Mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells
Introduction
Rhinoviruses (RVs) are the major cause of the common cold and the most common acute infection illnesses in humans, and are also associated with acute exacerbations of bronchial asthma (Nicholson et al., 1993) and chronic obstructive pulmonary disease (COPD) (Sethi, 2004). Several mechanisms have been proposed, and the manifestations of RV-induced pathogenesis are suggested to be the result of virus-induced mediators of inflammation (Sethi, 2004, Zhu et al., 1996). However, it is still uncertain how RV infections cause exacerbations in patients with asthma and COPD.
Sputum production is a common symptom in exacerbations of asthma and COPD, and mucus hypersecretion plays a central role in the pathogenesis of severe airway obstruction in exacerbations of asthma and COPD (Huber and Koessler, 1922, Sethi, 2004). Influenza infection affects water absorption through the inhibition of the sodium channel function (Kunzelmann et al., 2000). RV infection also increases epithelial cell permeability, although RV infection does not affect the sodium and chloride channel function, and does not induce epithelial cell detachment from culture vessels (Terajima et al., 1997). Although experimental RV infection increased nasal lavage fluid concentrations of mucoglycoprotein exocytosis (Yuta et al., 1998), it is uncertain which kinds of mucins are secreted or produced after RV infection. Furthermore, the mechanisms of RV infection-induced mucin production are also uncertain.
Airway mucins are produced mainly by goblet cells and submucosal gland cells. To date, at least nine mucin genes including MUC1–MUC8 have been detected in epithelial cells including human airway epithelial cells, airway submucosal cells or lung epithelial cell lines (Buisine et al., 1999, Ordonez et al., 2001, Rose et al., 2001). MUC5AC is known as a main content of hypersecreted mucins (Ordonez et al., 2001). Increased expression of MUC5AC mRNA in bronchial surface epithelial cells was reported in mice after respiratory syncytial virus (RSV) (Miller et al., 2003), and RV infection induces MUC5AC production in the human airway surface epithelium (He et al., 2004). However, it is still uncertain which signaling pathways are utilized for mucin production.
We therefore examined the effects of RV infection on MUC5AC mucin production in cultured human tracheal surface epithelium (Terajima et al., 1997) and submucosal glands cells (Yamaya et al., 1999). We also examined the effects of RV infection on the production of total mucin by an enzyme-linked immunosorbent assays (ELISA) using a pan-mucin antibody, 17Q2, that cross reacts with a carbohydrate epitope on human mucins (Lin et al., 1989, Park et al., 2005). Furthermore, we studied the mechanisms of mucin production after RV infection.
Section snippets
Human embryonic fibroblast cell culture
Human embryonic fibroblast cells were cultured in a Roux type bottle (Iwaki Glass, Chiba, Japan) sealed with a rubber plug in Eagle's minimum essential medium (MEM) (GIBCO-BRL Life Technologies, Palo Alto, CA) containing 10% fetal calf serum (FCS) (GIBCO) supplemented with 5 × 104 U/l penicillin and 50 mg/l streptomycin (Sigma Chemical, St. Louis, MO) (Terajima et al., 1997, Yamaya et al., 1999). Cells (1.5 × 105 cells/ml) were plated in plastic tubes with round bottoms (16 mm diameter and 125 mm
RV14 titers in culture supernatants of human tracheal epithelial cells
In culture supernatants of human tracheal surface epithelial cells (Fig. 1(A)) and submucosal gland cells (Fig. 1(B)), RV14 was not detectable at 1 h after infection, but was detectable at 4 h after infection, and the viral content progressively increased between 4 and 24 h after infection. RV14 infection of the epithelial cells was constant and the coefficient of variation of the viral titers in the supernatants during 1–3 days was small (7.8%; n = 25 in surface epithelial cells and 8.1%; n = 20 in
Discussion
In the present study, human tracheal surface epithelial cells expressed the mRNA of mucin genes MUC1–MUC4, MUC5AC, MUC5B, MUC6, and MUC8. RV14 infection up-regulated the mRNA expression of mucin genes MUC2, MUC3, MUC5AC, MUC5B and MUC6. Secretion and synthesis of MUC5AC, which is the major mucous glycoprotein of the airway epithelium (Miller et al., 2003), and total mucin also increased after RV14 infection in primary cultures of human tracheal surface epithelial cells. Time-course of increased
Acknowledgements
We thank Mr. Grant Crittenden for the English correction and Mr. Akira Ohmi and Ms. Michiko Okamoto, Ms. Minako Tada and Ms. Fusako Chiba for technical assistance. Source of financial support: Grant from The Ministry of Education and Scientific Technique (16590732), and The Ministry of Health, Labour and Welfare (17243601) of the Japanese government to MY.
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