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Increased IL-33 expression by epithelial cells in bronchial asthma

2010; Elsevier BV; Volume: 125; Issue: 3 Linguagem: Inglês

10.1016/j.jaci.2009.12.935

1097-6825

David Préfontaine, Jessica Nadigel, Fazila Chouiali, Séverine Audusseau, Abdelhabib Semlali, Jamila Chakir, James G. Martin, Qutayba Hamid,

Eosinophilic Esophagitis

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 membrane-bound ST2 protein.1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. 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: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2874) Google Scholar 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.1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. 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: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2874) Google Scholar The IL-33/ST2 pathway also contributes to allergen-induced airway inflammation and hyperresponsiveness,2Kearley J. Buckland K.F. Mathie S.A. Lloyd C.M. 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: 772-781Crossref PubMed Scopus (195) Google Scholar 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.2Kearley J. Buckland K.F. Mathie S.A. Lloyd C.M. 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: 772-781Crossref PubMed Scopus (195) Google Scholar, 3Hayakawa H. Hayakawa M. Kume A. Tominaga S. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation.J Biol Chem. 2007; 282: 26369-26380Crossref PubMed Scopus (438) Google Scholar Elevated IL-33 mRNA levels were measured in the lung tissue from subjects with asthma compared with healthy controls.4Préfontaine D. Lajoie-Kadoch S. Foley S. Audusseau S. Olivenstein R. Halayko A.J. et al.Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells.J Immunol. 2009; 183: 5094-5103Crossref PubMed Scopus (442) Google Scholar Together, these data support a role for IL-33 in the pathogenesis of allergic asthma. Although studies reported that structural cells, including endothelial cells,4Préfontaine D. Lajoie-Kadoch S. Foley S. Audusseau S. Olivenstein R. Halayko A.J. et al.Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells.J Immunol. 2009; 183: 5094-5103Crossref PubMed Scopus (442) Google Scholar, 5Carrière V. Roussel L. Ortega N. Lacorre D.A. 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: 282-287Crossref PubMed Scopus (791) Google Scholar fibroblasts, smooth muscle cells, and epithelial cells,6Moussion C. Ortega N. Girard J.P. 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)Crossref PubMed Scopus (911) Google Scholar express IL-33 in various organs,1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. 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: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2874) Google Scholar 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 Sacré-Coeur Hospital based on the criteria of the American Thoracic Society Workshop on Refractory Asthma, and patients' characteristics were reported previously.4Préfontaine D. Lajoie-Kadoch S. Foley S. Audusseau S. Olivenstein R. Halayko A.J. et al.Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells.J Immunol. 2009; 183: 5094-5103Crossref PubMed Scopus (442) Google Scholar 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) and subjects with asthma (n = 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 IgG1κ [Nessy-1]; Alexis Biochemicals, Axxora LCC, San Diego, Calif) and a peroxidase-based detection method.5Carrière V. Roussel L. Ortega N. Lacorre D.A. 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: 282-287Crossref PubMed Scopus (791) Google Scholar As reported in other organs,6Moussion C. Ortega N. Girard J.P. 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)Crossref PubMed Scopus (911) Google Scholar 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 4°C (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; mean age, 24 years; FEV1, 96.3 ± 6.3%; PC20, 3.6 ± 5.3 mg/mL) and healthy subjects without asthma (n = 4; mean age, 23.2 years; FEV1, 105.6 ± 4.3%; PC20, 115.2 ± 28.6 mg/mL) (mean ± SD). Subjects were recruited from the Institut Universitaire de Cardiologie et de Pneumologie de Québec (Québec 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.7Goulet F. Boulet L.P. Chakir J. Tremblay N. Dubé 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: 312-318Crossref PubMed Scopus (46) Google Scholar Epithelial cells were processed for gene expression profiling by RT-PCR and quantitative real-time PCR.4Préfontaine D. Lajoie-Kadoch S. Foley S. Audusseau S. Olivenstein R. Halayko A.J. et al.Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells.J Immunol. 2009; 183: 5094-5103Crossref PubMed Scopus (442) Google Scholar 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 IL-33 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 Santé du Québec, which enabled the use of bronchoalveolar lavage fluid (BALF) samples from age-matched subjects with mild and moderate asthma on the basis of American Thoracic Society criteria, and from healthy nonatopic controls without asthma (n = 10 per group). Subjects with asthma were all atopic, and displayed BAL eosinophilia: subjects with mild asthma had an FEV1 of 77.3 ± 2.9 (3/10 on bronchodilators; none on steroids), whereas subjects with moderate asthma had an FEV1 of 86.0 ± 1.6 (all on bronchodilators; 8/10 on 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 ± 22.4 pg/mL) compared with those with mild asthma (89.8 ± 26.4 pg/mL) or controls without asthma (89.4 ± 12.4 pg/mL; FEV1, 110.0 ± 5.2), respectively (Fig 2, B). This suggests that bronchial epithelium and other airway resident cells express elevated levels of IL-33 in asthma1Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. 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: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2874) Google Scholar, 4Préfontaine D. Lajoie-Kadoch S. Foley S. Audusseau S. Olivenstein R. Halayko A.J. et al.Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells.J Immunol. 2009; 183: 5094-5103Crossref PubMed Scopus (442) Google Scholar 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.8Hackett T.L. Knight D.A. The role of epithelial injury and repair in the origins of asthma.Curr Opin Allergy Clin Immunol. 2007; 7: 63-68Crossref PubMed Scopus (83) Google Scholar 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.9Cayrol C. Girard J.P. The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1.Proc Natl Acad Sci U S A. 2009; 106: 9021-9026Crossref PubMed Scopus (548) Google Scholar 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 IL-33 levels in BALF from subjects with moderate versus mild asthma (Fig 2) suggest other cell types, like smooth muscle,4Préfontaine D. Lajoie-Kadoch S. Foley S. Audusseau S. Olivenstein R. Halayko A.J. et al.Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells.J Immunol. 2009; 183: 5094-5103Crossref PubMed Scopus (442) Google Scholar 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 Stéphane Lajoie-Kadoch for technical advice as well as Dr Ron Olivenstein, Dr Michel Laviolette, and Dr Catherine Lemière for their help with recruitment of the patients and collection of tissues.