Increased IL-33 expression by epithelial cells in bronchial asthma

752 LETTERS TO THE EDITOR

J ALLERGY CLIN IMMUNOL MARCH 2010

testing the difference per person-year at risk (PYR) by means of a 2-sample t test. We assumed unequal variances after testing the homogeneity of variances using the Levene’s test. The adolescents with asthma in the follow-up survey had 2.72 more reported febrile days per PYR in early childhood (95% CI, 0.23-5.20; P 5 .03) than those who were healthy (Table I). The number of febrile days per PYR during gastroenteritis episodes did not differ between the groups (Table I). Likewise, the subjects with asthma at follow-up had 0.85 more days of paracetamol medication per PYR and 1.37 more days of any antipyretic medication per PYR than the healthy adolescents (differences not statistically significant, Table I). The adolescents with atopic eczema at follow-up had had 1.46 more febrile days per PYR in early childhood (95% CI, 0.10-2.83; P 5 .04) than the healthy adolescents (Table I). The greater number of febrile days in early childhood observed in the adolescents with asthma in our prospective study appears to be explained by increased morbidity from respiratory tract infections because no difference was observed in febrile episodes caused by gastroenteritis. Thus observational studies assessing the association of paracetamol medication in early life with the later development of allergic diseases are likely to be confounded by increased respiratory tract infection morbidity in asthma-susceptible children. The fact that even adolescents with atopic eczema appeared to have more febrile days in early childhood than the healthy adolescents is probably explained by susceptibility to wheezing episodes during infancy, leading to increased respiratory tract infection morbidity in these children as well.6 The increased morbidity of asthma-susceptible children, together with the concern shown by their parents, could easily have led to frequent temperature measurements and increased medication with symptom-relieving drugs, such as paracetamol. Nevertheless, the possibility of an increased number of febrile days has not been investigated in any previous study of the relation between paracetamol and the later development of asthma in children.2,3 The problems of paracetamol and asthma could ideally be resolved by means of a randomized controlled trial, as proposed by Farquhar et al.1 A long-term follow-up would be needed, however, and protocol violations would be probable because paracetamol is a common over-the-counter drug. In one previous randomized trial, short-term paracetamol medication during febrile illness in children aged 6 months to 12 years with asthma was found to be associated with more outpatient visits for asthma in the next 4 weeks than among children treated with ibuprofen.7 It could not be determined, however, whether this was due to an increased risk of asthma exacerbations after paracetamol or to a protective effect of ibuprofen because there was no placebo group. Thus a randomized controlled trial assessing the safety of paracetamol would require a placebo group, which would be difficult for parents to accept. We conclude that previous observational studies on the longterm effects of paracetamol medication on allergic disease morbidity in children appear to be confounded by an increase in the number of days with fever during respiratory tract infections in asthma-susceptible children. This might explain the previously reported effects of paracetamol on asthma. Terhi Tapiainen, MD, PhD Teija Dunder, MD, PhD Merja Mo¨tto¨nen, MD, PhD Tytti Pokka, BSc Matti Uhari, MD, PhD

From the Department of Pediatrics, University of Oulu, Oulu, Finland. E-mail: terhi. [email protected]. Disclosure of potential conflict of interest: T. Tapiainen has received research support from the Finnish Medical Foundation. The rest of the authors have declared that they have no conflict of interest.

REFERENCES 1. Farquhar H, Crane J, Mitchell EA, Eyers S, Beasley R. The acetaminophen and asthma hypothesis 10 years on: a case to answer. J Allergy Clin Immunol 2009; 124:649-51. 2. Beasley R, Clayton T, Crane J, von Mutius E, Lai CK, Montefort S, et al. Association between paracetamol use in infancy and childhood, and risk of asthma, rhinoconjunctivitis, and eczema in children aged 6-7 years: analysis from Phase Three of the ISAAC programme. Lancet 2008;372:1039-48. 3. Koniman R, Chan YH, Tan TN, Van Bever HP. A matched patient-sibling study on the usage of paracetamol and the subsequent development of allergy and asthma. Pediatr Allergy Immunol 2007;18:128-34. 4. Uhari M, Mo¨tto¨nen M. An open randomized controlled trial of infection prevention in child day-care centers. Pediatr Infect Dis J 1999;18:672-7. 5. Dunder T, Tapiainen T, Pokka T, Uhari M. Infections in child day care centers and later development of asthma, allergic rhinitis, and atopic dermatitis: prospective follow-up survey 12 years after controlled randomized hygiene intervention. Arch Pediatr Adolesc Med 2007;161:972-7. 6. Illi S, von Mutius E, Lau S, Nickel R, Gruber C, Niggeman B, et al. The natural course of atopic dermatitis from birth to age 7 years and the association with asthma. J Allergy Clin Immunol 2004;113:925-31. 7. Lesko SM, Louik C, Vezina RM, Mitchell AA. Asthma morbidity after the shortterm use of ibuprofen in children. Pediatrics 2002;109:e20. Available online February 8, 2010. doi:10.1016/j.jaci.2009.11.039

Increased IL-33 expression by epithelial cells in bronchial asthma To the Editor: Allergic asthma is a chronic inflammatory disease of the lungs characterized by TH2-type immune bias. Apart from the infiltrating immune cells, the epithelium is increasingly considered as an active immune player producing numerous inflammatory mediators. IL-33, also called IL-1F11 or nuclear factor from high endothelial venules (NF-HEV), is a member of the IL-1 cytokine family. IL-33 is an inducer of the TH2 branch of adaptive immunity and signals through a complex including the membranebound ST2 protein.1 Aside from TH2 cells, ST2 is present on mast cells, basophils, eosinophils, natural killer cells, and natural killer T cells. The IL-33/ST2 axis triggers the release of several proinflammatory mediators, such as chemokines and cytokines, and induces systemic TH2-type inflammation in vivo.1 The IL33/ST2 pathway also contributes to allergen-induced airway inflammation and hyperresponsiveness,2 both important features of asthma. IL-33 effects were antagonized by administration of soluble ST2 decoy receptor or blocking antibodies in vivo and in vitro.2,3 Elevated IL-33 mRNA levels were measured in the lung tissue from subjects with asthma compared with healthy controls.4 Together, these data support a role for IL-33 in the pathogenesis of allergic asthma. Although studies reported that structural cells, including endothelial cells,4,5 fibroblasts, smooth muscle cells, and epithelial cells,6 express IL-33 in various organs,1 this study is the first to investigate expression of IL-33 by the airway epithelium in bronchial asthma and compared that with controls. Adult subjects with and without asthma were recruited from the Montreal Chest Institute and the Sacre´-Coeur Hospital based on the criteria of the American Thoracic Society Workshop on Refractory Asthma, and patients’ characteristics were reported

J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 3

FIG 1. Epithelial IL-33 immunoreactivity in bronchial asthma compared with controls. Endobronchial tissue sections from subjects with mild asthma (A), subjects with severe asthma (B), and controls (C) were stained by immunocytochemistry. Preabsorption of primary antibody with recombinant IL-33 abolished staining on a severe asthma section (D). E, IL-33 staining intensity was blindly graded and revealed an increased immunoreactivity in subjects with severe asthma versus control subjects. Mod., Moderate.

previously.4 Study protocols were approved by ethics committee boards, and all subjects gave written informed consent. Endobronchial tissue specimens from healthy controls without asthma (n 5 5) and subjects with asthma (n 5 13: 4 with mild, 4 with moderate, and 5 with severe asthma) were fixed in formaldehyde, and paraffin sections were used for immunocytochemistry staining by using mAb against human IL-33 (0.01 mg/mL, mouse IgG1k [Nessy-1]; Alexis Biochemicals, Axxora LCC, San Diego, Calif) and a peroxidase-based detection method.5 As reported in other organs,6 bronchial epithelial cells express IL-33 protein (Fig 1, A-C). Specificity of IL-33 immunostaining was confirmed by preabsorbing the antihuman IL-33 antibody with recombinant human IL-33 for 1 hour at 48C (0.05 mg/mL; Axxora LLC), which resulted in a complete abolishment of the IL-33 staining across the tissue sections (Fig 1, D). IL-33 expression in controls without asthma appeared mostly localized in both the cell nuclei and cytoplasm of basal cells (Fig 1, C), which contrasted with widespread distribution of the signal throughout the epithelium in sections from asthmatic subjects (Fig 1, A and B, respectively). Semiquantitative assessment of epithelial cell IL-33 immunoreactivity was achieved through blind grading of both nuclear and cytoplasmic staining intensity based on a 0 to 8 scale where 0 corresponds to no staining (similar to preabsorbed antibody control) and 8 represents the darkest staining in which all epithelial cells were positive, both in the cytoplasm and cell nuclei. We observed an increased IL-33 staining intensity in subjects with severe asthma compared with healthy controls (P < .05), but not other subjects with asthma (Fig 1, E). IL-33 mRNA expression by epithelial cells was confirmed by using bronchial epithelial cells isolated from bronchial biopsies of subjects with asthma (n 5 5; mean age, 24 years; FEV1, 96.3 6 6.3%; PC20, 3.6 6 5.3 mg/mL) and healthy subjects without

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FIG 2. A, Cultured bronchial epithelial cells isolated from subjects with asthma exhibit elevated baseline IL-33/glyceraldehyde-3-phosphate (GAPDH) mRNA ratios versus cells from subjects without asthma (P < .05). B, Elevated IL-33 cytokine levels were detected in BALF from adults with moderate asthma compared with those from controls with mild asthma or without asthma (n 5 10 per group; P < .05; Bonferroni test). ELISA experiments were run in duplicate.

asthma (n 5 4; mean age, 23.2 years; FEV1, 105.6 6 4.3%; PC20, 115.2 6 28.6 mg/mL) (mean 6 SD). Subjects were recruited from the Institut Universitaire de Cardiologie et de Pneumologie de Que´bec (Que´bec City, Quebec, Canada). All subjects with asthma were atopic (positive skin test for house dust) and were nonsmokers, and none used corticosteroids. The isolated epithelial cells (99% purity) were subcultured in vitro between P3 and P5 and characterized by immunofluorescence staining using panepithelial type II cytokeratins (clone AE3) and antifibroblast vimentin antibodies.7 Epithelial cells were processed for gene expression profiling by RT-PCR and quantitative real-time PCR.4 We detected significantly increased IL-33/glyceraldehyde-3-phosphate transcript ratios in cells from subjects with asthma compared with those from controls without asthma (Fig 2, A). Detection of IL33 mRNA was also confirmed in epithelial cells isolated from frozen bronchial tissue sections from normal subjects and subjects with asthma by laser-capture microdissection and RT-PCR (data not shown). These data confirm that bronchial epithelium of subjects with asthma expresses elevated levels of IL-33 compared with controls, which supports the concept that bronchial epithelial cells in asthma exhibit altered and proinflammatory phenotype compared with cells from healthy controls. We then sought to determine whether IL-33 is secreted in the lumen of asthmatic airways. Our research proposal was approved by the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherche en Sante´ du Que´bec, which enabled the use of bronchoalveolar lavage fluid (BALF) samples from agematched subjects with mild and moderate asthma on the basis of American Thoracic Society criteria, and from healthy nonatopic controls without asthma (n 5 10 per group). Subjects with asthma were all atopic, and displayed BAL eosinophilia: subjects with mild asthma had an FEV1 of 77.3 6 2.9 (3/10 on bronchodilators; none on steroids), whereas subjects with moderate asthma had an FEV1 of 86.0 6 1.6 (all on bronchodilators; 8/10 on

754 LETTERS TO THE EDITOR

inhaled steroids). By using a human IL-33 ELISA kit (CytoSet; Invitrogen, Carlsbad, Calif), we detected increased IL-33 levels in the BALF supernatants from subjects with moderate asthma (average of 120.8 6 22.4 pg/mL) compared with those with mild asthma (89.8 6 26.4 pg/mL) or controls without asthma (89.4 6 12.4 pg/mL; FEV1, 110.0 6 5.2), respectively (Fig 2, B). This suggests that bronchial epithelium and other airway resident cells express elevated levels of IL-33 in asthma1,4 and may also be subjected to conditions favoring its release. In asthma, several reports documented intense epithelial cell turnover caused by cell damage/death and aberrant repair leading to thickening of epithelium.8 Recent studies on IL-33–expressing endothelial cells reported significant release of IL-33 in supernatants of damaged and/or necrotic endothelial cells compared with supernatants from live cells.9 It is therefore tempting to speculate that increased epithelial damage caused by chronic inflammation of the asthmatic bronchi contributes to the release of IL-33. However, discrepancies observed between the epithelial IL-33 immunoreactivity in subjects with moderate versus mild asthma (Fig 1, E) and the increased IL33 levels in BALF from subjects with moderate versus mild asthma (Fig 2) suggest other cell types, like smooth muscle,4 may also be significant IL-33 sources in asthma. Aside from the literature describing IL-33 as an ‘‘alarmin’’-type cytokine with proallergic properties, there is a lack of studies regarding the sources of this cytokine and the regulation of its release in airway diseases. This study proposes that bronchial epithelium is an important IL-33 reservoir in the lung and that its expression is elevated in bronchial asthma. Histologic observations support the intracellular and nuclear localization of IL-33 in epithelial cells, whereas we also document the increasing release of this cytokine in the airway lumen along with asthma severity (moderate > mild asthma and controls). This study sheds light on the relevance of further using IL-33 BALF levels and studying epithelial cells’ IL-33 expression as potential biomarkers in moderate and severe asthma, respectively. We thank Elsa Schotman and Dr Ste´phane Lajoie-Kadoch for technical advice as well as Dr Ron Olivenstein, Dr Michel Laviolette, and Dr Catherine Lemie`re for their help with recruitment of the patients and collection of tissues. David Pre´fontaine, MSca Jessica Nadigel, BSca Fazila Chouiali, MSca Se´verine Audusseau, MSca Abdelhabib Semlali, PhDb Jamila Chakir, PhDb James G. Martin, MD, DSca Qutayba Hamid, MD, PhDa From athe Meakins-Christie Laboratories, Faculty of Medicine, McGill University, Montreal; and bCentre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Que´bec, Universite´ Laval, Que´bec City, Quebec, Canada. E-mail: [email protected]. Supported by the Richard and Edith Strauss Canada Foundation (Montreal, Quebec, Canada), the Canadian Institutes of Health Research (Ottawa, Ontario, Canada), and the J. T. Costello Memorial Research Fund (Montreal, Quebec, Canada). The MeakinsChristie Laboratories and the McGill University Health Center–Research Institute are supported in part by a Center Grant from Fonds de la Recherche en Sante´ du Que´bec. D.P. is supported by a doctoral studentship from the Fonds de la Recherche en Sante´ du Que´bec and by an American Academy of Allergy, Asthma and Immunology’s Strategic Training in Allergy Research Award. A.S. is the recipient of a GSK/Canadian Institutes of Health Research fellowship. Disclosure of potential conflict of interest: The authors have declared that they have no conflict

J ALLERGY CLIN IMMUNOL MARCH 2010

REFERENCES 1. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005;23:479-90. 2. Kearley J, Buckland KF, Mathie SA, Lloyd CM. Resolution of allergic inflammation and airway hyperreactivity is dependent upon disruption of the T1/ST2-IL-33 pathway. Am J Respir Crit Care Med 2009;179:772-81. 3. Hayakawa H, Hayakawa M, Kume A, Tominaga S. Soluble ST2 blocks interleukin33 signaling in allergic airway inflammation. J Biol Chem 2007;282:26369-80. 4. Pre´fontaine D, Lajoie-Kadoch S, Foley S, Audusseau S, Olivenstein R, Halayko AJ, et al. Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells. J Immunol 2009;183:5094-103. 5. Carrie`re V, Roussel L, Ortega N, Lacorre DA, Americh L, Aguilar L, et al. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc Natl Acad Sci U S A 2007;104:282-7. 6. Moussion C, Ortega N, Girard JP. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel ‘‘alarmin’’? PLoS One 2008;3:e3331. 7. Goulet F, Boulet LP, Chakir J, Tremblay N, Dube´ J, Laviolette M, et al. Morphologic and functional properties of bronchial cells isolated from normal and asthmatic subjects. Am J Respir Cell Mol Biol 1996;15:312-8. 8. Hackett TL, Knight DA. The role of epithelial injury and repair in the origins of asthma. Curr Opin Allergy Clin Immunol 2007;7:63-8. 9. Cayrol C, Girard JP. The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proc Natl Acad Sci U S A 2009;106:9021-6. Available online February 12, 2010. doi:10.1016/j.jaci.2009.12.935

Polymorphisms of chitinases are not associated with asthma To the Editor: One of the most exciting findings recently in the pathophysiology of asthma is that mammalian chitinases might play an important role in the pathogenesis of asthma. Some researchers hypothesize that mammalian chitinases and a chitinase homologue might contribute to the pathogenesis of type 2 helper immune responses, which play an important role in asthma.1,2 Chitinases are enzymes that cleave chitin, a polysaccharide that is present in fungal cells, crustaceans, insects, and parasitic nematodes.3 Although chitin does not exist in human subjects, 2 chitinases, acidic mammalian chitinase (CHIA) and chitotriosidase (CHIT1), have been described in human subjects.3 A third protein, chitinase-like protein YKL-40 (also known as human cartilage glycoprotein 39 and chitinase 3–like 1 [CHI3L1]), also appears to be important in asthma.1 The objectives of this study were to assess whether single nucleotide polymorphisms (SNPs) in CHIT1, CHIA, and CHI3L1 and one CHIT1 duplication are associated with asthma, changes in lung physiology that are associated with asthma, or allergyrelated phenotypes. We used data from the Childhood Asthma Management Program (CAMP), a multicenter trial that enrolled children between the ages of 5 and 12 years with mild-tomoderate persistent asthma and their parents.4 Subjects were followed every 2 to 4 months for 4 years to study the long-term use of budesonide, nedocromil, and placebo.4 SNPs in CHIT1, CHIA, and CHI3L1 (see Fig E1 in this article’s Online Repository at www.jacionline.org for linkage disequilibrium plots) were genotyped by using the Infinium HumanHap550 genotyping at Illumina (San Diego, Calif). Genotyping quality was evaluated by using the program PLINK (version 1.01). SNPs with low Illumina GenCall scores, poor completion rates, or 4 or more parent-offspring genotyped inconsistencies were dropped. Using the Basic Local Alignment Search Tool, SNPs