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Lipocalin2 (lcn2) is Upregulated and May Serve as a Novel Protective Factor in Allergic Airway Disease
S182 Abstracts
Ribonomic Analysis of Airway Epithelial Cell Gene Expression: Identification of Chemokine Transcripts as Targets of the RNA-Binding Protein HuR C. Stellato1, S. L. Curry1, U. X. Atasoy2, M. Gorospe3, J. Fan1; 1Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD, 2University of Missouri-Columbia, Columbia, MO, 3National Institute of Aging, NIH, Baltimore, MD. RATIONALE: We previously identified CCL11/eotaxin as a target of the RNA-binding protein HuR, which associates with adenylate-uridylaterich elements (ARE) in the 3’-untranslated regions (UTR) of mRNAs, promoting mRNA stability and translation of key immune genes. The present array study aims at identifying HuR-regulated genes in resting and cytokine-challenged human airway epithelial cells, which are important players in the pathophysiology of allergic inflammation. METHODS: Following culture of the human bronchial epithelial cell line BEAS-2B with medium or with TNF in combination with IFN or IL-4, cytoplasmic lysates were prepared using procedures that preserve mRNA-protein complexes. HuR-bound target mRNAs were then isolated by immunoprecipitation using specific anti-HuR antibodies and the associated mRNAs eluted, reverse-transcribed, and hybridized to two different array platforms. HuR targets were validated by biotin pull-down assay in which protein extracts were incubated with biotin-labeled RNA probes spanning the 3’UTRs or coding regions of selected targets, followed by HuR Western Blot analysis. RESULTS: A cluster of chemokines including CCL2, CCL8, CCL13, CXCL1, and CXCL2 were among the 15 genes (4% of total gene number) identified on both arrays and validated as specific HuR targets; all of them contained a predicted AU-rich HuR motif. CONCLUSIONS: HuR may regulate the coordinate expression of subsets of epithelial ARE-bearing genes that are functionally related by their participation in the inflammatory cascade. Identification of these genes will reveal the impact of HuR as mediator of inflammation, and shed new light on a novel inflammatory pathway in the posttranscriptional regulation of chemokine expression. Funding: AAAAI/Aventis 2004 Women Physicians in Allergy Junior Faculty Development Award; NIH
707
SUNDAY
Lipocalin2 (lcn2) is Upregulated and May Serve as a Novel Protective Factor in Allergic Airway Disease A. M. Dittrich, D. Quarcoo, M. Krokowski, A. Awagjan, B. Ahrens, E. H. Hamelmann; Pediatric Pneumology and Immunology, Charité University Medicine, Berlin, GERMANY. RATIONALE: Allergen-induced bronchial asthma is a chronic airway disease that involves the interplay of various genes with environmental factors triggering numerous inflammatory pathways. We sought to identify novel common inflammatory mediators playing an important role in the pathogenesis of allergic airway disease independently of atopic susceptibility. METHODS: In a mouse model of allergic airway inflammation whole lung RNA expression was compared between mice with increased allergic receptiveness, (BALB/c) and mice with lower allergic susceptibility (C57/Bl6) by using microarray based RNA expression analysis. Results were confirmed by western blot analysis of mouse bronchoalveolar lavage fluid. The location of the target proteins was determined by immune-histological staining (IHC) of murine airway tissues. Functional relevance of the target was assessed using a blocking antibody directed against the LCN2. RESULTS: The expression of LCN2, a protein with yet an undefined role in the pathogenesis of allergic airway inflammation, was significantly increased in both strains after allergen sensitization and airway provocation on the RNA and protein level. LCN2 expression in lung tissue was localized to epithelial cells by IHC. Intranasal application of a blocking antibody against LCN2 resulted in increased eosinphilic airway inflammation, suggesting a protective role for LCN2 in allergic airway disease. CONCLUSIONS: These data suggest that LCN2 is a new mediator in allergic airway disease with a potential protective role. Due to its known
708
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
role in mediating LPS-induced effects on immune cells, LCN2 possibly constitutes another molecule linking innate and adaptive immune responses in allergic airway disease. Funding: National Genomforschungsnetzwerk (NGFN) Role of Resistin-Like Molecule (RELM)- in Allergic Airway Inflammation A. Haczku1, S. Ying2, S. Kierstein1, Q. Meng2, L. D. McVay1, C. J. Corrigan2, G. D. Wu1; 1Medicine, University of PA School of Medicine, Philadelphia, PA, 2Guy’s, King’s & St. Thomas’ School of Medicine, London, UNITED KINGDOM. RATIONALE: RELM-, a novel protein with both cytokine and hormone-like activities is produced under Th2-type immune regulation in the gut in mice. We hypothesized that this molecule is also involved in the pathogenesis of the allergic airway response. METHODS: In situ hybridization and immunocytochemistry was performed on endobronchial biopsies from asthmatic patients and normal controls. Human airway epithelial cells (A549) were cultured in the presence and absence of IL-13. Mice deficient in RELM- and their wild-type littermates were sensitized and challenged with Aspergillus fumigatus (Af). Lung function, inflammatory cell and cytokine profile, expression of RELM- protein and mRNA was investigated 12, 24 and 48h later. RESULTS: RELM- mRNA and protein expression in the airway epithelium of asthmatic patients was markedly elevated. IL-13 treatment of A549 cells also induced RELM- production. Af provocation of sensitized mice stimulated mRNA activation and release of IL-4, IL-5 and IL-13 (but not IFN-) within 24h, and release of RELM- 48h later. In addition to a massive influx of neutrophils and eosinophils after challenge, sensitized wild-type mice had a significant airway hyperresponsiveness to methacholine. Interestingly, in spite of the presence of airway inflammation, RELM- deficient mice were impaired in their methacholine responsiveness after allergen challenge. CONCLUSIONS: RELM- is increased in the airway epithelium of asthmatic patients and in mice sensitized and challenged with Af. Since there was diminished methacholine responsiveness but no reduction in inflammation in RELM- deficient mice, this protein may play a direct role in altered airway smooth muscle function. Funding: R01 AI055593 (AH), ALA RG144N (AH), R01 AI39368 (GDW)
709
Aspirine Sensitive Rhinosinusitis is Associated with Reduced E-prostanoid 2(EP2) Receptor Expression on Nasal Mucosal Inflammatory Cells S. Ying1, Q. Meng1, G. Scadding2, A. Parikh2, C. J. Corrigan1, T. H. Lee1; 1Asthma, Allergy & Respiratory Science, GKS Medical School, London, UNITED KINGDOM, 2Royal National Throat, Nose and Ear Hospital, London, UNITED KINGDOM. RATIONALE: Impaired “braking” of inflammatory cell cysteinyl leukotriene production by PGE2 has been implicated in the pathogenesis of aspirin exacerbated airways disease, but the mechanism is obscure. PGE2 acts via G-protein-coupled receptors, EP1-4, but there is little information on the expression of PGE2 receptors in this condition. To address the hypothesis that expression of one or more EP receptors on nasal mucosal inflammatory cells is deficient in patients with aspirin sensitive (AS), as compared with non-aspirin sensitive (NAS) polypoid rhinosinusitis. METHODS: Using specific antibodies, immunohistochemistry and image analysis, we measured, the expression of EP1-4 in sections of nasal biopsies from patients with AS (n=12) and NAS (n=10) polypoid rhinosinusitis and normal controls (n=9). Double staining was employed to phenotype inflammatory leukocytes expressing EP1 and EP2. RESULTS: Global mucosal expression of EP1 and EP2, but not EP3 or EP4 immunoreactivity was significantly elevated in AS and NAS rhinosinusitis as compared with controls (p<0.03). This was attributable principally to elevated epithelial expression. In contrast, the percentages of neutrophils, mast cells, eosinophils and T cells expressing EP2, but not
710
Ribonomic Analysis of Airway Epithelial Cell Gene Expression: Identification of Chemokine Transcripts as Targets of the RNA-Binding Protein HuR C. Stellato1, S. L. Curry1, U. X. Atasoy2, M. Gorospe3, J. Fan1; 1Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD, 2University of Missouri-Columbia, Columbia, MO, 3National Institute of Aging, NIH, Baltimore, MD. RATIONALE: We previously identified CCL11/eotaxin as a target of the RNA-binding protein HuR, which associates with adenylate-uridylaterich elements (ARE) in the 3’-untranslated regions (UTR) of mRNAs, promoting mRNA stability and translation of key immune genes. The present array study aims at identifying HuR-regulated genes in resting and cytokine-challenged human airway epithelial cells, which are important players in the pathophysiology of allergic inflammation. METHODS: Following culture of the human bronchial epithelial cell line BEAS-2B with medium or with TNF in combination with IFN or IL-4, cytoplasmic lysates were prepared using procedures that preserve mRNA-protein complexes. HuR-bound target mRNAs were then isolated by immunoprecipitation using specific anti-HuR antibodies and the associated mRNAs eluted, reverse-transcribed, and hybridized to two different array platforms. HuR targets were validated by biotin pull-down assay in which protein extracts were incubated with biotin-labeled RNA probes spanning the 3’UTRs or coding regions of selected targets, followed by HuR Western Blot analysis. RESULTS: A cluster of chemokines including CCL2, CCL8, CCL13, CXCL1, and CXCL2 were among the 15 genes (4% of total gene number) identified on both arrays and validated as specific HuR targets; all of them contained a predicted AU-rich HuR motif. CONCLUSIONS: HuR may regulate the coordinate expression of subsets of epithelial ARE-bearing genes that are functionally related by their participation in the inflammatory cascade. Identification of these genes will reveal the impact of HuR as mediator of inflammation, and shed new light on a novel inflammatory pathway in the posttranscriptional regulation of chemokine expression. Funding: AAAAI/Aventis 2004 Women Physicians in Allergy Junior Faculty Development Award; NIH
707
SUNDAY
Lipocalin2 (lcn2) is Upregulated and May Serve as a Novel Protective Factor in Allergic Airway Disease A. M. Dittrich, D. Quarcoo, M. Krokowski, A. Awagjan, B. Ahrens, E. H. Hamelmann; Pediatric Pneumology and Immunology, Charité University Medicine, Berlin, GERMANY. RATIONALE: Allergen-induced bronchial asthma is a chronic airway disease that involves the interplay of various genes with environmental factors triggering numerous inflammatory pathways. We sought to identify novel common inflammatory mediators playing an important role in the pathogenesis of allergic airway disease independently of atopic susceptibility. METHODS: In a mouse model of allergic airway inflammation whole lung RNA expression was compared between mice with increased allergic receptiveness, (BALB/c) and mice with lower allergic susceptibility (C57/Bl6) by using microarray based RNA expression analysis. Results were confirmed by western blot analysis of mouse bronchoalveolar lavage fluid. The location of the target proteins was determined by immune-histological staining (IHC) of murine airway tissues. Functional relevance of the target was assessed using a blocking antibody directed against the LCN2. RESULTS: The expression of LCN2, a protein with yet an undefined role in the pathogenesis of allergic airway inflammation, was significantly increased in both strains after allergen sensitization and airway provocation on the RNA and protein level. LCN2 expression in lung tissue was localized to epithelial cells by IHC. Intranasal application of a blocking antibody against LCN2 resulted in increased eosinphilic airway inflammation, suggesting a protective role for LCN2 in allergic airway disease. CONCLUSIONS: These data suggest that LCN2 is a new mediator in allergic airway disease with a potential protective role. Due to its known
708
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
role in mediating LPS-induced effects on immune cells, LCN2 possibly constitutes another molecule linking innate and adaptive immune responses in allergic airway disease. Funding: National Genomforschungsnetzwerk (NGFN) Role of Resistin-Like Molecule (RELM)- in Allergic Airway Inflammation A. Haczku1, S. Ying2, S. Kierstein1, Q. Meng2, L. D. McVay1, C. J. Corrigan2, G. D. Wu1; 1Medicine, University of PA School of Medicine, Philadelphia, PA, 2Guy’s, King’s & St. Thomas’ School of Medicine, London, UNITED KINGDOM. RATIONALE: RELM-, a novel protein with both cytokine and hormone-like activities is produced under Th2-type immune regulation in the gut in mice. We hypothesized that this molecule is also involved in the pathogenesis of the allergic airway response. METHODS: In situ hybridization and immunocytochemistry was performed on endobronchial biopsies from asthmatic patients and normal controls. Human airway epithelial cells (A549) were cultured in the presence and absence of IL-13. Mice deficient in RELM- and their wild-type littermates were sensitized and challenged with Aspergillus fumigatus (Af). Lung function, inflammatory cell and cytokine profile, expression of RELM- protein and mRNA was investigated 12, 24 and 48h later. RESULTS: RELM- mRNA and protein expression in the airway epithelium of asthmatic patients was markedly elevated. IL-13 treatment of A549 cells also induced RELM- production. Af provocation of sensitized mice stimulated mRNA activation and release of IL-4, IL-5 and IL-13 (but not IFN-) within 24h, and release of RELM- 48h later. In addition to a massive influx of neutrophils and eosinophils after challenge, sensitized wild-type mice had a significant airway hyperresponsiveness to methacholine. Interestingly, in spite of the presence of airway inflammation, RELM- deficient mice were impaired in their methacholine responsiveness after allergen challenge. CONCLUSIONS: RELM- is increased in the airway epithelium of asthmatic patients and in mice sensitized and challenged with Af. Since there was diminished methacholine responsiveness but no reduction in inflammation in RELM- deficient mice, this protein may play a direct role in altered airway smooth muscle function. Funding: R01 AI055593 (AH), ALA RG144N (AH), R01 AI39368 (GDW)
709
Aspirine Sensitive Rhinosinusitis is Associated with Reduced E-prostanoid 2(EP2) Receptor Expression on Nasal Mucosal Inflammatory Cells S. Ying1, Q. Meng1, G. Scadding2, A. Parikh2, C. J. Corrigan1, T. H. Lee1; 1Asthma, Allergy & Respiratory Science, GKS Medical School, London, UNITED KINGDOM, 2Royal National Throat, Nose and Ear Hospital, London, UNITED KINGDOM. RATIONALE: Impaired “braking” of inflammatory cell cysteinyl leukotriene production by PGE2 has been implicated in the pathogenesis of aspirin exacerbated airways disease, but the mechanism is obscure. PGE2 acts via G-protein-coupled receptors, EP1-4, but there is little information on the expression of PGE2 receptors in this condition. To address the hypothesis that expression of one or more EP receptors on nasal mucosal inflammatory cells is deficient in patients with aspirin sensitive (AS), as compared with non-aspirin sensitive (NAS) polypoid rhinosinusitis. METHODS: Using specific antibodies, immunohistochemistry and image analysis, we measured, the expression of EP1-4 in sections of nasal biopsies from patients with AS (n=12) and NAS (n=10) polypoid rhinosinusitis and normal controls (n=9). Double staining was employed to phenotype inflammatory leukocytes expressing EP1 and EP2. RESULTS: Global mucosal expression of EP1 and EP2, but not EP3 or EP4 immunoreactivity was significantly elevated in AS and NAS rhinosinusitis as compared with controls (p<0.03). This was attributable principally to elevated epithelial expression. In contrast, the percentages of neutrophils, mast cells, eosinophils and T cells expressing EP2, but not
710