Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro

BACKGROUND: Mucus 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. METHODS: To 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)]. RESULTS: The 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. CONCLUSION: Given 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

Antimicrobial activity of innate immune molecules against <it>Streptococcus pneumoniae, Moraxella catarrhalis </it>and nontypeable <it>Haemophilus influenzae</it>

<p>Abstract</p> <p>Background</p> <p>Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and β defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable <it>Haemophilus influenzae </it>(NTHi) 12, <it>Moraxella catarrhalis </it>035E, and <it>Streptococcus pneumoniae </it>3, and 6B.</p> <p>Methods</p> <p>Two assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM) was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Student's <it>t</it>-test was performed.</p> <p>Results</p> <p>Results of the radial diffusion assay showed that β defensin-2 was active against all four OM pathogens tested, while treatment with β defensin-1 appeared to only affect <it>M. catarrhalis</it>. The radial assay results also showed that lysozyme was quite effective against <it>S. pneumoniae </it>3 and 6B and was partially bacteriostatic/bactericidal against <it>M. catarrhalis</it>. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive <it>S. pneumoniae </it>as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that lysozyme and β defensin-2 could act synergistically against <it>S. pneumoniae </it>6B. Moreover, in the liquid broth assay, β defensin-1 showed a modest inhibitory effect on the growth of <it>S. pneumoniae </it>6B. As assessed by ultrastructural analysis, lysozyme and β defensin-2, and to a much lesser extent, β defensin-1, appeared to be able to cause damage to the bacterial membranes.</p> <p>Conclusions</p> <p>Here we report that lysozyme and the β defensins can inhibit the growth of clinical isolates of otitis media pathogens – namely NTHi strain 12, <it>S. pneumoniae </it>strains 3 and 6B and <it>M. catarrhalis </it>strain 035E – and cause ultrastructural damage to these pathogens. Moreover, we demonstrate that lysozyme and β defensin-2 can act synergistically against <it>S. pneumoniae</it>. These findings are consistent with the concept that secreted antimicrobial peptides and other components of innate immunity constitute the first line of defense protecting host mucosal surfaces, including the tubotympanal (eustachian tube and middle ear cavity) mucosa, against pathogens.</p