ABSTRACT
Airways function as an innate immune organ against airborne bacteria that are inhaled and deposited in airways. One of the mechanisms of host defense is to recruit neutrophils into airways to clear the invaders. Airway epithelial cells produce neutrophil chemoattractant interleukin (IL)-8 in response to invading bacteria. In this study we show a signaling pathway on the plasma surface of human airway epithelial NCI-H292 cells that regulate IL-8 production in response to a model inflammatory stimulus, phorbol 12-myristate 13-acetate, and a pathophysiological stimulus, gram-negative bacterial lipopolysaccharide. First, we show that EGF receptor (EGFR) and MAP kinase ERK1/2 are involved in IL-8 expression by these stimuli. Second, we show that EGFR ligand transforming growth factor (TGF)-alpha mediates IL-8 production. Third, we show that tumor necrosis factor-alpha-converting enzyme (TACE) is required for IL-8 production by cleaving EGFR proligand proTGF-alpha into soluble TGF-alpha, activating EGFR. Last, we show that dual oxidase 1 (Duox1), a homolog of NADPH oxidase in airways, mediates TACE activation and IL-8 expression via generation of reactive oxygen species. In summary, we describe a signaling pathway, Duox1-TACE-TGF-alpha-EGFR, on the surface of airway epithelial (NCI-H292) cells that mediates airway epithelial defense against bacterial infection by producing IL-8. This pathway, which also regulates mucin production in human airways, provides mechanisms for killing foreign organisms and for their clearance.
Subject(s)
Interleukin-8/genetics , Respiratory Mucosa/physiology , Signal Transduction/physiology , Carcinoma, Squamous Cell , Cell Line, Tumor , DNA Primers , ErbB Receptors/antagonists & inhibitors , Gene Expression Regulation , Humans , Lung Neoplasms , Phosphorylation , RNA, Small Interfering/genetics , TransfectionABSTRACT
Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the U.S. Because cigarette smoking is so importantly implicated in the pathogenesis of COPD and because mucus hypersecretion plays such an important role in COPD, understanding of the mechanisms of smoking-induced mucus hypersecretion could lead to new therapies for COPD. Cigarette smoke causes mucin overproduction via EGF receptor (EGFR) in airway epithelial cells, but the cellular mechanism remains unknown. Airway epithelial cells contain EGFR proligands on their surfaces, which can be cleaved by metalloprotease and subsequently bind to EGFR resulting in mucin production. We hypothesize that TNF-alpha-converting enzyme (TACE) is activated by cigarette smoke, resulting in increased shedding of EGFR proligand, leading to EGFR phosphorylation and mucin induction in human airway epithelial (NCI-H292) cells. Here we show that cigarette smoke increases MUC5AC production in NCI-H292 cells, an effect that is prevented by an EGFR-neutralizing antibody and by specific knockdown of transforming growth factor-alpha (TGF-alpha) using small interfering RNA (siRNA) for TGF-alpha, implicating TGF-alpha-dependent EGFR activation in the responses. Cigarette smoke increases TGF-alpha shedding, EGFR phosphorylation, and mucin production, which are prevented by metalloprotease inhibitors (GM-6001 and TNF-alpha protease inhibitor-1) and by specific knockdown of TACE with TACE siRNA, implicating TACE in smoking-induced responses. Furthermore, pretreatment with antioxidants prevents smoking-induced TGF-alpha shedding and mucin production, suggesting that reactive oxygen species is involved in TACE activation. These results implicate TACE in smoking-induced mucin overproduction via the TACE-proligand-EGFR signal pathway in NCI-H292 cells.
Subject(s)
Metalloendopeptidases/metabolism , Mucins/genetics , Respiratory Mucosa/physiology , Smoking/adverse effects , ADAM Proteins , ADAM17 Protein , Carcinoma, Mucoepidermoid , Cell Line, Tumor , ErbB Receptors/metabolism , Gene Expression , Humans , Ligands , Lung Neoplasms , Mucin 5AC , Phosphorylation , Protein Precursors/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Reactive Oxygen Species/metabolism , Respiratory Mucosa/cytology , Transforming Growth Factor alpha/metabolism , Tumor Necrosis Factor-alpha/metabolism , Up-RegulationABSTRACT
OBJECTIVE: To determine the mechanism of mucus production by nasal glands. STUDY DESIGN: Because neutrophilic inflammation is associated with mucus hypersecretion in disease states, here we examine the role of neutrophil recruitment in mucous cell degranulation and regranulation in rat nasal glands. METHODS: N-formyl-methionyl-leucyl-phenylalanine (fMLP) was aerosolized intranasally in rats (n = 5), and its effects on degranulation and regranulation of submucosal glands were evaluated by Alcian blue/periodic acid-Schiff (AB/PAS) staining and by immunolocalization of neutrophils and epidermal growth factor receptor (EGF-R). RESULTS: In control subjects, glands were filled with mucin. After fMLP inhalation, degranulation, 31.7 +/- 0.8% (P <.01), was maximal at 2 to 4 hours. By 24 to 48 hours after fMLP inhalation, degranulation had decreased to 10.3 +/- 0.6% (P <.05), indicating that regranulation of mucous glycoconjugates was occurring. After fMLP inhalation, neutrophils around submucosal glands increased within 0.5 hours from 1.4 +/- 0.1 to 9.5 +/- 0.3 per 0.0032 mm2 (P <.05). In control subjects, EGF-R protein was expressed near acinar ducts, 16.4 +/- 0.7% of gland area, and increased to 30.9 +/- 0.9% (P <.05) 24 to 48 hours after fMLP inhalation. Nasal pretreatment with a selective EGF-R tyrosine kinase inhibitor (BIBX1522, 15 mg/kg bid) prevented regranulation at 24 hours after fMLP inhalation (degranulation 27.8 +/- 0.3%, P <.05, compared to 24 hours after fMLP alone), indicating that inhibition of EGF-R activation had prevented regranulation after fMLP inhalation. CONCLUSIONS: Degranulation of rat nasal glands by fMLP is followed by regranulation; regranulation depends on a neutrophil-associated EGF-R cascade.
