Mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells

doi:10.1016/j.resp.2005.11.006

Respiratory Physiology & Neurobiology

Volume 154, Issue 3, December 2006, Pages 484-499

https://doi.org/10.1016/j.resp.2005.11.006 Get rights and content

Abstract

Mucus hypersecretion relates to exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD) caused by rhinovirus (RV) infection. We examined the mechanisms of RV infection-induced mucin production in human tracheal surface epithelial cells and submucosal gland cells. RV14 up-regulated the mRNA expression of MUC2, MUC3, MUC5AC, MUC5B and MUC6, and increased MUC5AC and total mucin concentration in supernatants and lysates of the surface cells. An inhibitor of the nuclear factor κB caffeic acid phenylethyl ester, inhibitors of selective p44/42 mitogen-activated protein kinase–kinase PD98059 and U0126, and a selective Src inhibitor PP1 attenuated MUC5AC mRNA expression, and secretion and production of MUC5AC and total mucin glycoprotein in the surface cells. In the gland cells, RV14 also increased mRNA expression of MUC2, MUC5AC, MUC5B and MUC7, and the inhibitors attenuated the secretion of total mucin glycoprotein. Src-related p44/42 mitogen-activated protein kinase pathway may be associated with RV-induced mucin hypersecretion in human airways.

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.

References (39)

M.C. Copin et al.
Mucinous bronchioloalveolar carcinomas display a specific pattern of mucin gene expression among primary lung adenocarcinomas
Hum. Pathol.
(2001)
A. Dohrman et al.
Mucin gene (MUC 2 and MUC 5AC) upregulation by Gram-positive and Gram-negative bacteria
Biochim. Biophys. Acta
(1998)
J.M. Greve et al.
The major human rhinovirus receptor is ICAM-1
Cell
(1989)
J.A. Park et al.
Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase Cδ-mediated mechanism
Am. J. Pathol.
(2005)
Q. Wang et al.
Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells
J. Biol. Chem.
(2003)
ABI Prism 7700 Sequence Detection System Livak Applied Biosystems User Bulletin #2 (part number 4303859), 1997....
C. Basbaum et al.
Control of mucin transcription by diverse injury-induced signaling pathways
Am. J. Respir. Crit. Care Med.
(1999)
M.P. Buisine et al.
Developmental mucin gene expression in the human respiratory tract
Am. J. Respir. Cell Mol. Biol.
(1999)
D.R. DeSilva et al.
Inhibition of mitogen-activated protein kinase blocks T cell proliferation but does not induce or prevent anergy
J. Immunol.
(1998)
K. Guzman et al.
Quantitation of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC mRNA levels by retinoids
Am. J. Physiol.
(1996)

Z. Han et al.

c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis

J. Clin. Invest.

(2001)

S.H. He et al.

Induction of mucin secretion from human bronchial tissue and epithelial cells by rhinovirus and lipopolysaccharide

Acta Pharmacol. Sin.

(2004)

C.A. Heid et al.

Real time quantitative PCR

Genome Res.

(1996)

C.A. Hewson et al.

Rhinovirus induces respiratory epithelial cell mucin synthesis and secretion via NF-kappaB

Am. J. Respir. Crit. Care Med.

(2003)

H.L. Huber et al.

The pathology of bronchial asthma

Arch. Intern. Med.

(1922)

Y.D. Kim et al.

Interleukin-1beta induces MUC2 and MUC5AC synthesis through cyclooxygenase-2 in NCI-H292 cells

Mol. Pharmacol.

(2002)

K. Kunzelmann et al.

Influenza virus inhibits amiloride-sensitive Na⁺ channels in respiratory epithelia

Proc. Natl. Acad. Sci. U.S.A.

(2000)

J.D. Li et al.

Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease

Proc. Natl. Acad. Sci. U.S.A.

(1997)

J.D. Li et al.

Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells

Proc. Natl. Acad. Sci. U.S.A.

(1998)

Cited by (79)

Tacrolimus impairs airway mucociliary clearance of rats
2024, Transplant Immunology
Tacrolimus (TAC) is the most widely used immunosuppressive agent after lung transplantation. Considering that the ciliary beat frequency (CBF) mainly depends on the cytoplasmic calcium concentration and that TAC can affect this due to its binding with the intracellular immunophilin FKBP12, we hypothesized that TAC could also impair the airway mucociliary clearance of rats.
Sixty rats were divided into two groups (n = 30 each): Control = water; TAC = tacrolimus. After 7, 15 or 30 days of treatment, ten animals from each group were euthanized and the following parameters were studied: mucus transportability, CBF, mucociliary transport velocity (MCTV), and neutral and acid mucus production.
There was a significant decrease in CBF (Control vs TAC: 7 days, p = 0.008; 15 days, p = 0.007; 30 days, p = 0.001) and MCTV (Control vs TAC: 7 days, p = 0.004; 15 days, p < 0.001; 30 days, p < 0.001) in all immunosuppressed animals. TAC therapy also caused an increase in acid mucus production at all treatment times (Control vs TAC: 7 days, p = 0.001; 15 days, p = 0.043; 30 days, p = 0.001).
TAC impairs airway mucociliary clearance of rats.
Anti-inflammatory effects of medications used for viral infection-induced respiratory diseases
2023, Respiratory Investigation
Citation Excerpt :
RV infection induces the production of proinflammatory mediators, including IL-1β, IL-6, IL-8, RANTES, IP-10, and granulocyte-macrophage (GM) colony-stimulating factor (CSF), in the human bronchial epithelial cell line BEAS-2B, primary HTE cells, human bronchial cells, HNE cells, and human tracheal submucosal gland cells [28,29,34,70,82–85] by activating NF-κB [29] and toll-like receptor (TLR) [86,87]. RV infection induces the production of mucin (MUC5AC) in the cells of the human airway, bronchial epithelium, and tracheal submucosal gland [30,88,89] (Fig. 1), partly by activating MAPK, SAM-pointed domain-containing Ets-like factor (SPDEF)-regulated genes, or the action of adenosine triphosphate (ATP), which is released from RV-infected cells via purinergic P2 receptors [30,88,89]. Serum levels of IP-10 and granulocyte CSF (G-CSF) were elevated in RS-infected children with wheezing [79].
Respiratory viruses like rhinovirus, influenza virus, respiratory syncytial virus, and coronavirus cause several respiratory diseases, such as bronchitis, pneumonia, pulmonary fibrosis, and coronavirus disease 2019, and exacerbate bronchial asthma, chronic obstructive pulmonary disease, bronchiectasis, and diffuse panbronchiolitis. The production of inflammatory mediators and mucin and the accumulation of inflammatory cells have been reported in patients with viral infection-induced respiratory diseases. Interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, and regulated on activation normal T-cell expressed and secreted are produced in the cells, including human airway and alveolar epithelial cells, partly through the activation of toll-like receptors, nuclear factor kappa B and p44/42 mitogen-activated protein kinase. These mediators are associated with the development of viral infection-induced respiratory diseases through the induction of inflammation and injury in the airway and lung, airway remodeling and hyperresponsiveness, and mucus secretion. Medications used to treat respiratory diseases, including corticosteroids, long-acting β₂-agonists, long-acting muscarinic antagonists, mucolytic agents, antiviral drugs for severe acute respiratory syndrome coronavirus 2 and influenza virus, macrolides, and Kampo medicines, reduce the production of viral infection-induced mediators, including cytokines and mucin, as determined in clinical, in vivo, or in vitro studies. These results suggest that the anti-inflammatory effects of these medications on viral infection-induced respiratory diseases may be associated with clinical benefits, such as improvements in symptoms, quality of life, and mortality rate, and can prevent hospitalization and the exacerbation of chronic obstructive pulmonary disease, bronchial asthma, bronchiectasis, and diffuse panbronchiolitis.
Glutathione S-transferases and their implications in the lung diseases asthma and chronic obstructive pulmonary disease: Early life susceptibility?
2021, Redox Biology
Our lungs are exposed daily to airborne pollutants, particulate matter, pathogens as well as lung allergens and irritants. Exposure to these substances can lead to inflammatory responses and may induce endogenous oxidant production, which can cause chronic inflammation, tissue damage and remodeling. Notably, the development of asthma and Chronic Obstructive Pulmonary Disease (COPD) is linked to the aforementioned irritants. Some inhaled foreign chemical compounds are rapidly absorbed and processed by phase I and II enzyme systems critical in the detoxification of xenobiotics including the glutathione-conjugating enzymes Glutathione S-transferases (GSTs). GSTs, and in particular genetic variants of GSTs that alter their activities, have been found to be implicated in the susceptibility to and progression of these lung diseases. Beyond their roles in phase II metabolism, evidence suggests that GSTs are also important mediators of normal lung growth. Therefore, the contribution of GSTs to the development of lung diseases in adults may already start in utero, and continues through infancy, childhood, and adult life. GSTs are also known to scavenge oxidants and affect signaling pathways by protein-protein interaction. Moreover, GSTs regulate reversible oxidative post-translational modifications of proteins, known as protein S-glutathionylation. Therefore, GSTs display an array of functions that impact the pathogenesis of asthma and COPD.
In this review we will provide an overview of the specific functions of each class of mammalian cytosolic GSTs. This is followed by a comprehensive analysis of their expression profiles in the lung in healthy subjects, as well as alterations that have been described in (epithelial cells of) asthmatics and COPD patients. Particular emphasis is placed on the emerging evidence of the regulatory properties of GSTs beyond detoxification and their contribution to (un)healthy lungs throughout life. By providing a more thorough understanding, tailored therapeutic strategies can be designed to affect specific functions of particular GSTs.
NOD-like receptor family, pyrin domain containing 3 (NLRP3) contributes to inflammation, pyroptosis, and mucin production in human airway epithelium on rhinovirus infection
2019, Journal of Allergy and Clinical Immunology
Citation Excerpt :
Nevertheless, mechanisms controlling the differential cell death pathways (apoptosis, pyroptosis, and necroptosis) within the virus-infected airway epithelial cells and the critical factors regulating certain “thresholds” for cell death patterns require further clarification. hRV can induce mucus production in the airway epithelium,17 but the underlying mechanism is largely unclear. Only one study demonstrated that the Toll-like receptor 3–epidermal growth factor receptor pathway was involved in hRV-induced airway mucin production.15
The airway epithelium maintains mucosal homeostasis and effectively responds to pathogens. The roles of the epithelial NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in human rhinovirus (hRV) infection and its effects mediating epithelial functional changes remain poorly understood.
We investigated the mechanisms and cellular functions mediated by the epithelial NLRP3 inflammasome on hRV infection.
Using models of primary human nasal epithelial progenitor cells and differentiated human nasal epithelial cells (hNECs) infected by hRV, we functionally examined key factors for NLRP3 inflammasome activation, cell death, and mucus production. Furthermore, NLRP3 and IL-1β in human epithelium from nasal mucosal inflammation induced by hRV were evaluated.
The inflammasome-mediated IL-1β secretion and pyroptosis in human nasal epithelial progenitor cells and hNECs on hRV infection were dependent on the DDX33/DDX58–NLRP3–caspase-1–GSDMD axis. In differentiated hNECs hRV could also promote major airway epithelial mucin (MUC5AC) production through this axis. Our results further confirmed that the NLRP3 inflammasome signaling pathway was responsible for suppressing hRV replication in airway epithelium. Finally, hRV infection in chronically inflamed nasal mucosa was associated with epithelial mucus hyperproduction, whereas NLRP3 and IL-1β expression levels were significantly increased in hRV-infected epithelium with goblet cell hyperplasia compared with normal epithelium without viral infection.
The current study showed that the NLRP3 inflammasome signaling axis could functionally mediate hRV-induced inflammation, pyroptosis, and mucus production in airway epithelium, which might be an essential mechanism associated with hRV-induced airway remodeling.
c- Src and its role in cystic fibrosis
2016, European Journal of Cell Biology
Citation Excerpt :
The activated NF-κB in turn binds to a site in the 5′-flanking region of the MUC2 gene inducing its transcription (Li et al., 1998). A different c-Src-related mitogen-activated protein kinase pathway (MAPK p44/42, also known as extracellular signal-regulated kinase 1 and 2, ERK1/2) is involved in the rhinovirus-induced mucin hypersecretion in cultured human airway epithelial cells (rhinovirus produce the common cold) (Inoue et al., 2006). In contrast, the LPS of Helicobacter pylori induces gastric tissue inflammation and dedifferentiation, which eventually leads to gastric cancer (Song et al., 2013), by a process which is accompanied by inhibition of gastric mucin (Slomiany and Slomiany, 2004a) (instead of hypersecretion).
Cystic fibrosis (CF) is a lethal inherited disease produced by mutations in the gene encoding the CFTR chloride channel. Loss of function in the CFTR gene is associated with a not much noticed increased expression and activity of the non-receptor protein-tyrosine kinase c-Src. CF is therefore the result from the loss of CFTR chloride transport function and its consequences, including a chronic and excessive c-Src signaling. On the other hand, c-Src, encoded by the SRC gene, is involved in diverse signaling mechanisms that regulate key cellular functions such as cell proliferation, apoptosis, oxidative stress, inflammation, and innate immunity. These c-Src-regulated cellular functions are also affected in CF; however, studies exploring a direct role of c-Src in the regulation of these cellular functions in CF are yet scarce and often controversial. Here we describe the c-Src regulation and functions, with emphasis in those altered in CF, and describe the role of CFTR as a “signaling molecule” that negatively modulates c-Src expression and activity. It is also discussed the emerging role of intracellular Cl⁻ and IL-1β as intermediate signaling effectors between CFTR and c-Src.
Virus-induced modulation of lower airway diseases: Pathogenesis and pharmacologic approaches to treatment
2015, Pharmacology and Therapeutics
Uncomplicated upper respiratory viral infections are the most common cause of days lost from work and school and exert a major economic burden. In susceptible individuals, however, common respiratory viruses, particularly human rhinoviruses, also can have a major impact on diseases that involve the lower airways, including asthma, chronic obstructive pulmonary diseases (COPD) and cystic fibrosis (CF). Respiratory virus-induced wheezing illnesses in early life are a significant risk factor for the subsequent development of asthma, and virus infections may also play a role in the development and progression of airway remodeling in asthma. It is clear that upper respiratory tract virus infections can spread to the lower airway and trigger acute attacks of asthma, COPD or CF. These exacerbations can be life-threatening, and exert an enormous burden on health care systems. In recent years we have gained new insights into the mechanisms by which respiratory viruses may induce acute exacerbations of lower airway diseases, as well as into host defense pathways that may regulate the outcomes to viral infections. In the current article we review the role of viruses in lower airway diseases, including our current understanding on pathways by which they may cause remodeling and trigger acute exacerbations. We also review the efficacy of current and emerging therapies used to treat these lower airway diseases on the outcomes due to viral infection, and discuss alternative therapeutic approaches for the management of virus-induced airway inflammation.

View all citing articles on Scopus

View full text

Mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells

Abstract

Introduction

Section snippets

Human embryonic fibroblast cell culture

RV14 titers in culture supernatants of human tracheal epithelial cells

Discussion

Acknowledgements

Hum. Pathol.

Biochim. Biophys. Acta

Cell

Am. J. Pathol.

J. Biol. Chem.

Control of mucin transcription by diverse injury-induced signaling pathways

Am. J. Respir. Crit. Care Med.

Developmental mucin gene expression in the human respiratory tract

Am. J. Respir. Cell Mol. Biol.

Inhibition of mitogen-activated protein kinase blocks T cell proliferation but does not induce or prevent anergy

J. Immunol.

Quantitation of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC mRNA levels by retinoids

Am. J. Physiol.

c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis

J. Clin. Invest.

Induction of mucin secretion from human bronchial tissue and epithelial cells by rhinovirus and lipopolysaccharide

Acta Pharmacol. Sin.

Real time quantitative PCR

Genome Res.

Rhinovirus induces respiratory epithelial cell mucin synthesis and secretion via NF-kappaB

Am. J. Respir. Crit. Care Med.

The pathology of bronchial asthma

Arch. Intern. Med.

Interleukin-1beta induces MUC2 and MUC5AC synthesis through cyclooxygenase-2 in NCI-H292 cells

Mol. Pharmacol.

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Proc. Natl. Acad. Sci. U.S.A.

Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease

Proc. Natl. Acad. Sci. U.S.A.

Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells

Proc. Natl. Acad. Sci. U.S.A.

Influenza virus inhibits amiloride-sensitive Na⁺ channels in respiratory epithelia