)d,g 11.5 (9.7-19.6)c,d 6.8 (5.0-8.3)c,d
8.3 (1.6) 16.6 (1.3) 68 48.0 80 230.6 (81.9-514.0)d,g 523.3 (349.8)d,g 32.6 (10.9-214.5)d,g 22.0 (11.3-37.5)d,g 3.3 (2.3-6.3) 11.1 (8.9-16.5)
8.9 (1.7) 22.0 (3.7)d 57 NA NA 68.0 (12.8-96.7) 143.3 (89.8) 13.8 (7.6-21.3) 12.5 (10.3-15.5) 11.7 (7.5-23.2)d 8.2 (5.3-10.5)d
7.8 (1.8) 16.3 (1.1) 43 NA NA 36.5 (17.6-64.3) 133.3 (70.2) 9.8 (4.5-15.0) 9.0 (7.3-17.8) 4.0 (1.9-5.7) 15.5 (8.1-20.8)
.20 <.001 .78e .75f .44f <.001 <.001 <.001 <.001 <.001 <.001
88.9 (11.6)d,g 93.3 (7.7) 81.5 (6.9)d 11.4 (5.3)d,g 79c,d,g 26.8 (13.3)c,d,g 3.6 (2.9)
90.3 (10.0)d 93.1 (8.6) 83.9 (3.3)d 10.3 (7.2)d 44d,g 16.4 (11.2)d,g 3.4 (2.3)
94.3 (11.5)d 98.2 (11.5) 86.4 (4.1)d 0.2 (3.9) 14 6.7 (4.7) NA
104.1 (9.9) 98.4 (8.5) 90.7 (2.2) 1.5 (3.8) 10 3.8 (6.1) NA
.02 .64 <.001 <.001 <.001 <.001 .77h
Abbreviations: BMI, body mass index; ECP, eosinophil cationic protein; EIB, exercise-induced bronchoconstriction; FEV1, forced expiratory volume in 1 second; ICS, inhaled corticosteroid; NA, not applicable; PB, peripheral blood; PC20, provocative concentration of methacholine inducing a 20% decrease in FEV1. a Data are presented as absolute numbers, means (SDs), or medians (interquartile ranges). b Kruskal-Wallis test. c P < .05 vs normal-weight asthmatic patients (post hoc comparisons with the Tamhane test). d P < .05 vs healthy patients (post hoc comparisons with the Tamhane test). e Chi-square test. f Fisher’s exact test. g P < .05 vs obese nonasthmatic patients (post hoc comparisons with the Tamhane test). h Mann-Whitney test.
(Table 1). Baseline FEV1 values were lower in obese asthmatic patients compared with obese nonasthmatic patients. The bronchodilator response of asthmatic patients was significantly greater than that of nonasthmatic patients, as were EIB frequencies and maximum percentage change in FEV1 after exercise. Further significant differences in EIB frequencies and maximum percentage change in FEV1 after exercise were found between obese and normal-weight asthmatic patients. However, no significant differences were found in PC20 between the 2 asthmatic groups. Effects of Exercise on Urinary LTE4 and 9a,11b-PGF2 Urinary LTE4 levels significantly increased after exercise in both obese (P < .001) and normal-weight (P ¼ .006) asthmatic patients. However, no significant increase was found in urinary LTE4 excretion after exercise in either obese nonasthmatic patients or healthy controls (Table 2 and Fig 1A). Urinary logarithmic D (the difference between baseline and after exercise) LTE4 values were significantly higher in obese asthmatic patients compared with normal-weight
asthmatic patients (1.67 [0.37] vs 1.27 [0.63] pg/mg creatinine; P ¼ .02). Urinary logarithmic D LTE4 values were significantly higher in normal-weight asthmatic patients compared with obese nonasthmatic patients (1.27 [0.63] vs 0.84 [0.83] pg/mg creatinine; P ¼ .04) and healthy controls (1.27 [0.63] vs 0.71 [0.95] pg/mg creatinine; P ¼ .04). No significant differences were found in urinary logarithmic D LTE4 between obese nonasthmatic patients and healthy controls (Fig 2A). Urinary 9a,11b-PGF2 levels increased significantly after exercise in both obese asthmatic patients (P < .001) and normal-weight (P ¼ .002) asthmatic patients, whereas urinary 9a,11b-PGF2 levels did not change significantly in either obese nonasthmatic patients or healthy controls (Table 2 and Fig 1B). Urinary logarithmic D 9a,11b-PGF2 was significantly higher in obese asthmatic patients compared with normal-weight asthmatic patients (1.66 [0.52] vs 1.28 [0.41] pg/mg creatinine; P ¼ .02). Urinary logarithmic D 9a,11bPGF2 was significantly higher in normal-weight asthmatic patients compared with obese nonasthmatic patients (1.28 [0.41] vs 0.80
Table 2 Baseline and postexercise urinary LTE4 and 9a,11b-PGF2 Levelsa Variable
Asthmatic patients Obese (n ¼ 19)
Urinary LTE4, pg/mg creatinine Baseline After exercise Urinary 9a,11b- PGF2, pg/mg creatinine Baseline After exercise
P valueb
Nonasthmatic patients Normal weight (n ¼ 25)
Obese (n ¼ 23)
Normal weight (n ¼ 20)
87.3 (98.7-114.8) 138.1 (41.8-175.4)c
72.1 (33.8-97.3) 90.9 (50.9-140.8)d
67.3 (35.9-106.2) 84.7 (58.8-126.8)
60.4 (42.6-82.4) 60.5 (41.5-97.3)
.18 .002
97.9 (75.0-166.0) 129.6 (81.9-263.0)c
84.1 (72.1-110.0) 100.5 (78.3-132.2)d
95.4 (77.3-137.6) 95.3 (87.4-133.9)
89.8 (73.6-132.9) 87.0 (65.0-108.8)
.44 .001
Abbreviations: 9a,11b-PGF2, 9a,11b-prostaglandin F2; LTE4, leukotriene E4; and pg/mg creatinine, picograms per milligram of creatinine. a Data are presented as medians (interquartile ranges). b Kruskal-Wallis test. c P < .001 compared with baseline (Wilcoxon signed-rank test). d P < .01 compared with baseline (Wilcoxon signed-rank test).
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Associations Between Serum Leptin Levels and Maximum Percentage Change in FEV1 and Change in Urinary LTE4 and 9a,11b-PGF2 After Exercise Logarithmic serum leptin values were significantly associated with the logarithmic maximum percentage change in FEV1 from baseline to after exercise in both obese (0.416% increase for each doubling of biomarker levels from baseline, P ¼ .01) and normalweight (0.566% increase, P ¼ .02) asthmatic patients (Table 3). Logarithmic serum leptin values were significantly associated with logarithmic urinary LTE4 change from baseline to after exercise in both obese (0.459% increase, P ¼ .04) and normal-weight (0.992% increase, P ¼ .04) asthmatic patients. Logarithmic serum leptin values were also significantly associated with logarithmic urinary 9a,11b-PGF2 change from baseline to after exercise in both obese (0.675% increase, P ¼ .03) and normalweight (0.704% increase, P ¼ .04) asthmatic patients. Logarithmic serum adiponectin values were inversely associated with the logarithmic maximum percentage change in FEV1 from baseline to after exercise in both obese (1.115% decrease, P ¼ .03) and normal-weight (0.497% decrease, P ¼ .03) asthmatic patients. In contrast, logarithmic serum adiponectin values were not significantly associated with the logarithmic urinary LTE4 change or logarithmic urinary 9a,11b-PGF2 change in asthmatic children. Discussion
Figure 1. Leukotriene E4 (LTE4) (A) and 9a,11b-prostaglandin F2 (9a,11b-PGF2) (B) levels in urine measured at baseline and then 30 minutes after exercise in obese and normal-weight asthmatic patients, obese nonasthmatic patients, and healthy controls. NS indicates nonsignificant. *P < .05; **P < .01.
[0.75] pg/mg creatinine; P ¼ .02) and healthy controls (1.28 [0.41] vs 0.70 [0.79] pg/mg creatinine; P ¼ .03). No significant differences were found in urinary logarithmic D 9a,11b-PGF2 between obese nonasthmatic patients and healthy controls (Fig 2B).
Recent studies have advanced our understanding of EIB as a distinct syndrome in asthma that is related to indirect airway hyperresponsiveness and is notable for increased production of bronchoconstrictive eicosanoids, such as cysteinyl-leukotrienes and prostaglandin D2, and shedding of epithelial cells into the airway lumen.2,19e21 Increased urinary cysteinyl-leukotrienes levels may also be detected after exercise in patients with EIB6 but not always.4 In this study, there was a significant increase in the urinary LTE4 levels from baseline to after exercise in both obese and normal-weight asthmatic patients. In addition, logarithmic urinary D LTE4 values were significantly higher in obese asthmatic patients compared with normal-weight asthmatic patients. O’Sullivan et al4 reported an increase in urinary levels of the mast cell marker 9a,11b-PGF2 in association with EIB. We documented that urinary 9a,11b-PGF2 levels increased significantly after exercise compared with baseline in both obese and normal-weight asthmatic patients and urinary logarithmic D 9a,11b-PGF2 was significantly higher in obese compared with normal-weight asthmatic patients. We reported previously that serum leptin and adiponectin levels were significantly correlated with BHR induced by exercise in both obese and normal-weight asthmatic patients22; our findings are in agreement. Shore et al9,10 observed the effects of leptin and adiponectin on mouse airway responsiveness. They found that
Figure 2. Differences in urinary leukotriene E4 (LTE4) (A) and 9a,11b-prostaglandin F2 (9a,11b-PGF2) (B) from baseline to after exercise challenge in obese and normal-weight asthmatic patients, obese nonasthmatic patients, and healthy controls. NS indicates nonsignificant. *P < .05.
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Table 3 Multiple linear regression models for change in FEV1 and Urinary LTE4 and 9a,11b-PGF2 after exercise challenge in asthmatic children Variable
Age Estimate 95% CI P valuea Sex Estimate 95% CI P valuea Atopy Estimate 95% CI P valuea BMI Estimate 95% CI P valuea Log Leptin Estimate 95% CI P valuea Log Adiponectin Estimate 95% CI P valuea
Log D FEV1
Log D LTE4
Log D 9a,11b-PGF2
Obese asthmatic patients (n ¼ 19)
Normal-weight asthmatic patients (n ¼ 25)
Obese asthmatic patients (n ¼ 19)
Normal-weight asthmatic patients (n ¼ 25)
Obese asthmatic patients (n ¼ 21)
Normal-weight asthmatic patients (n ¼ 21)
0.070 0.176 to 0.036 .17
0.038 0.130 to 0.054 .40
0.053 0.075 to 0.055 .39
0.041 0.230 to 0.148 .65
0.064 0.36 to 0.241 .64
0.100 0.301 to 0.101 .30
0.097 0.176 to 0.370 .46
0.141 0.412 to 0.130 .29
0.100 0.360 to 0.559 .64
0.227 0.362 to 0.815 .42
0.142 0.835 to 0.550 .65
0.527 1.055 to 0.001 .06
0.109 0.424 to 0.206 .47
0.337 0.56 to 0.109 .11
0.094 0.588 to 0.400 .68
0.214 0.844 to 0.417 .48
0.229 1.085 to 0.627 .56
0.030 0.330 to 0.391 .56
0.026 0.077 to 0.025 .28
0.035 0.139 to 0.070 .498
0.054 0.134 to 0.026 .17
0.149 0.402 to 0.130 .22
0.028 0.137 to 0.082 .53
0.023 0.228 to 0.182 .72
0.416 0.122 to 0.711 .01
.57 0.081 to 1.051 .02
.46 0.024 to 0.894 .04
.99 0.012 to 1.971 .04
.68 0.022 to 1.327 .03
.70 0.017 to 1.392 .04
1.115 2.063 to 0.167 .03
0.497 0.943 to 0.051 .03
0.752 1.962 to 0.458 .20
0.462 1.832 to 0.968 .495
0.832 3.581 to 1.917 .52
0.516 1.777 to 0.746 .39
Abbreviations: 9a,11b-PGF2, 9a,11b-prostaglandin F2; CI, confidence interval; FEV1, forced expiratory volume in 1 second; LTE4, leukotriene E4. P values were computed using a regression model to evaluate the difference of the estimate (slope) from zero.
a
leptin treatment augmented allergen-induced BHR but did not affect eosinophil influx or TH2 cytokine expression, suggesting that leptin augments BHR through a mechanism independent of TH2 inflammation.9 They also found in mice that adiponectin administration resulted in almost complete suppression of allergeninduced BHR and airway inflammation in the lung.10 Nair et al23 found that leptin did not stimulate proinflammatory cytokine release from airway smooth muscle cells and concluded that the proinflammatory effects of obesity in asthma were unlikely to be due to a direct effect of leptin on airway smooth muscle. Mancuso et al12 reported that leptin augmented alveolar macrophage leukotriene synthesis by increasing phospholipase activity. Hallstrand et al24 reported that group X secretory phospholipase A2 was overexpressed in airway epithelial cells and bronchial macrophages of asthmatic patients and was released in response to exercise. They suggested that group X secretory phospholipase A2 functions as a key regulator of eicosanoid formation in the airways; furthermore, this enzyme was strongly implicated in features of airway hyperresponsiveness, such as EIB. Misso et al25 demonstrated that plasma secretory phospholipase A2 activity in patients with acute asthma was increased and associated with increased BMI. We postulate that high leptin levels in asthmatic patients lead to the synthesis and release of leukotrienes and/or prostaglandins from activated mast cells, epithelial cells, or eosinophils through increasing secretory phospholipase A2 activity during exercise and ultimately enhance airway responsiveness; however, further work is necessary to demonstrate a causative relationship. Leptin levels were significantly associated with urinary LTE4 release induced by exercise in both obese and normal-weight asthmatic patients. Our results are similar to those of an earlier study, which reported that leptin and adiponectin had positive and negative associations, respectively, with urinary LTE4⁄creatinine in a selected cohort of asthmatic patients.26 We found that leptin levels were significantly associated with urinary levels of the mast cell marker 9a,11b-PGF2 induced by exercise in both obese and normal-weight asthmatic patients. Taildeman et al27 reported expression of leptin receptors on human
lung mast cells, which suggests that leptin has paracrine or autocrine immunomodulatory effects on mast cells. In contrast, adiponectin levels were not significantly associated with urinary LTE4 release or 9a,11b-PGF2 release induced by exercise in our study. In asthmatic patients, EIB is related to the presence of eosinophils. Those with EIB tend to have a greater concentration of eosinophils in sputum than those without EIB.19,28 An association between peripheral blood eosinophil counts and EIB severity has been reported.29 Fractional exhaled nitric oxide is correlated to total eosinophil count in asthmatic patients.30 In this study, no significant differences were seen in peripheral blood eosinophil count and fractional exhaled nitric oxide levels between obese and normal-weight asthmatic patients. Postexercise FEV1 decreased markedly in both the obese patients with asthma and normal-weight patients with asthma, although the obese patients without asthma, who had higher leptin and lower adiponectin levels, did not have significant exerciseinduced BHR. Because BHR to exogenous stimuli is a characteristic feature of asthma and we excluded individuals with positive methacholine challenge results for BHR from the groups without asthma, the obese patients without asthma might not have EIB. These findings suggest that leptin levels are associated with exercise-induced BHR in a synergistic manner in asthmatic patients but not in patients who are only obese. Leptin levels might play a role in augmenting exercise-induced BHR in patients with asthma, as demonstrated in the mouse study by Shore et al.9 Many unresolved questions regarding the mechanisms involved in leptin-related effects on inflammation and BHR remain. One limitation of our study was the small sample size. Because of the lack of a group of asthmatic patients with frequent exacerbations or asthma symptoms, we cannot state how our findings relate to poor asthma control. Nevertheless, our study design had strengths. To our knowledge, this is the first controlled observational study of the relationship between serum leptin levels and urinary LTE4 and 9a,11b- PGF2 release after exercise in asthmatic children. The finding of the mouse study of Shore et al9 that leptin treatment augmented allergen-induced BHR raises the possibility that the leptin levels are
H.-S. Baek et al. / Ann Allergy Asthma Immunol 111 (2013) 112e117
causative of EIB and/or BHR. Our study suggests that leptin might augment BHR through the mechanisms involved in LTE4 and 9a,11bPGF2 release during exercise-induced bronchoconstriction in asthmatic children, although we did not obtain any direct evidence for a cause-and-effect relationship. In summary, serum leptin levels were significantly associated with BHR and urinary LTE4 and 9a,11bPGF2 release after exercise in asthmatic children.
[15]
Acknowledgments
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We thank our pediatric patients and their parents for generous donation of study samples, Mr Soon-Ho Koo for technical assistance, and Professor Young-Gwan Im and Mr Han-Sang Baek for data management. We also thank Aerocrine for their kind support of the NIOX MINO for exhaled nitric oxide measurement. References [1] Joos GF, O’Connor B, Anderson SD, et al. Indirect airway challenges. Eur Respir J. 2003;21:1050e1068. [2] Hallstrand TS, Moody MW, Wurfel MM, Schwartz LB, Henderson WR Jr, Aitken ML. Inflammatory basis of exercise-induced bronchoconstriction. Am J Respir Crit Care Med. 2005;172:679e686. [3] Carroll NG, Mutavdzic S, James AL. Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur Respir J. 2002;19:879e885. [4] O’Sullivan S, Roquet A, Dahlen B, et al. Evidence for mast cell activation during exercise-induced bronchoconstriction. Eur Respir J. 1998;12:345e350. [5] Nagakura T, Obata T, Shichijo K, et al. GC/MS analysis of urinary excretion of 9alpha,11beta-PGF2 in acute and exercise-induced asthma in children. Clin Exp Allergy. 1998;28:181e196. [6] Reiss TF, Hill JB, Harman E, et al. Increased urinary excretion of LTE4 after exercise and attenuation of exercise-induced bronchospasm by montelukast, a cysteinyl leukotriene receptor antagonist. Thorax. 1997;52:1030e1035. [7] Sin DD, Sutherland ER. Obesity and the lung, 4: obesity and asthma. Thorax. 2008;63:1018e1023. [8] Jartti T, Saarikoski L, Jartti L, et al. Obesity, adipokines and asthma. Allergy. 2009;64:770e777. [9] Shore SA, Schwartzman IN, Mellema MS, Flynt L, Imrich A, Johnston RA. Effect of leptin on allergic airway responses in mice. J Allergy Clin Immunol. 2005; 115:103e109. [10] Shore SA, Terry RD, Flynt L, Xu A, Hug C. Adiponectin attenuates allergeninduced airway inflammation and hyperresponsiveness in mice. J Allergy Clin Immunol. 2006;118:389e395. [11] Johnston RA, Zhu M, Rivera-Sanchez YM, et al. Allergic airway responses in obese mice. Am J Respir Crit Care Med. 2007;176:650e658. [12] Mancuso P, Canetti C, Gottschalk A, Tithof PK, Peters-Golden M. Leptin augments alveolar macrophage leukotriene synthesis by increasing phospholipase activity
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