Differential Mucin Subtype Regulation and Anti-inflammatory Effects of Inducible Nitric Oxide Synthase in Stimulated Airway Epithelial Cells in Vitro

Abstract

This dissertation focuses on two critical functions mediated by the airway epithelium: mucus production and inflammatory mediation. Both processes are important for pulmonary defense, but may be deleterious to the surrounding airway if regulated inappropriately. In the first study, a novel monoclonal antibody developed against guinea pig Muc2 mucin and a commercially available anti-MUC5AC mucin antibody were characterized for use in guinea pig studies. Muc2 and Muc5AC mucin production was then measured in guinea pig tracheal epithelial (GPTE) cells stimulated with pro-inflammatory cytokines, TNF-α, IL-1β, and IFN-γ (cytomix). It was demonstrated that Muc2, but not Muc5AC, mucin secretion increased over constitutive production in cytomix-stimulated cells, but intracellular protein and mRNA increased similarly for both mucin subtypes. It was concluded that differential mechanisms for mucin subtype secretion are present in the guinea pig airway epithelium. This differential regulation of mucin subtype expression is an important finding, as it may affect the interactive properties of mucus in both normal and diseased airway.

In the second study, the anti-inflammatory role of inducible nitric oxide synthase (iNOS) was evaluated in normal human bronchial epithelial (NHBE) cells. Using iNOSspecific inhibitor, cGMP-dependent kinase inhibitor, and exogenous nitric oxide and cGMPanalogue application, it was demonstrated that an iNOS/cGMP/PKG-mediated pathway suppresses granulocyte macrophage colon stimulating factor (GM-CSF), but not interleukin-8, expression in cytomix-stimulated NHBE cells.

The third study demonstrated that an anti-inflammatory action of the β2-adrenergic agonist, (R)-albuterol, is dependent on iNOS. Through the use of small interfering RNA targeted against iNOS, it was demonstrated that (R)-albuterol, and not its inert enantiomer, (S)-albuterol, suppressed GM-CSF message and protein release in IL-1β/IFN-γ-stimulated NHBE cells by augmenting iNOS expression. In addition, through the use of various kinasespecific inhibitors and activators, it was demonstrated that (R)-albuterol-mediated iNOS augmentation requires protein kinase Cδ, but not cAMP or cGMP-dependent kinase, activity. Overall, this study identifies a novel pathway in which β2-adrenergic agonists may exhibit anti-inflammatory effects in the airway epithelium and surrounding milieu.