Phospho- and nonphospho-specific ERK1/2 and anti-rabbit-IgG HRP antibody were purchased from Cell Signaling Technology, Inc (Beverly, MA). goblet cells compared with normally differentiated cells, and neutralization with anti-ST2R antibody attenuated IL-33-induced apical CXCL8/IL-8 secretion from goblet cells (p 0.02). Conclusions and Clinical Relevance Goblet cells secrete CXCL8/IL-8 and this is increased Timonacic by IL-33 through ST2R-ERK pathway suggesting a mechanism for enhanced airway inflammation in the asthmatic airway with goblet cell metaplasia. and [2, 8C13]. IL-33 is usually a member of the IL-1 family that is a ligand for the orphan IL-1 family receptor ST2, and an inducer of Th2 immunity. IL-33 signals through a complex including the membrane bound ST2 (ST2L) protein . ST2 is present on Th2 cells as well as mast cells, basophils, eosinophils, natural killer T cells , and bronchial epithelial cells . The IL-33/ST2 axis triggers the release of proinflammatory chemokines and cytokines, and stimulates Th2 inflammation . The IL-33/ST2 pathway also contributes to allergen-induced airway inflammation and hyperresponsiveness . A likely source of IL-33 is the airway epithelium. IL-33 expression is increased in biopsies from your airways of subjects with asthma compared to healthy individuals and is greater still in subjects with severe asthma . IL-33 expression is usually refractory to the effects of corticosteroids . These data support a role for IL-33 in the pathogenesis of severe asthma. Neutrophils are found in the airways of subjects with severe asthma and the extent of neutrophilia is related to the disease severity . IL-8, now known as cysteine-X-cysteine chemokine 8 (CXCL8), is an important chemoattractant for neutrophil recruitment into airways of patients with severe asthma [20C22]. Normal human bronchial epithelial (NHBE) cells treated with IL-13 over 7 days have increased CXCL8/IL-8 secretion  and IL-13 exposure increases CXCL8/IL-8 in airway epithelial cells from healthy subjects and asthmatics . IL-33 uncovered mice have hypertrophy of the airway epithelium with large amounts of mucus and inflammatory infiltrates of neutrophils, mononuclear cells, and eosinophils in lung tissue and bronchoalveolar lavage fluid [14, 25]. We speculated that IL-33 would stimulate cultured NHBE cells toward goblet cell differentiation and stimulate CXCL8/IL-8 production in these goblet HBEGF cells by activating the ST2 receptor (ST2R) pathway. Materials and Methods Reagents Recombinant human IL-13 (rhIL-13), human ST2/IL-1 R4 antibody as well as anti-goat-IgG horseradish peroxidase (HRP) antibody were obtained from R&D Systems Inc. (Minneapolis, MN). Recombinant human IL-33 (rhIL-33) was obtained from Pepro Tech Inc. (Rocky Hill, NJ). Anti MUC5AC monoclonal antibody (45M1) was obtained from Lab Vision (Fremont, CA). PD98059 (2-amino-3-methoxyfl avone), an MAPK/ERK kinase (MEK) inhibitor (an upstream kinase of ERK1/2) was obtained from Calbiochem (La Jolla, CA). Phospho- and nonphospho-specific ERK1/2 and anti-rabbit-IgG HRP antibody were purchased from Cell Signaling Technology, Inc (Beverly, MA). Demethylsulfoxide (DMSO), anti–actin, anti-mouse-IgG-HRP antibody and all other reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO) unless normally indicated. Culture and differentiation Timonacic of NHBE cells NHBE cells (Clonetics? Lonza, Basel, Switzerland) were plated at 3,500 cells/cm2 in small airway epithelial cell growth medium (SAGM, Clonetcs? Timonacic Lonza) and cultured at 37 C in a 5% CO2 incubator. Second passage NHBE cells were seeded at a density of 2.0105/cm2 onto polyester inserts (6.5-mm diameter, 0.4-m pore size and 10-m thickness; Costar Transwell? Clear; Costar, Cambridge, MA) coated with type I rat tail collagen (Sigma, St. Louis, MO), and then cultured in serum-free DMEM/F12 medium made up of Insulin-Transferrin-Selenium-Sodium Pyruvate (ITS-A) (1.0%; Invitrogen, Carlsbad, CA), triiodothyronine (10 ng/ml; ICN, Irvine, CA), epidermal growth factor (recombinant human EGF, 0.5 ng/mL; Invitrogen), all-trans retinoic acid (all-trans RA, 10?7 test. Multiple comparisons were made by one-way analysis variance (ANOVA) except for ST2 mRNA expression over time, which was analyzed by two-way repeated measure ANOVA. A analysis for multiplicity was performed by using the Bonferronis method. Conventionally, p 0.05 was considered significant. Results IL-13-induced goblet cell differentiation NHBE cells cultured at ALI exhibited a well-differentiated morphology with ciliated cells at the surface of epithelial layers (referred to normally differentiated cells for convenience) (Figs. 1ACC, 2A, B, E, F, I, J, M, N, R, S) and weakly stained with PAS (Figs. 1B,.