Abstract
BackgroundMucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Expression of two mucin genes, MUC2 and MUC5AC, and their protein products (mucins), is modulated in certain disease states. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways.MethodsTo study the differential expression of Muc2 and Muc5ac, a novel monoclonal antibody recognizing guinea pig Muc2 and a commercially-available antibody against human MUC5AC were optimized for recognition of specific guinea pig mucins by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). These antibodies were then used to analyze expression of Muc2 and another mucin subtype (likely Muc5ac) in guinea pig tracheal epithelial (GPTE) cells stimulated with a mixture of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interferon- γ (IFN-γ)].ResultsThe anti-Muc2 (C4) and anti-MUC5AC (45M1) monoclonal antibodies specifically recognized proteins located in Muc2-dominant small intestinal and Muc5ac-dominant stomach mucosae, respectively, in both Western and ELISA experimental protocols. IHC protocols confirmed that C4 recognizes murine small intestine mucosal proteins while 45M1 does not react. C4 and 45M1 also stained specific epithelial cells in guinea pig lung sections. In the resting state, Muc2 was recognized as a highly expressed intracellular mucin in GPTE cells in vitro. Following cytokine exposure, secretion of Muc2, but not the mucin recognized by the 45M1 antibody (likely Muc5ac), was increased from the GPTE cells, with a concomitant increase in intracellular expression of both mucins.ConclusionGiven the tissue specificity in IHC and the differential hybridization to high molecular weight proteins by Western blot, we conclude that the antibodies used in this study can recognize specific mucin subtypes in guinea pig airway epithelium and in proteins from GPTE cells. In addition, Muc2 is highly expressed constitutively, modulated by inflammation, and secreted differentially (as compared to Muc5ac) in GPTE cells. This finding contrasts with expression patterns in the airway epithelium of a variety of mammalian species in which only Muc5ac predominates.
Highlights
Mucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis
C4 monoclonal antibody recognizes Muc2 To verify that the novel monoclonal antibody (C4) developed in this study would recognize guinea pig Muc2 apomucin, a dilution series of purified Muc2 protein fragment was examined by enzyme-linked immunosorbent assay (ELISA)
It may be that the early increase in Muc2 mRNA is a secondary consequence of the cytomix-induced preferential secretion of stored Muc2, rather than a direct induction of Muc2 transcription by the cytokines. These results suggest that exposure to pro-inflammatory cytokines can induce an increase in Muc2 gene expression in guinea pig tracheal epithelial (GPTE) cells, with some increase in Muc5ac mRNA possible with extended exposure
Summary
Mucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways. MUC2 and MUC5AC expression are altered in inflamed airways [10,11,12,13] and, may contribute to the pathogenesis of several respiratory diseases. These mucins exhibit cell- and tissue-specific expression in mammals where, in addition to their airway expression, Muc is expressed primarily in gastrointestinal epithelium and Muc5ac in gastric epithelium [14,15]. Differential regulation of mucin subtype expression may affect mucus composition in disease states, little is known regarding mechanisms that modulate such expression [16,17,18,19,20]
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