Thorax Online First, published on August 23, 2006 as 10.1136/thx.2006.060657 Thorax: first published as 10.1136/thx.2006.060657 on 23 August 2006. Downloaded from

Induced sputum inflammatory measures correlate with disease severity in children with obstructive sleep apnoea

Albert M. Li, Emily Hung, Tony Tsang, Jane Yin, Hung K So, Eric Wong1, Tai F Fok, Pak C Ng

Department of Paediatrics and 1Centre for Epidemiology and Biostatistics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong SAR.

http://thorax.bmj.com/ Correspondence to: Dr Albert Martin Li Associate Professor Department of Paediatrics Prince of Wales Hospital The Chinese University of Hong Kong Shatin, Hong Kong SAR on September 27, 2021 by guest. Protected copyright. Tel: (852) 2632 2982 Fax: (852) 2636 0020 E-mail: [email protected]

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Objective: To establish the association between airway inflammation and severity of OSA in children. Methods: Consecutive children presented with symptoms suggestive of OSA were recruited. They completed a sleep apnoea symptom questionnaire, underwent physical examination, spirometry, sputum induction and an overnight polysomnography. Adequate sputum contained < 50% squamous epithelial cells and OSA was diagnosed if obstructive apnoea index (OAI) > 1. Results: Seventy-three children with a median (IQR) age of 11.3 (10.0–13.2) years were recruited. There were 21 girls and the median body mass index of the group was 24.0 (18.0-27.0). The commonest presenting symptoms were habitual snoring, mouth breathing and prone sleeping position. Sputum induction was successful in 43 subjects (59%) of whom 14 were found to have OSA. Subjects with OSA had significantly greater percentage sputum neutrophil than non-OSA counterparts [18.5 (IQR 8.0-42.0) vs 4 (IQR 3.0-11.3), p=0.006]. On multiple regression analysis, percentage sputum neutrophil was significantly associated with OSA (OR=1.1, p=0.013). Conclusion: Children with OSA had airway inflammation characterized by a significant increase in neutrophils. Further studies are needed to confirm our findings and to better define the downstream cellular interactions and molecular pathogenesis in childhood OSA.

Keywords: sputum, neutrophils, child, obstructive sleep apnoea

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Introduction Obstructive sleep apnoea (OSA) is characterised by repeated episodes of upper airway occlusion during sleep that are associated with daytime behavioural changes and abnormalities in cardiovascular function.1-3 In children, the commonest cause for OSA is adenotonsillar hypertrophy.4 The condition is being increasingly recognised, and the prevalence among paediatric population could be as high as 10.3%.5 In adults, evidence suggests that both local airway and systemic inflammation are implicated in the pathophysiology of this seemingly all-mechanical condition. Surgically removed tissues from OSA patients undergoing uvulopalatopharyngoplasty and tonsillectomy showed marked subepithelial oedema6 and inflammatory cell infiltration.7 The number of leucocytes is substantially increased in the upper airway mucosa of adults with OSA, especially in the muscular layer of the soft palate and tonsillar pillars.8 Polymorphonuclear leucocytes, bradykinin and vasoactive intestinal peptide are found at increased levels in nasal lavage fluid of patients with OSA.9 Exhaled markers of airway inflammation and oxidative stress such as interleukin-6 and 8-isopentane are also found at elevated levels in breath condensate of OSA adults.10,11 Serum tumour necrosis factor- α, interleukin-6 and C-reactive protein (CRP) have all been reported to present at higher levels in adult OSA patients compared with controls.12,13 Treatment of OSA is known not only to improve daytime symptoms of the patients but also to decrease the risk of cardiovascular complications. These treatment effects are postulated to be related to decreases in systemic inflammation. In support of this hypothesis, serum CRP has been found to reduce significantly after 1 month of nasal continuous positive airway pressure (CPAP) therapy.14 There is accumulating evidence to suggest local and systemic inflammatory response also exists in children with OSA.15,16 CRP levels are elevated in children with OSA, which may suggest on-going systemic inflammation contributing to the http://thorax.bmj.com/ proliferation of upper airway lymphoid tissues.15 Cysteinyl leukotrienes are major mediators of inflammation, potent neutrophil chemoattractants and activator.17 Upregulation of cysteinyl leukotriene receptor has been found in the tonsils of children undergoing tonsillectomy for OSA.18 Montelukast, a cysteinyl leukotriene receptor antagonist induced reductions in adenoidal size and respiratory-related sleep disturbances.19 There is, however, no available data on airway inflammation and its on September 27, 2021 by guest. Protected copyright. correlation with OSA severity in children. If such a relationship does exist, this will generate further interest to examine for the role of anti-inflammatory therapy in childhood OSA. Sputum induction has gained popularity over the past few years as a non-invasive method in assessing airway inflammation. The technique is well established and safe to use in children.20 In this study, we investigated airway inflammation in children with OSA by utilising the analysis of differential cell counts in induced sputum.

Methods Subjects Children with symptoms suggestive of OSA and aged 6 to 18 years were consecutively recruited from our Paediatric Respiratory, Sleep Disorder and Obesity Clinic during the period between January 2004 and June 2005. Patients were excluded from the study if they had an intercurrent respiratory tract infection, a neuromuscular

3 Thorax: first published as 10.1136/thx.2006.060657 on 23 August 2006. Downloaded from disorder such as Duchenne muscular dystrophy, craniofacial anomalies or a syndromic disorder for example Down syndrome, or if they had previously undergone upper airway surgery. The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong and informed written consent from the subjects and their parents was obtained at the beginning of the assessment. Each subject on the day of admission underwent physical examination and overnight polysomnography (PSG). Lung function test and sputum induction were carried in the morning after the overnight PSG.

Anthropometry and physical assessment The weight and standing height of the subjects were measured with a calibrated weighing scale and stadiometer by standard anthropometric methods.21 Body mass index (BMI) was calculated as weight / height2 (kg/m2). The sizes of the tonsils were graded according to a standardized scale from 0 (absent) to 4 (kissing tonsils).22,23

Lung function This was performed using a standard technique to measure the forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC) (Spirolab II, MIR, Italy).24 The best of three efforts was compared with local age- and sex-matched reference values.25

Sputum induction and processing Sputum induction (SI) involved the incremental inhalation of a 4.5% hypertonic saline (4.5% HS) solution via an ultrasonic nebuliser, as described previously.20 SI was only performed if the subject’s FEV1 was > 65% predicted. Sputum differential cell counts were evaluated by a laboratory investigator blinded to the clinical data of the subjects. Specimens containing squamous epithelial cells of less than 50% of the total http://thorax.bmj.com/ inflammatory cell number were considered as successful inductions. At least 400 inflammatory cells were counted for each specimen and the percentage of eosinophils, neutrophils, macrophages and lymphocytes were recorded.

Sleep Study An overnight PSG was performed on each subject using Siesta ProFusion II PSG on September 27, 2021 by guest. Protected copyright. monitor (Compumedics Telemed PTY Ltd. Abbotsford, Australia), as described previously.26 Obstructive apnoea (OA) was defined as absence of airflow with persistent respiratory effort lasting longer than 2 baseline breaths, irrespective of SaO2 changes. Obstructive Apnoea Index (OAI) was defined as the number of OA per hour of sleep. Central Apnoea (CA) was defined as absence of respiratory effort associated with absence of airflow. Those of greater than 20 seconds with or without oxygen desaturation or arousals, and those of any duration but associated with oxygen desaturation of at least 4% and/or arousals are quantified. Hypopnoea was defined as a reduction of 50% or more in the amplitude of the airflow signal. It was only quantified if longer than 2 baseline breaths and associated with oxygen desaturation of at least 4% and/or arousals. Apnoea hypopnoea index (AHI) was defined as the total number of apnoeic and hypopnoeic episodes per hour of sleep. Oxygen saturation nadir and the percentage of total sleep time where oxygen saturation was below 90% were noted. Arousal was defined as an abrupt shift in EEG frequency during sleep, which may include theta, alpha and/or frequencies

4 Thorax: first published as 10.1136/thx.2006.060657 on 23 August 2006. Downloaded from greater than 16 Hz but not spindles, with 3 to 15 seconds in duration. In REM sleep, arousals were scored only when accompanied by concurrent increases in submental EMG amplitude. We defined OSA as OAI > 1.0. Subjects who were taking as required anti- histamine were asked to withhold their medication for 48 hours prior to PSG.

Statistical analysis The results were presented as medians with interquartile ranges (IQR). Potential factors including demographic data, polysomnographic parameters, lung function measures and sputum differential cell counts were evaluated for their association with OSA with Mann-Whitney test and chi-square test. Risk factors with a p-value less than 0.25 were then analyzed by univariate and multivariate logistic regression analysis, using a forward stepwise selection strategy. The most significant factor (i.e., the one that would result in the largest likelihood ratio statistic) was added to the model at each step and the process continued until no further significant contributing factor could be added. Whenever two or more potential factors were highly correlated or had P values that were similar, the factor that was more clinical or biological important was selected for entry. Relationship between airway inflammation and severity of OSA was presented after logarithmic transformation. Analyses were performed using SPSS for Windows statistical software (version 13.0). The level of significance was set at 5% in all comparisons.

Results Seventy-three consecutive subjects were invited and agreed to participate in this study. There were 52 boys and the median (IQR) age of the subjects was 11.3 (10.0-13.2) years. Forty-nine subjects had allergic rhinitis for which they used antihistamine on an as required basis, and 6 were using regular corticosteroids nasal spray. One subject had asthma and seventeen had a combination of allergic rhinitis and asthma, but none of the http://thorax.bmj.com/ asthmatics were taking regular inhaled corticosteroids and their asthma had been well controlled prior to and during this study. All subjects had normal physical examination and pulmonary function test; FEV1 and FVC were 94% predicted (83.3-100.8) and 93% predicted (84.3-100.8) respectively. The three most common OSA related presenting complaints were habitual snoring (snoring for > 3 nights per week), mouth breathing and prone sleeping position. All subjects completed the sputum induction protocol and an on September 27, 2021 by guest. Protected copyright. adequate sputum sample was obtained in 43 (59%). None of the subjects complained of any adverse effects from the procedure. The characteristics of the subjects with and without successful sputum induction are shown in table 1. Those who were able to produce an adequate sputum sample were significantly older and taller. Twenty-four subjects satisfied the diagnostic criterion for OSA and of whom 14 had successful sputum induction. Amongst subjects with successful sputum induction, the characteristics of those with and without OSA were compared in table 2. There were no significant differences in age, gender distribution, anthropometric measures, pulmonary function and tonsil sizes between the two groups. Subjects with OSA had significantly higher percentage sputum neutrophils [18.5 (8.0-42.0) vs. 4.0 (3.0-11.3), p = 0.006] and lower percentage sputum macrophages [76.0 (56.0-87.0) vs. 84.0 (77.8-89.0), p = 0.015] and eosinophils [0.4 (0.0-3.0) vs. 2.0 (0.8-4.2), p = 0.023] their non-OSA counterparts. There were no significant differences in the number of subjects with and without asthma and allergic rhinitis between those with and without OSA.

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Table 3 shows univariate analysis demonstrating significantly different parameters between OSA and non-OSA groups. On multiple regression analysis, only percentage sputum neutrophil [OR (95%CI) 1.10 (1.02-1.18), p = 0.013) and oxygen saturation nadir [0.85 (0.75-0.96), p = 0.007] remained as significant predictors for OSA. Scatter-plot showing the relationships between OAI / AHI and sputum neutrophils after logarithmic transformation is shown in figure 1.

Discussion We have demonstrated for the first time that in children with obstructive sleep apnoea, percentage sputum neutrophils were significantly increased, and that the degree of neutrophilic airway inflammation correlated significantly and positively with the severity of OSA. Using specific histological parameters and surrogate biomarkers in nasal lavage fluid and exhaled breath, previous studies have collectively shown that nasal and oropharyngeal mucosal inflammation is present in patients with OSA.6-11 Recurrent vibration in the upper airway of children with OSA has been suggested to promote such inflammatory changes in tonsillar tissue and upper airway mucosa.27 The stimulation of any part of the respiratory tract can have ripple effects along the entire airway and reinforces the concept of “united airways”.28 A case was recently reported involving a healthy cystic fibrosis patient who developed recurrent cough with worsening lower airway obstruction that were reversed following treatment for OSA.29 This observation could be explained by the “united airway” hypothesis whereby OSA causes airway inflammation that adversely affects the lung function. Neutrophilic airway inflammation has been demonstrated in adult patients affected by OSA.30 In the study involving 16 OSA patients, the authors found significantly higher percentage of neutrophils in the induced sputum of patients compared to healthy volunteers [mean (SD), 66.7 (18.9) vs. http://thorax.bmj.com/ 25.8 (15.6), p < 0.001]. The percentage sputum neutrophil count obtained in our current study is not as great as that demonstrated in adult patients, and differences in the chronicity and severity of disease among adult subjects could explain this discrepancy. Adult patients tend to have had prolonged disease prior to diagnosis and they typically have more severe OSA compared to their paediatric counterparts. In another study that involved 19 male OSA patients, circulating neutrophil levels were higher in affected on September 27, 2021 by guest. Protected copyright. patients than in control subjects.31 In addition, receptors of cysteinyl leukotrienes which are potent neutrophil chemoattractants and activator are found at increased levels in the tonsils of children undergoing tonsillectomy for OSA.17,18 There are limited data evaluating the effects of anti-inflammatory therapy on upper airway inflammation in OSA. Topical nasal corticosteroids produced only modest therapeutic effect in children with OSA, without any significant change in the size of the adenoid or tonsillar tissues.32 This may at least be partly explained by the fact that corticosteroids are better in controlling eosinophilic than neutrophilic inflammation. It has recently been reported that montelukast, a cysteinyl leukotriene receptor antagonist induced significant reductions in adenoidal size and respiratory-related sleep disturbances in children suffering from OSA.19 Taken together, there is accumulating evidence to support an inflammatory neutrophilic reaction in the airways of patients with OSA. Current research has highlighted the potential use of sputum induction as a non- invasive method of assessing airway inflammation. The availability of both sputum

6 Thorax: first published as 10.1136/thx.2006.060657 on 23 August 2006. Downloaded from cellular and fluid components for identifying types of inflammatory cells and pattern of cytokines and chemokines has allowed a better delineation of the underlying inflammatory process.33,34 The success rate of sputum induction in children has been reported to vary from 68% – 100% and the procedure is generally well tolerated.20 If our findings can be confirmed in subsequent studies involving larger sample size, then sputum examination may be used as an objective assessment of the severity of OSA or a guide for the evaluation of success of both medical and surgical interventions. There are a few potential limitations to our study. First, our study was a cross- sectional assessment at a single time point. Longitudinal data including interventions would have provided more interesting data for the association and complex interaction between OSA and airway inflammation. Second, we have selected a biased sample as we recruited our subjects from attendants to our Paediatric Respiratory, Sleep Disorder and Obesity Clinic. Boys are more likely to be obese and this explains the male preponderance seen in this study. We had excluded those under the age of 6 years as in our experience children under this age were unlikely to success in sputum induction. Further studies involving the more typical OSA children will be needed to confirm the association between airway inflammation and severity of OSA seen in the current study. Our study has provided a new insight into the pathophysiology of OSA in children, highlighting the presence of neutrophilic inflammation in the lower airways. Further studies are needed to confirm our findings and to better define the downstream cellular interactions and molecular pathogenesis in childhood OSA.

Funding - Direct Grant for Research, CUHK (Reference no. 2005.2.028). http://thorax.bmj.com/

Competing interests - None declared.

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18. Goldbart AD, Goldman GL, Li RC, et al. Differential expression of cysteinyl leukotriene receptors 1 and 2 in tonsils of children with obstructive sleep apnea and recurrent infection. Chest 2004;126:13-18. 19. Goldbart AD, Goldman GL, Veling MC, et al. Leukotriene modifier therapy for mild sleep-disordered breathing in children. Am J Respir Crit Care Med 2005;172:364-370. 20. Li AM, Tsang TWT, Chan DFY, Lam HS, So HK, Sung RYT, Fok TF. Sputum induction in children with asthma: A tertiary center experience. Pediatr Pulmonol 2006;41(8):720-725. 21. Tanner JM. Physical growth and development. In: Forfar JO, Arneil GC, eds. Textbook of Paediatrics. Edinburgh: Churchill Livingstone;1984:304-305. 22. Brooks LJ, Stephens BM, Bacevice AM: Adenoid size is related to severity but not the number of episodes of obstructive apnea in children. J Pediatr 1998;132:682-686. 23. Leach J, Olson J, Hermann J, Manning S: Polysomnographic and clinical findings in children with obstructive sleep apnea. Arch Otolaryngol 1992;118:741-744. 24. Medical Section of the American Lung Association. Standardization of spirometry, 1994 update. Am J Respir Crit Care Med 1995;152:1107-1136. 25. Ip MS, Karlberg EM, Karlberg JP, Luk KD, Leong JC. Lung function reference values in Chinese children and adolescents in Hong Kong. Am J Respir Crit Care Med 2000;162:424-429. 26. Li AM, Wing YK, Cheung A, Chan D, Ho C, Hui S, Fok TF. Is a 2-night polysomnographic study necessary in childhood sleep-related disordered breathing? Chest 2004;126:1467-1472. 27. Cohn M, Hesla PE, Kiel M, et al. Vibration frequency of snoring in obstructive sleep apnea syndrome. Chest 1986;89:529S. http://thorax.bmj.com/ 28. McCusker CT. Use of mouse models of allergic rhinitis to study the upper and lower airway link. Curr Opin Allergy Clin Immunol 2004;4:11-16. 29. Hayes D. Obstructive sleep apnea syndrome: a potential cause of lower airway obstruction in cystic fibrosis. Sleep Med 2006;7:73-75. 30. Salerno FG, Carpagnano E, Guido P, Bonsignore MR, Roberti A, Aliani M, Vignola AM, Spanevello A. Airway inflammation in patients affected by on September 27, 2021 by guest. Protected copyright. obstructive sleep apnea syndrome. Resp Med 2004;98:25-28. 31. Ryan S, Taylor CT, McNicholas WT. Selective activation of inflammatory pathways by intermittent hypoxia in obstructive sleep apnea syndrome. Circulation 2005;112(17):2660-2667. 32. Brouillette RT, Manoukian JJ, Ducharme FM, Oudjhane K, Earle LG, Ladan S, Morielle A. Efficacy of fluticasone nasal spray for pediatric obstructive sleep apnea. J Pediatr 2001;138:838-844. 33. Pavord ID, Pizzichini MM, Pizzichini E, Hargreave FE. The use of induced sputum to investigate airway inflammation. Thorax. 1997 Jun;52(6):498-501. 34. Gibson PG, Henry RL, Thomas P. Noninvasive assessment of airway inflammation in children: induced sputum, exhaled nitric oxide, and breath condensate. Eur Respir J. 2000 Nov;16(5):1008-15.

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Table 1. Characteristics of subjects with and without successful sputum induction With sputum (n=43) No sputum (n=30) p-value

Age (years) 11.8 (1.08-13.7) 10.6 (8.8-11.6) 0.005

Male / Female 30 / 13 22 / 8 0.742

Weight (kg) 53.4 (40.5-66.0) 42.0 (29.6-59.9) 0.071

Height (m) 1.5 (1.4-1.6) 1.4 (1.3-1.5) 0.008

BMI (kg/m2) 24.3 (20.7-27.4) 22.8 (15.2-26.8) 0.239

Tonsil size (left) 1.0 (1.0-3.0) 1.0 (1.0-2.0) 0.617

Tonsils size (right) 1.0 (0-3.0) 1.0 (1.0-2.0) 0.981

FEV1 (% predicted) 93.5 (84.0-100.0) 97.0 (82.5-101.0) 0.846

FVC (% predicted) 92.5 (85.0-100.0) 95.0 (81.0-102.8) 0.871

FEV1 / FVC 90.0 (85.0-94.0) 91.0 (90.0-94.0) 0.247

OAI 0.2 (0.0-1.3) 0.3 (0.0-1.4) 0.592

AHI 2.0 (0.7-5.5) 1.4 (0.2-5.4) 0.340 http://thorax.bmj.com/

% TST with SaO2 < 90% 1.0 (0-0.03) 2.5 (0.2-6.7) 0.561

SaO2 nadir (%) 87.5 (83.0-91.0) 89.0 (77.5-90.3) 0.991

Arousal index 6.7 (4.9-11.2) 6.0 (3.9-10.4) 0.349

Asthma (yes / no) 12 / 31 6 / 24 0.444 on September 27, 2021 by guest. Protected copyright.

Allergic rhinitis (yes / no) 30 / 13 19 / 11 0.567 Median (IQR) % TST with SaO2 < 90% – percentage of total sleep time with oxygen saturation < 90%.

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Table 2. Characteristics of subjects with and without OSA Non-OSA (n=29) OSA (n=14) p-value

Age (years) 11.6 (10.8-13.8) 11.8 (11.0-13.6) 0.678

Male / Female 22 / 7 8 / 6 1.000

Weight (kg) 52.3 (39.7-64.4) 53.4 (48.3-66.0) 0.577

Height (m) 1.5 (1.4-1.5) 1.5 (1.5-1.6) 0.508

BMI (kg/m2) 24.3 (19.8-27.3) 24.3 (20.9-30.0) 0.836

Tonsil size (left) 1.0 (1.0-2.0) 2.0 (1.0-3.0) 0.126

Tonsils size (right) 1.0 (0.0–2.0) 1.5 (1.0-4.0) 0.089

FEV1 (% predicted) 94.0 (87.0-102.5) 87.0 (82.8-96.0) 0.187

FVC (% predicted) 95.0 (85.0-100.3) 89.0 (84.8-99.0) 0.531

FEV1 / FVC 90.0 (88.0-94.3) 86.0 (84.0-93.5) 0.130

Sputum eosinophils (%) 2.0 (0.8-4.2) 0.4 (0.0-3.0) 0.023

Sputum neutrophils (%) 4.0 (3.0-11.3) 18.5 (8.0-42.0) 0.006 http://thorax.bmj.com/ Sputum macrophages (%) 84.0 (77.8-89.0) 76.0 (56.0-87.0) 0.015

Sputum lymphocytes (%) 6.8 (2.0-8.0) 2.5 (1.0-9.7) 0.413

% TST with SaO2 < 90% 0.1 (0.0-0.2) 1.6 (0.2-3.7) 0.008 on September 27, 2021 by guest. Protected copyright. SaO2 nadir (%) 90.5 (84.3-93.0) 82.0 (72.0-87.0) <0.001

Arousal index (per hr) 5.7 (4.4-8.3) 9.1 (6.8-15.3) 0.010

Asthma (yes / no) 10 / 19 2 / 12 0.279

Allergic rhinitis (yes / no) 21/ 8 9 / 5 1.000 Median (IQR)

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Table 3. Univariate logistic regression Non-OSA

(n=29) OSA (n=14) OR (95%CI) p-value 1.51 (0.91-2.48) Tonsil size (left) 1.0 (1.0-2.0) 2.0 (1.0-3.0) 0.110 1.62 (0.98-2.98) Tonsils size (right) 1.0 (0.0–2.0) 1.5 (1.0-4.0) 0.098 94.0(87.0- 0.96 (0.89-1.03) FEV (% predicted) 87.0 (82.8-96.0) 0.233 1 102.5) 0.67 (0.44-1.03) Sputum eosinophil (%) 2.0 (0.8-4.2) 0.4 (0.0-3.0) 0.067 1.08 (1.02-1.16) Sputum neutrophil (%) 4.0 (3.0-11.3) 18.5 (8.0-42.0) 0.014 0.93 (0.88-0.99) Sputum macrophages (%) 84.0 (77.8-89.0) 76.0 (56.0-87.0) 0.017 1.79 (1.01-3.18) % TST with SaO2 < 90% 0.1 (0.0-0.2) 1.6 (0.2-3.7) 0.046 0.87 (0.78-0.96) SaO2 nadir (%) 90.5 (84.3-93.0) 82.0 (72.0-87.0) 0.004 1.25 (1.04-1.51) Arousal index 5.7 (4.4-8.3) 9.1 (6.8-15.3) 0.020 3.18 (0.59-16.96) Asthma: yes/no 10 / 19 2 / 12 0.180

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Figure 1. Scatterplot showing relationship between OAI / AHI and sputum neutrophil

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