Leptin: Does it have any role in childhood asthma?

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1 Leptin: Does it have any role in childhood asthma? Nermin Guler, MD, a Emel Kirerleri, MD, a Ulker Ones, MD, a Zeynep Tamay, MD, a Nihal Salmayenli, PhD, b and Feyza Darendeliler, MD c Istanbul, Turkey Background: Although there is evidence of a positive association between asthma and obesity in adults and children, very little is known about the role of leptin in asthmatic children. Objectives: The aims of this study were to evaluate the relation between leptin and parameters of atopy and asthma in children. Methods: Body mass index (BMI) and serum leptin levels were measured in 102 (37 female, 65 male; mean age, years) asthmatic and 33 (14 female, 19 male; mean age, years) healthy children. Skin prick tests, total serum IgE, and pulmonary function tests were performed and were completed. Results: A significant difference was observed in serum leptin levels between asthmatic and healthy children. Median (interquartile range) levels were 3.53 ( ) ng/ml and 2.26 ( ) ng/ml, respectively (P =.008). Subgroup analysis revealed that this difference in leptin levels was confined entirely to boys: 3.09 ( ) ng/ml in boys with asthma versus 1.52 ( ) ng/ml in boys without asthma (P =.003). By logistic regression analysis, we found that leptin was a predictive factor for having asthma (odds ratio, 1.98; CI, ; P =.021), whereas sex, age, or BMI were not. In a stepwise multiple regression analysis including sex (P =.001), age (P =.016), BMI (P <.001), and asthma (P =.022), all of these variables were found to affect log leptin levels (R 2 = 0.404). There was no significant sex difference in serum leptin levels among asthmatic children, whereas healthy boys had significantly lower leptin levels than healthy girls (P =.019). Atopic asthmatic subjects had significantly higher leptin levels than nonatopic asthmatic subjects (P =.038) with similar BMI. A significant, but weak, correlation was observed between leptin levels and IgE in the overall group of asthmatic children (r = 0.231; P =.019). Again, this correlation was confined entirely to boys (r = 0.319; P =.010). There was no relation between leptin levels and skin prick tests, pulmonary function tests, passive smoking, birth weight, and duration of breast-feeding. Conclusion: Our findings suggest that leptin may play a role in atopic asthma. High serum leptin levels in asthmatic boys may From a the Department of Pediatrics, Division of Allergy and Chest Diseases; b the Department of Biochemistry; and c the Department of Pediatrics, Division of Endocrinology, Istanbul Medical Faculty. Disclosure of potential conflict of interest: The authors have no relevant commercial associations that might pose a conflict of interest. Supported by GlaxoSmithKline. Received for publication October 24, 2003; revised March 19, 2004; accepted for publication March 29, Reprint requests: Nermin Guler, MD, Hasırcı Veli sok No 16, Cxıragan- Besxiktasx, Istanbul, Turkey. eztamay@yahoo.com /$30.00 Ó 2004 American Academy of Allergy, Asthma and Immunology doi: /j.jaci partly explain the higher prevalence of childhood asthma in male sex. (J Allergy Clin Immunol 2004;114:254-9.) Key words: Leptin, atopy, asthma, children The worldwide prevalence of asthma continues to rise in children and young adults. 1,2 Several studies have reported an association between increased body mass index (BMI) and asthma prevalence. 3,4 Whether patients with asthma gain weight as a result of limitations in activity or whether obesity increases the risk of developing asthma is uncertain. 5 It can be assumed that impairment in pulmonary mechanics related to obesity increases airway resistance and gives way to dysfunction in small airways. On the other hand, asthma is a disease characterized by inflammation, and there is increasing evidence in the literature that the obesity is an inflammatory state. 6-8 The effects of increased BMI on asthma may be mediated by upregulation of inflammatory mechanisms in the airway epithelium. Leptin is a proteohormone produced by adipocytes and is thought to act primarily through specific receptors at the hypothalamus. 9 It decreases appetite and increases energy expenditure. 10 Serum leptin levels show a positive correlation with BMI and reflect the body fat mass. 11 Leptin receptors also exist in human lung tissue, and leptin may have stimulatory effects on the proliferation of cells of a human cell line through its specific leptin receptor. 12 Leptin may provide a link between inflammation and T-cell function in asthma. Glucocorticoids have been reported to stimulate leptin production in both rodents 13 and human beings. 14 Although there is evidence of a positive association between asthma and obesity in adults and children, very little is known about the role of leptin in asthmatic children. The factors leading to the initiation and maintenance of the asthmatic inflammatory response are presumed to begin early in life, even in utero. The relation of some perinatal risk factors such as birth weight, duration of breast-feeding, and parental smoking on the serum leptin levels in childhood asthma also need to be investigated. In our preliminary study, we assessed serum leptin levels in a small number of asthmatic and healthy children before the initiation of corticosteroid therapy. The study group consisted of randomly selected asthmatic patients and healthy children in the same age group. We found higher serum levels of leptin in asthmatic children than in healthy controls. Significant correlations with serum

2 J ALLERGY CLIN IMMUNOL VOLUME 114, NUMBER 2 Guler et al 255 Abbreviation used BMI: Body mass index leptin levels and BMI, FEV 1 reversibility, and serum total IgE were observed. 15 The aims of this study were to evaluate serum leptin levels in a larger group of asthmatic children before the initiation of corticosteroid therapy and to examine the relationship between leptin and parameters of atopy and some risk factors in asthmatic children. METHODS Our study was performed at the outpatient asthma clinic of the Department of Pediatrics, Medical School of Istanbul University. One hundred two prepubertal asthmatic children (64% boys) consecutively evaluated in the outpatient asthma clinic were included in the study. The patients had been previously diagnosed with moderate-persistent asthma according to the Global Strategy for Asthma Management and Prevention Classification. 16 The patients were evaluated before the initiation of inhaled corticosteroid therapy. Children with a history of attacks requiring systemic corticosteroids within 6 months were excluded. Thirty-three prepubertal children (58% boys) who were referred to our outpatient clinic for routine physical examination and vaccination, who were without a history of wheezing or any other recurrent or chronic airway or inflammatory disease, and who were without a history of infection during the previous 2 weeks served as controls. These children had neither parental history of atopy or asthma nor a history of atopic dermatitis, food allergy, or allergic rhinoconjunctivitis. Skin prick tests were performed by using a panel of common allergens including house dust mite, mold mix, tree mix, grass mix, weed mix, cat, dog, cockroach, cow s milk, and egg (Stallergenes, Paris, France). Skin prick tests were performed only on the asthmatic children. A positive skin prick test was defined as a wheal with a diameter that exceeded the negative control by at least 3 mm. Children with a positive reaction to any of the allergens tested or with high IgE levels above 100 ku/l were considered atopic. The heights of all children were measured at the study center by the same examiner by using a wall-mounted Harpenden stadiometer (Holtain Limited, Crymych, Dyfeed, United Kingdom). Weight was determined in underwear by using a calibrated electronic scale (Seca, Hamburg, Germany). BMI was calculated as weight (kg)/height (m 2 ). Blood samples were obtained between 6 AM and 9 AM after an overnight fast. After clotting at 48C, the serum was separated by centrifugation and stored at ÿ708c until assay. Serum leptin concentrations were measured with the double antibody sandwich ELISA assay by using a mab specific for human leptin (Active Human Leptin ELISA DSL ; Diagnostic Systems Laboratories, Webster, Tex). In the assay, standards, controls, and unknown serum or plasma samples were incubated in microtitration wells that had been coated with antihuman leptin detection antibody labeled with the enzyme horseradish peroxidase. After a second incubation and washing step, the wells were incubated with the substrate tetramethylbenzidine. An acidic stopping solution was then added, and the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement at 450 and 620 nm. The absorbance measured was directly proportional to the concentration of human leptin present. A set of human leptin standards was used to plot a standard curve of absorbance versus human leptin concentration from which the human leptin concentrations in the unknowns could be calculated. All performance characteristics were stated in nanograms per milliliter. The minimum detection limit was 0.05 ng/ml. Total IgE values were measured by the CAP radioallergosorbent technique according to the manufacturer s instructions (UniCAP; Pharmacia and Upjohn, Uppsala, Sweden) in asthmatic children. Only 1/3 (35/102) of asthmatic children cooperated for spirometry (Sensor Medics, Yorba Linda, Calif) because of the young age group included in the study. Potential confounding factors for atopy and childhood asthma, such as family history of atopy, passive smoking, birth weight, and duration of breast-feeding, were also evaluated by using a questionnaire. The study was approved by the Medical Ethical Committee of our institution. Statistical analysis Numerical variables were reported as means ± SDs. Numerical parameters with nonnormal distribution (serum leptin and IgE levels) were reported as medians and interquartile ranges and were logtransformed before analysis. Bivariate analyses were evaluated by using v 2 and Mann-Whitney or Student t tests. Correlations between log leptin and the various other parameters were assessed by using the Pearson correlation coefficient. For multivariate analysis, a multiple logistic regression model was used for estimating the associations between sex (1, male; 0, female), age, BMI, leptin, and the dichotomous dependent variable asthma (1, asthmatic subjects; 0, nonasthmatic subjects). To predict independent factors for serum leptin levels, stepwise linear regression analysis was performed. Variables that were significant in analyses, ie, sex, age, BMI, and presence of asthma, were included as independent variables in this model. Statistical analysis was performed with the SPSS software, version 7.5 for Windows (SPSS, Chicago, Ill). P <.05 was considered significant. RESULTS Clinical characteristics of asthmatic children are summarized in Table I. Growth parameters of the asthmatic patients and the controls are shown in Table II. The mean values for age, weight, height, and BMI did not show any difference between the groups of asthmatic and healthy children. There was no significant sex difference with respect to these parameters between and within the groups, either. A significant difference (P =.008) was observed in the mean levels of serum leptin between asthmatic and healthy children in spite of a similar BMI in both groups. This significant difference was also apparent between the asthmatic and healthy boys with similar BMI values (P =.003), whereas a similar difference was not observed between asthmatic and healthy girls. No significant sex difference was detected in serum leptin levels among asthmatic children. Healthy boys had significantly lower serum leptin than healthy girls (P =.019; Table II). Log leptin levels of both asthmatic and healthy children showed positive correlations with BMI, whereas only in asthmatic girls, significant correlation existed with age (Table III). In a stepwise multiple regression analysis including sex (P =.001), age (P =.016), BMI (P =.000), and asthma (P =.022), all of these variables were found to affect log leptin levels (R 2 = 0.404; Table IV). By logistic regression analysis with sex, age, BMI, and

3 256 Guler et al J ALLERGY CLIN IMMUNOL AUGUST 2004 TABLE I. Clinical characteristics of asthmatic children N 102 Sex, female/male 37/65 Age at examination, y, mean ± SD 5.99 ± 3.46 Parental history of allergic diseases, n (%) 72 (71%) Birth weight, g, mean ± SD 3240 ± 0.60 Breast-feeding for >6 months, n (%) 76 (75%) Positive skin prick tests to 1 or 64 (63%) more allergens, n (%) Serum total IgE (>100 ku/l), n (%) 66 (65%) Number of atopic asthmatics, n (%) 78 (78%) Positive history for passive smoking, n (%) 61 (60%) FEV 1 % predicted, mean ± SD* ± Percent increase in FEV 1 after 8.35 ± 6.65 bronchodilator, mean ± SD* *In the children who were able to perform spirometry (n = 35). log leptin, we found that leptin was a predictive factor for having asthma, whereas sex, age, and BMI were not (Table V). The median (interquartile range) leptin levels were 3.77 ( ) ng/ml in atopic asthmatic subjects and 2.92 ( ) ng/ml in nonatopic asthmatic subjects; the difference was significant (P =.038), although the BMI values did not show a significant difference (17.58 ± 3.00 and ± 2.80; P =.756). Atopic asthmatic subjects showed significantly higher serum leptin levels than healthy children (P =.03), whereas a similar difference was not observed for nonatopic asthmatic subjects (P =.374; Fig 1). A sex difference was evident only for boys with atopic asthma who showed significantly higher serum leptin levels than healthy boys (P =.001). A significant but weak correlation was observed between log IgE and log leptin levels among asthmatic children (r = 0.231; P =.019). Leptin levels in asthmatic boys correlated positively with total log IgE (r = 0.319; P =.010), but a similar correlation was not found for asthmatic girls (r = ÿ0.108; P =.523; Fig 2). There was no statistically significant difference in the leptin levels between asthmatic children on the basis of skin test reactivity. No statistical differences were observed in leptin levels between the groups of asthmatic children with positive or negative results for family history of atopy and passive smoking. Leptin levels did not correlate with pulmonary function tests, birth weight, and duration of breast-feeding. DISCUSSION Investigations show that obesity increases the risk of asthma in both sexes and in different ethnic groups. 3,17 However, some evidence points toward the association between asthma and obesity being of a recent origin, and this may be explained by obesity being a marker of recent lifestyle differences associated with both asthma and being overweight. 18 The aim of this study was to investigate serum leptin levels and its relationship with TABLE II. Growth parameters and serum leptin levels in asthmatic children and healthy controls* Patients (n = 102)(female/ male = 37/65) Controls (n = 33)(female/ male = 14/19) Age (y) Total 5.99 ± ± Girls 5.41 ± ± Boys 6.31 ± ± Weight (kg) Total ± ± Girls ± ± Boys ± ± Height (cm) Total ± ± Girls ± ± Boys ± ± BMI Total ± ± Girls ± ± Boys ± ± Leptin (ng/ml) Total 3.53 ( ) 2.26 ( ).008 Girls 4.68 ( ) 2.70 g gà.447 Boys 3.09 ( ) 1.52 ( ).003 *Age, weight, height, and BMI are given as arithmetic means ± SDs; the Student t test is used. Leptin is given as median (interquartile range); the Mann-Whitney U test is used for comparison. P =.208. àp =.019. parameters of atopy and some risk factors in asthmatic children. Our results show that serum leptin levels of asthmatic children and especially of asthmatic boys were higher than those of healthy controls in spite of no difference in BMI levels. The lack of difference in leptin levels between asthmatic and healthy girls could be a result either of a true modifying effect of sex on leptin or a small sample size to detect a difference. Significantly higher levels of leptin in asthmatic boys but not in girls may imply that leptin might be involved in the pathophysiologic mechanisms of childhood asthma, which is also seen more frequently in boys than in girls. 19,20 Serum leptin levels are relatively low in prepubertal ages, showing a gradual rise in both sexes before the onset of puberty, followed by a significant increase during puberty in girls and a decrease in boys. 21,22 Although leptin is low and shows a weak correlation with age during childhood, it still shows a gradual increase throughout prepuberty, which may explain the effect of age on leptin levels in our study. Serum leptin levels show positive correlation with BMI in healthy prepubertal children, as has also been shown in our study in both the asthmatic children and the healthy children. 21,23 Leptin levels were found in similar concentrations in both sexes over the prepubertal years in some studies, 21 whereas a sex difference was noted in other studies, 22,23 with girls having higher leptin levels than boys despite the absence of difference in body weight, as has also been shown in healthy P

4 J ALLERGY CLIN IMMUNOL VOLUME 114, NUMBER 2 Guler et al 257 TABLE III. Correlations of log leptin levels with age and BMI in asthmatic and healthy children Total group Girls Boys r P r P r P Patients (n = 102) Age NS BMI < < <.001 Healthy controls (n = 33) Age NS NS ÿ0.009 NS BMI < TABLE IV. Multiple regression analysis in the study group* Unstandardized coefficients Standardized coefficients B SE Beta t Statistic P value 95% CI Constant ÿ ÿ3.704 <.001 ÿ2.582, ÿ0.784 BMI < , Sex ÿ ÿ0.233 ÿ ÿ0.762, ÿ0.202 Age , Asthma , *In all analyses, the dependent variable was log leptin (R 2 = 0.404); regression df, 4;F = ; P <.001. children in our study. This sex difference, which disappeared in asthmatic children, was probably a result of the higher levels of leptin in asthmatic boys, which might underlie the intrinsic pathology between asthma and leptin. Although the relationship among obesity, asthma, and leptin cannot be adequately addressed in this study because of the small sample size, the results of multivariant analysis implied that leptin may play a role in asthmatic inflammation. As body weight increases, more leptin is produced. 24 Although its primary effect is regulating fat mass, leptin is also proinflammatory. 25 In animal models, exogenous leptin has been shown to increase phagocytosis by macrophages as well as to upregulate production of TNF-a, IL-6, and IL In asthma, levels of circulating TNF-a are increased, and, on exposure to allergens, the production of TNF-a further increases. IL-6 is a proinflammatory cytokine, and the IL- 6 that is produced in adipose tissue is estimated to account for as much as 25% of the IL-6 released into circulation. 27 IL-6 production is increased in asthma, and it is thought that it may be a key modulator of airway remodeling. 28 IL- 12 plays a major part in antagonism of T H 2 differentiation and in induction of antiviral host defense. Genetically determined attenuation of IL-12 response capacity may provide an immunologic pathway to disease severity in asthmatic children. 29 The relationship between obesity and asthma is complex and involves interactions among obesity, inflammation, and sex. Additional studies for other biological mechanisms are still required. In the current study, there was a positive correlation between leptin and IgE levels. Although leptin levels of atopic asthmatics were significantly higher than those of nonatopic asthmatic subjects and healthy children, there was no statistically significant difference in the leptin TABLE V. Prediction analysis for asthma P value Odds ratio 95% CI Log leptin Sex Age BMI levels between asthmatic children on the basis of skin test reactivity. This would imply that aspect of atopy that is responsible for the difference is based on IgE levels alone. It seems that leptin may have some effects on the regulation of allergic inflammation in the airways. However, in the European Community Respiratory Health Survey, information collected and analyzed from 15,454 young adults to determine the association of BMI with respiratory allergies and atopy, no association was found between BMI and total or specific IgE levels. 30 In a crosssectional, population-based study of teenagers in Taiwan, BMI was found to be a significant predictor of atopy defined by positive skin reaction, allergic symptoms, and bronchial hyperreactivity in teenage girls, but not in boys. 31 In the National Health and Nutrition Examination Study, von Mutius et al 32 reported that the prevalence of asthma and atopy rose significantly with increasing BMI, but they found neither an independent relation between BMI and atopy assessed as skin test reactivity nor an association between eosinophil counts and BMI, suggesting that the effects of increased BMI may be mediated through factors other than the induction of allergic eosinophilic inflammation. Although we did not find any relationship between skin test positivity and leptin, the positive correlation between leptin and IgE

5 258 Guler et al J ALLERGY CLIN IMMUNOL AUGUST 2004 FIG 1. Serum leptin levels of atopic and nonatopic asthmatics and healthy controls. Data represent median values. P values are given for comparison between groups by using the Mann-Whitney U test. found in our study may highlight the positive relation between BMI and atopy observed in their study. Low birth weight is associated with asthma risk. 33 Small airway size is likely to explain the association between lower birth weight and asthma. Studies have noted that low birth weight is also associated with increased percentage of body fat in children at older ages. 34 Umbilical cord leptin levels are increased both in neonates large for gestational age and in human beings with growth retardation. 35 It has been postulated that infants exposed to poor nutrition during the early trimesters may be programmed for enhanced leptin production and subsequent adipose tissue deposition. In our group of asthmatic children, there was no association between birth weight and serum leptin levels. Perinatal factors have been found to be involved in the pathogenesis of obesity and its complications. There is increasing evidence that perinatal and early childhood events affect the subsequent development of asthma. 36 Higher leptin concentrations were observed in breast-fed infants in the first months of life. 37 It is not clear whether the presence of leptin in breast milk is important for children. Infants who are exclusively breast-fed for a period are protected from becoming obese. 38 Leptin seems to modulate some immune system functions in infants, thus producing a metabolic advantage related to growth. Effects of leptin on immunomodulation may provide an additional physiologic basis for reduced systemic illness such as asthma in breast-fed infants. We did not observe any relationship with leptin and breastfeeding duration in asthmatic children. To understand better the role of leptin in asthma, cohort studies from infancy to adulthood are necessary. FIG 2. Relationship between serum leptin and total IgE concentrations (log scale). Leptin levels were significantly (r = 0.319; P =.010) related to total IgE in boys (A) but not in girls (B). Serum leptin and total IgE levels are determined as described in the Methods section. The contributions of the distribution of adipose tissue and obesity to pulmonary function impairment by using FEV 1 are not established in either asthmatic or nonasthmatic children. Therefore, interpreting the results of pulmonary function in asthmatic children is very difficult. The most consistent alteration in lung function found in obesity is a reduction in functional residual capacity caused by the effect of the abdominal contents on the position of the diaphragm. 39 It has been reported that obese asthmatic children miss more school days, receive more medications, and have lower peak expiratory flow rates than nonobese asthmatics. 17 These findings may be a result not of the obesity per se but of more severe disease. In this study, we did not find any correlation between leptin levels and spirometric parameters. In our preliminary study, we had found a significant positive correlation between leptin and FEV 1 reversibility to bronchodilator. This result must be interpreted with caution, because FEV 1 is a lung volume index, which is a function of height, a parameter that is also included in the derivation of BMI. Although FEV 1 is considered a measure of asthma severity, it is not consistently related to inflammation or symptoms and is affected by multiple factors. In conclusion, our findings suggest that leptin may play a role in atopic asthma. High serum leptin levels in

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