Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor ␣ (TGF␣), causes MUC5AC expression in airway epithelial cells both in in vitro and in vivo. We found that a MUC5AC-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively; EGF-R gene expression was stimulated further by tumor necrosis factor ␣ (TNF␣). EGF-R ligands increased the expression of MUC5AC at both gene and protein levels, and this effect was potentiated by TNF␣. Selective EGF-R tyrosine kinase inhibitors blocked MUC5AC expression induced by EGF-R ligands. Pathogenfree rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNF␣ induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blueperiodic acid-Schiff staining (ref lecting mucous glycoconjugates), and MUC5AC gene expression, whereas TNF␣, EGF, or TGF␣ alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNF␣-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways.

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-α increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-l-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-α) were without effect, and no TGF-α protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-α, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.

Ectodomain shedding of epidermal growth factor receptor (EGFR) ligands [e.g., transforming growth factor type ␣ (TGF-␣)] and EGFR phosphorylation are implicated in mucin production in airway epithelial cells. Tumor necrosis factor ␣-converting enzyme (TACE) is reported to cleave precursor of TGF-␣, with release of soluble

Sputum from patients with cystic fibrosis, bronchiectasis, and chronic bronchitis contains neutrophils and neutrophil proteases, which have been implicated in the pathophysiology of mucus hypersecretion in airways. We asked whether interleukin-8 (IL-8), a potent neutrophil chemoattractant, might be involved in recruiting neutrophils into airways of patients with cystic fibrosis, bronchiectasis, and chronic bronchitis. We found significant neutrophil chemotactic activity in sputum obtained from these patients. The IL-8 concentrations that we measured in sputum of patients with cystic fibrosis (7.1 +/- 1.0 x 10(-9) M, mean +/- SE), bronchiectasis (9.6 +/- 2.9 x 10(-9) M), and chronic bronchitis (2.8 +/- 1.0 x 10(-9) M) have been reported to cause significant chemotaxis in vitro and in airways in vivo, whereas concentrations measured in induced sputum from healthy subjects (1.1 +/- 0.3 x 10(-10) M) do not. A monoclonal antibody to IL-8 significantly inhibited the chemotactic activity in patients' sputum by 75-98%, but not in induced sputum from healthy subjects (9%). We conclude that IL-8 is an important chemotactic factor in sputum of patients with cystic fibrosis, bronchiectasis, and chronic bronchitis, and we suggest that IL-8 accounts, at least in part, for neutrophil recruitment into airways of patients with these diseases.

The increase in airway responsiveness induced by O3 exposure in dogs is associated with airway epithelial inflammation, as evidenced by an increase in the number of neutrophils (polymorphonuclear leukocytes) found in epithelial biopsies and in bronchoalveolar lavage fluid. We investigated in 10 healthy, human subjects whether O3-induced hyperresponsiveness was similarly associated with airway inflammation by examining changes in the types of cells recovered in bronchoalveolar lavage fluid obtained after exposure to air or to O3 (0.4 or 0.6 ppm). We also measured the concentrations of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in lavage fluid. We measured airway responsiveness to inhaled methacholine aerosol before and after each exposure and performed bronchoalveolar lavage 3 h later. We found more neutrophils in the lavage fluid from O3-exposed subjects, especially in those in whom O3 exposure produced an increase in airway responsiveness. We also found significant increases in the concentrations of prostaglandins E2, F2 alpha, and thromboxane B2 in lavage fluid from O3-exposed subjects. These results show that in human subjects O3-induced hyperresponsiveness to methacholine is associated with an influx of neutrophils into the airways and with changes in the levels of some cyclooxygenase metabolites of arachidonic acid.