key: cord-0035753-qixhpefs
authors: Kim, Victor; Kato, Kosuke; Kim, K. Chul; Lillehoj, Erik P.
title: Role of Epithelial Cells in Chronic Inflammatory Lung Disease
date: 2013-07-13
journal: Smoking and Lung Inflammation
DOI: 10.1007/978-1-4614-7351-0_4
sha: 4b4bda22a0ab593e91b97f7fdd1bdde0050b6290
doc_id: 35753
cord_uid: qixhpefs

Airborne pathogens entering the lungs first encounter the mucus layer overlaying epithelial cells as a first line of host defense [1, 2]. In addition to serving as the physical barrier to these toxic agents, intact epithelia also are major sources of various macromolecules including antimicrobial agents, antioxidants and antiproteases [3, 4] as well as proinflammatory cytokines and chemokines that initiate and amplify host defensive responses to these toxic agents [5]. Airway epithelial cells can be categorized as either ciliated or secretory [6]. Secretory cells, such as goblet cells and Clara cells, are responsible for the production and secretion of mucus along the apical epithelial surface and, in conjunction with ciliated cells, for the regulation of airway surface liquid viscosity. In addition, submucosal mucus glands connect to the airway lumen through a ciliated duct that propels mucins outward. These glands are present in the larger airways between bands of smooth muscle and cartilage. See Fig. 1.

Airborne pathogens entering the lungs fi rst encounter the mucus layer overlaying epithelial cells as a fi rst line of host defense [ 1 , 2 ] . In addition to serving as the physical barrier to these toxic agents, intact epithelia also are major sources of various macromolecules including antimicrobial agents, antioxidants and antiproteases [ 3 , 4 ] as well as proinfl ammatory cytokines and chemokines that initiate and amplify host defensive responses to these toxic agents [ 5 ] . Airway epithelial cells can be categorized as either ciliated or secretory [ 6 ] . Secretory cells, such as goblet cells and Clara cells, are responsible for the production and secretion of mucus along the apical epithelial surface and, in conjunction with ciliated cells, for the regulation of airway surface liquid viscosity. In addition, submucosal mucus glands connect to the airway lumen through a ciliated duct that propels mucins outward. These glands are present in the larger airways between bands of smooth muscle and cartilage. See Fig. 1 .

Initially, inhaled toxic agents encounter a mucus layer overlying the respiratory epithelium, become trapped, and are subsequently neutralized by macromolecules. Elimination of these toxic agents depends on mucociliary clearance and cough.

Continuous ciliary movement propels secretions proximally at about 1 mm per minute [ 7 ] , and this mucosal velocity is modifi ed by hydration of the mucus layer [ 7 , 8 ] and adrenergic and cholinergic stimuli [ 7 , 9 -11 ] . Effi cacy of cough in the elimination of mucus depends on inspiratory muscle strength and expiratory fl ow velocity, which must detach the mucus from the airway surface and expel the secretions proximally.

A second layer of defense is provided by cell surface receptors (e.g. Toll-like receptors, TLRs) on epithelial cells and resident leukocytes. They bind to various components of the harmful agents and stimulate the production of proinfl ammatory cytokines (e.g. tumor necrosis factor-α, TNF-α) and chemokines (e.g. interleukin-8). Finally, a third layer of protection is mediated by leukocytes recruited to the lumen of the airways by chemotactic molecules that attack pathogens by direct phagocytosis, as well as through the release anti-microbial proteases (e.g. neutrophil elastase) and oxygen radicals. Details of the role of airway epithelium in the host immune responses are described in a recent review [ 12 ] . This review is focused on the role of airway mucus, particularly MUC1 mucin, in the context of chronic infl ammatory lung diseases.

Mucus is overproduced in several infl ammatory lung diseases, a process detrimental to the normal lung defense against environmental toxins. To make matters worse, diffi culty in secretion clearance secondary to ineffective cough or poor mucociliary transport leads to mucostasis and paradoxically predisposes to bacterial colonization and infection [ 13 , 14 ] . Dyspnea, cough and sputum production result from the physical obstruction of the airways by mucus and stimulation of intrapulmonary vagal afferent nerves [ 15 , 16 ] . Lung diseases such as COPD, bronchiectasis, and cystic fi brosis are characterized by mucus hypersecretion, chronic bacterial colonization, and repeated lower respiratory tract infections [ 17 -21 ] .

Airway disease is a crucial pathologic component of multiple infl ammatory lung diseases (See Table 1 ). Pathologic changes in airway epithelium of COPD patients include squamous metaplasia, infl ammatory cell infi ltration, goblet cell hyperplasia, and mucus metaplasia, a process in which mucus is overproduced in response to infl ammatory stimuli (Figs. 2 and 3 ) [ 22 ] . These abnormalities are seen in both the larger central airways as well as smaller respiratory bronchioles [ 23 -26 ] . Airway infl ammation from smoking begins early in the course of the disease, and leads to persistent and progressive airway remodeling, even after smoking cessation [ 27 ] . Niewoehner et al. discovered infl ammatory changes in the peripheral airways of young smokers who died suddenly, suggesting that airway disease developed before the diagnosis of COPD could be established [ 28 ] . As further evidence of this concept, epithelial layer thickness and mucous metaplasia increase incrementally with disease severity [ 26 , 29 ] . These alterations in the epithelium increase airfl ow obstruction by several mechanisms: (1) excess mucus occludes the airway lumen [ 30 ] ; (2) epithelial layer thickening encroaches on the airway lumen, thereby reducing inner diameter [ 31 ] ; and (3) increased mucus alters surface tension of the airway, predisposing it to collapse during expiration [ 32 ] . Hogg et al. found inverse relationships between infl ammatory cell infi ltration and luminal occlusion of the small airways and lung function [ 29 ] , strongly supporting the notion that small airway pathology is responsible for severity of illness. Airway disease also has prognostic signifi cance in COPD. Mucus metaplasia in COPD has been associated with worse physiologic response to lung volume reduction surgery [ 33 ] as well as greater mortality [ 34 ] .

Although it is established that quantity of emphysema correlates well with clinical disease staging in COPD, the relationship between airway pathology, physiology and symptom severity is weak at best. Chronic bronchitis exists in 26-45 % of all smokers, but COPD develops in only 15-20 % [ 35 , 36 ] . Large airway mucous metaplasia correlates poorly with the degree of airfl ow obstruction [ 37 ] and amount of mucus expectoration [ 38 ] . Small airway disease has been found in surgical lung specimens from those with advanced emphysema, with no clinical or radiographic evidence to suggest its presence preoperatively [ 29 , 33 , 39 ] . More importantly, the degree of small airway mucous metaplasia is diffi cult to detect clinically by burden of cough or sputum [ 40 ] .

Despite the disconnect between symptoms and airway pathology, chronic cough and sputum production in COPD have multiple consequences, including an accelerated decline in lung function, [ 41 , 42 ] increased exacerbation frequency [ 43 -47 ] , greater respiratory symptoms [ 43 , 48 ] , worse health related quality of life [ 43 ] , and higher mortality [ 35 , 49 ] . These phenomena are without a doubt a result of increased airway infl ammation and worsened airfl ow obstruction, in addition to the aforementioned mechanisms. In a long term study of over 9,000 adults, an excess yearly rate of FEV 1 decline of 12.6-22.8 mL was attributed to chronic mucus hypersecretion [ 21 ] . We have found chronic cough and sputum production in patients with severe COPD were associated with higher dyspnea scores and more upper airway symptoms [ 43 , 48 ] . In multiple studies, patients with chronic bronchitis and COPD were found to be at a 1.20-1.92-fold increased risk for COPD exacerbation compared to those without chronic bronchitis [ 43 -47 ] . The cause of the observed increase in allcause mortality, however, is still a matter of debate. It is hypothesized that the increased lung infl ammation associated with chronic bronchitis causes greater systemic infl ammation, resulting in numerous downstream consequences, including coronary artery disease, dyslipidemia, osteoporosis, and skeletal muscle weakness [ 50 ] . In the Tucson Epidemiological Survey of Airway Obstructive Disease, chronic bronchitis was associated with a 2.2-fold greater risk of all-cause mortality in those under the age of 50, and was also associated with higher serum levels of IL-8 and C-reactive protein at enrollment [ 49 ] .

In asthma, chronic infl ammation and thickening of the small airway epithelium, submucosal space, and smooth muscle has been noted in several pathologic studies [ 51 -53 ] . In addition, shedding of the epithelial layer has been noted in postmortem studies, bronchoalveolar lavage fl uid, and sputum samples [ 54 ] , most likely as a result of weakened attachment of epithelial cells to the basement membrane. Large airway goblet cell hyperplasia and smooth muscle hypertrophy are prominent pathologic features of asthma. Goblet cell hyperplasia is more consistently seen in asthma compared to COPD, where clinical and pathologic phenotype is a highly variable combination of airway disease and emphysema. In asthma, mucus hypersecretion leads to obstruction of the majority of distal airways and ultimately respiratory failure during fatal asthma exacerbations [ 55 ] . Diffuse occlusion of the small and medium sized airways by mucus and cellular debris has been demonstrated in multiple autopsy studies of patients who died from asthma [ 56 , 57 ] . Goblet cell hyperplasia is also seen in less severe cases as well; Ordonez et al. found a greater number of goblet cells and secreted mucins in subjects with mild to moderate asthma compared to control subjects [ 58 ] .

Similar to asthma, mucus hypersecretion in cystic fi brosis leads to airfl ow obstruction and small airway occlusion [ 59 ] . However, cystic fi brosis is caused by dysfunction of an epithelial chloride channel, which results in sodium and water infl ux to the epithelial layer and therefore depletion of the airway surface liquid [ 19 ] .

The increased viscosity and tenacity of secretions makes detachment from the epithelium and propulsion outward during cough exceedingly diffi cult. Excess mucus production combined with airway surface liquid dessication results in mucostasis, causing colonization by pathogenic bacteria such as Pseudomonas aeruginosa , Staphylococcus aureus , and Burkholderia cepacia [ 18 -20 ] .

Mucus, or the airway surface liquid, is a complex mixture of ions, salts, peptides, proteins, glycoconjugates and water. Strict regulation of mucus production is indispensable for normal lung function. The protective function of mucus depends on its proper composition of constituent components, particularly mucin glycoproteins. Mucins are high molecular weight proteins with O-glycosidic linkages between the fi rst GalNAc residue of the oligosaccharide side chains and serine and threonine amino acids of the polypeptide backbone. Over 20 mucin (MUC in human, Muc in animals) genes have been cloned, 12 of which are expressed in the lung [ 2 , 60 ] . The airway mucin include secreted gene products (MUC2, 5AC, 5B, 7, 8, and 19) and membrane-tethered mucins (MUC1, 4, 11, 13, 16, and 20) . MUC5AC and MUC5B are the two major secretory mucins in the respiratory tract. The levels of these mucins in mucus have been shown to signifi cantly increase, and to directly correlate with, the number of goblet cells under the pathological conditions of goblet cell metaplasia or goblet cell hyperplasia. Although the exact roles of MUC5AC and MUC5B in the airways remain to be fully elucidated, it has been suggested that MUC5AC expression is inducible during airway infl ammation, whereas MUC5B expression is constitutive [ 61 ] . A recent report supports this notion by demonstrating that MUC5AC levels correlated with the degree of airway obstruction in COPD patients [ 62 ] . Cystic fi brosis, in contrast, is characterized by greater MUC5B levels compared to MUC5AC [ 63 , 64 ] , suggesting that impaired mucociliary clearance is the principal mechanism responsible for the overwhelming burden of mucus in these patients.

TLRs, and related molecules, on airway epithelial cells comprise a second line of defense against inhaled microbial pathogens [ 65 ] . These pattern recognition receptors (PRRs) constitute an evolutionary conserved family of gene products that interact with pathogen-associated molecular patterns (PAMPs) to initiate downstream signal transduction and innate infl ammatory responses. In general, all TLRs possess a leucine-rich repeat region in their ectodomains and an intracellular Toll/interleukin-1 receptor (TIR) domain. TLR signaling is activated by agonist-induced receptor homodimerization, recruitment of cytoplasmic adaptor proteins (MyD88, TIRAP, TRIF) to the TIR domain, and activation of protein kinases (IRAKs, TRAF6) [ 66 ] . Although all of the 10 known human TLRs are expressed by airway epithelial cells, TLR2 and TLR5 are the predominant respiratory PRRs [ 67 , 68 ] . TLR5 engages fl agellin, the major protein component of the bacterial fl agellum, while TLR2 recognizes a diverse array of components from Gram-positive and Gram-negative bacteria, including lipoproteins and peptidoglycan. It remains unclear how a single receptor (TLR2) can recognize such a broad diversity of stimuli, but a possible explanation is the ability of TLR2 to form heterodimers with TLR1 and TLR6. For example, bacterial peptidoglycan interacted with the TLR2/6 co-receptor complex on airway epithelial cells to activate NF-κB and stimulate production of TNF-α [ 69 ] . The magnitude of the response generated by the TLR2/6 heterodimer was greater than that produced by TLR2 alone. While TLR5 homodimers are clearly capable of binding to fl agellin, Mizel et al. [ 70 ] reported that nitric oxide production by airway epithelial cells in response to fl agellin was dependent on interaction of TLR5 with TLR4. TLR2 also was shown to be involved in signaling induced by fl agellin in human airway epithelial cells, suggesting a possible TLR2/TLR5 heterodimer interaction [ 67 ] . Biotinylation of surface proteins of airway epithelial cells followed by co-immunoprecipitation experiments demonstrated that both TLR2 and TLR5 were associated with the ganglioside, asialoGM1, in the plasma membrane [ 71 ] . IRAK1 and TRAF6 were also found in the co-receptor complex, whereas TLR4 was not. Furthermore, treatment of airway epithelial cells with Pseudomonas aeruginosa pili or fl agella mobilized asialoGM1, TLR2, and TLR5 to the apical surface of the cells leading to Ca +2 -associated activation of mitogen-activated protein kinases (MAPKs), nuclear translocation of NF-κB, and production of IL-8 [ 67 ] . These combined results indicate that TLRs link the asialoGM1 glycoconjugate to intracellular signal transduction leading to a proinfl ammatory host response following interaction with bacterial components. Other groups, however, have questioned the role of asialoGM1 as a cell surface receptor for P. aeruginosa , particularly clinical isolates of the bacterium [ 72 ] .

Neutrophils and macrophages constitute a third layer of defense in the clearance of bacteria from the lungs. The anti-microbial function of these immune cells is directly mediated through phagocytosis, and indirectly by the release of antimicrobial agents [ 73 ] . Among the soluble mediators released by neutrophils is the serine protease, neutrophil elastase (NE). Studies using NE knockout mice showed that this protease is required for host defense against experimental infection by Gram-negative bacteria [ 74 ] . However, the role of NE in the normal lung response to spontaneous bacterial infection needs to be more fi rmly established. Some evidence suggests that NE promotes neutrophil migration into the lung by degradation of the extracellular matrix, but this remains controversial [ 75 ] . In general, NE is considered as a proinfl ammatory molecule, and NE chemical inhibitors decrease infl ammation and lung edema in animal models [ 76 ] . Part of the mechanism through which NE mediates its anti-microbial effects is up-regulation of mucin secretion by goblet cells [ 77 ] . Using a co-culture system containing neutrophils and primary tracheal epithelial cells, Kim et al. [ 78 ] demonstrated that activation by fMLP/cytochalasin B resulted not only in increased NE production by neutrophils, but also greater mucin release from the epithelial cells. Both effects were blocked in a dosedependent fashion by pretreatment with α1-protease inhibitor, implicating a proteolytic effect of NE on the epithelial cells. Kohri et al. [ 79 ] reported that NE treatment of NCI-H292 airway epithelial cells stimulated the production of MUC5AC mucin through transforming growth factor-α (TGF-α)-dependent activation of the epidermal growth factor receptor (EGFR). Park et al. [ 80 ] showed that NE treatment of well-differentiated primary normal human bronchial epithelial (NHBE) cells cultured at an air-liquid interface (ALI) increased the release of MUC5AC and MUC5B mucins via an intracellular signaling pathway involving protein kinase Cδ (PKCδ). To date, NE is the most potent mucin secretagogue described.

Given the intricate and diverse host airway infl ammatory mechanisms, a critical balance between these processes and the counter-regulating anti-infl ammatory pathways is absolutely required to maintain a homeostatic environment in the airways. This balance ensures that harmful environmental insults are effectively neutralized without excessive bystander tissue damage. Although a large body of literature has characterized the microbial-stimulated pro-infl ammatory pathways summarized above, relatively less is known about the compensatory anti-infl ammatory responses. Nevertheless, it is hypothesized that failure to down-regulate airway infl ammation results in the development of acute or chronic respiratory diseases, including COPD, CF, ARDS, and asthma [ 25 ] . A number of anti-infl ammatory molecules have been shown to play an important role in controlling the normal infl ammatory response in the lung, including IL-10, transforming growth factor-β (TGF-β), peroxisome proliferator activating receptor (PPAR)-γ, and Mucin-1 (MUC1) [ 25 , 81 , 82 ] . However, what is less clear is whether defective expression and/or structure/function of these, or related, anti-infl ammatory mediators is responsible for the etiopathogenesis of infl ammatory lung diseases. The following sections briefl y describe each of these key anti-infl ammatory mediators with the goal of stimulating further basic and clinical research on their role in airway infl ammatory diseases.

IL-10 down-regulates the expression of proinfl ammatory cytokines, including interferon-γ (IFN-γ), IL-2, and TNF-α, major histocompatibility complex (MHC) class II antigens, and leukocyte co-stimulatory molecules [ 83 ] . IL-10 also enhances B cell survival, proliferation, and antibody production. These pleiotropic effects are mediated through interaction of the IL-10 homodimer with its cognate IL-10 receptor α subunit (IL-10Rα), and subsequent binding of this ligand-receptor complex to the IL-10R2 co-receptor. An accumulating body of evidence points toward a role for IL-10 in chronic infl ammation during COPD and asthma [ 84 , 85 ] , although a direct causal effect for IL-10 in the pathogenesis of these disorders is unclear. In the case of CF, airway secretions from affl icted patients, as well as CFTR −/− mice, have decreased IL-10 levels compared with secretions from normal individuals or CFTR +/+ mice [ 82 ] .

TGF-β is an anti-infl ammatory cytokine that exists in three isoforms, TGF-β1, -β2 and -β3. TGF-β knockout mice are embryonic lethal as a result of profound multiorgan infl ammation. TGF-β +/− heterozygous mice have reduced levels of the cytokine and exhibit exacerbated airway infl ammation compared with wild type animals, suggesting a role for endogenous TGF-β in suppressing the development of allergic airway disease [ 86 ] . Additional evidence supporting an anti-infl ammatory role for TGF-β comes from the observation that intratracheal delivery of TGF-β suppressed allergen-induced airway infl ammation in a murine model of asthma [ 87 ] . Increased airway infl ammation also was evident upon inhibition of TGF-β-dependent intracellular signaling [ 88 ] . Genetic studies have demonstrated an association between gene polymorphisms of the TGF-β locus and COPD [ 89 ] . Finally, a possible role for TGF-β in CF comes from the report that CF human cell lines and cells from CFTR −/− mice have decreased Smad3 levels and decreased responses to TGF-β [ 90 ] .

PPAR-α, -β, and -γ are members of the steroid hormone receptor family of ligandactivated transcription factors [ 82 ] . PPARs form heterodimers with retinoid X receptors that regulate gene transcription. PPARγ is expressed as two isoforms, PPARγ1 and PPARγ2, that differ by the presence of a unique 30 amino acid segment in the latter [ 91 ] . PPARγ2 is primarily expressed in adipose tissue, while PPARγ1 is expressed in the lung, heart, skeletal muscle, intestine, kidney, pancreas, spleen, breast, and lymphoid tissues [ 92 ] . Both PPARγ molecules are activated by prostanoids, a subclass of eicosanoids consisting of prostaglandins, thromboxanes, and prostacyclins. Synthetic PPARγ ligands, such as the thiazolidinediones [ 93 ] , have been developed that suppress infl ammation both in vitro and in vivo [ 94 , 95 ] , including in response to lung infection with Pseudomonas aeruginosa [ 96 ] , the major bacterial species that is responsible for the morbidity and mortality of CF. In the case of CF, at least three lines of evidence have been reported for an anti-infl ammatory role for PPAR-γ. First, PPAR-γ inhibits airway infl ammation by competitively inhibiting NF-κB binding to gene promoters, thereby blocking the activation of proinfl ammatory cytokines [ 97 ] . Second, PPAR-γ expression is decreased in lung of CFTR −/− mice compared with CFTR +/+ mice [ 98 ] . Finally, CF airway epithelial cell lines have reduced PPAR-γ levels compared with normal cells [ 99 ] . Thus, decreased PPAR-γ expression likely contributes to defective NF-κB signaling that favors increased airway infl ammation in CF, and possibly other infl ammatory airway diseases. However, the exact mechanisms by which PPARγ down-regulates infl ammatory responses in CF and other lung diseases remain to be clarifi ed.

Of the 20 known mucin genes, MUC1 was the fi rst to be cloned [ 100 , 101 ] . MUC1 is a single pass, transmembrane glycoprotein located on the apical surface of airway epithelial cells and is composed of two polypeptide chains, a large molecular weight (>250 kDa) subunit containing glycosylated variable number of tandem repeats (VNTR) and a SEA ( s ea urchin sperm protein, e nterokinase, a grin) domain, and a 25 kDa subunit comprised of the transmembrane and intracellular COOH-terminus (CT) regions of the molecule [ 25 ] . The two polypeptide structure of MUC1 arises as a consequence of proteolysis within the SEA domain [ 102 ] . MUC1 is unique among the membrane-bound mucins because its CT region constitutes a signal transduction domain. The CT contains multiple amino acid sequence motifs predicted as binding sites for Shc, c-Src, Grb-2, β-catenin, and phosphoinositide 3-kinase (PI3K) [ 25 ] . These motifs are evolutionarily conserved and undergo tyrosine phosphorylation. The presence of CT phosphorylation sites associated with signaling cascades that have been characterized for other membrane receptors has suggested that MUC1 is functionally analogous to cytokine and growth factor receptors [ 103 ] .

Identifi cation of a functional role for MUC1 in the airways was made possible by the generation of Muc1 knockout (Muc1 −/− ) mice [ 104 ] . Early experiments demonstrated that Muc1 −/− mice were predisposed to developing spontaneous eye infl ammation due to infections by Staphylococcus , Streptococcus , or Corynebacterium compared with wild type animals with an intact Muc1 gene [ 105 ] . Subsequent studies by Lu et al. [ 106 ] using an experimental model of bacterial lung infection showed that Muc1 −/− mice exhibited reduced lung colonization by P. aeruginosa , greater recruitment of leukocytes and higher levels of TNF-α and KC (mouse IL-8) in BALF compared with their wild type littermates. In vitro and in vivo mechanistic studies have indicated that MUC1/Muc1 plays an anti-infl ammatory role during P. aeruginosa airway infection by suppressing TLR5 signaling [ 107 -110 ] .

More interestingly, the anti-infl ammatory effect of MUC1/Muc1 was not limited to TLR5, but also included TLR2, 3, 4, 7 and 9, suggesting that this cell surface mucin may be a universal, negative regulator of TLR signaling [ 110 ] . Given that the host responses to lung pathogens involves the expression of multiple PAMPs, which must be activated and regulated in response to infection, this fi nding suggests a crucial role for MUC1/Muc1 in the resolution of infl ammation, and perhaps in the genesis of chronic infl ammatory disorders, such as COPD, CF and asthma.

Given the anti-infl ammatory role of MUC1 in the airways, it is crucial to understand the mechanisms by which MUC1 gene expression is regulated. Several proinfl ammatory cytokines have been shown to up-regulate MUC1 expression. Noteworthy in this regard is TNF-α. Skerrett et al. [ 111 ] reported that TNFR1 −/− mice treated intranasally with P. aeruginosa showed signifi cantly increased airway infl ammation compared with wild type mice, as measured by enhanced bacterial clearance from the lungs, increased numbers of neutrophils in BALF, and higher levels of TNF-α in BALF. Subsequently, TNF-α was demonstrated to stimulate MUC1 expression in A549 lung epithelial cells [ 107 , 112 ] . The molecular mechanism of TNF-α-induced MUC1 up-regulation has been described in detail using a combination of biochemical, pharmacological, and molecular biological approaches [ 107 ] . The requirement for TNF-α in increased MUC1 expression has also been observed in A549 cells infected with respiratory syncytial virus (RSV) [ 109 ] , as well as in mice infected with P. aeruginosa [ 113 ] . Thus, these results suggest that TNF-α may play a key role in controlling infl ammation during airway infection, from the initiation phase of bacterial exposure to the fi nal resolution of infl ammation, the latter likely by inducing the expression of key anti-infl ammatory molecules, such as MUC1, with possible assistance by IL-10 and/or PPAR-γ.

Based on the accumulated published literature, we propose the following model to account for MUC1, TLRs, and TNF-α in the airway epithelial response to respiratory infection [ 25 ] . Normally, transiently inspired pathogens are quickly removed by mucociliary clearance and phagocytosis by resident leukocytes in the airway lumen. With abnormally high pathogen load, for example due to a predisposing condition such as CF, microbial PAMPs activate TLRs resulting in the production of proinfl ammatory mediators (IL-8 and TNF-α), thereby promoting leukocyte infl ux into the airways. During the early stage of infection, MUC1 expression is suffi ciently low and TLR signaling is not antagonized. However, after invading pathogens have been cleared, increased levels of infl ammatory products such as neutrophil elastase and TNF-α up-regulate MUC1 expression which, in turn, suppresses the release of TNF-α, thus inhibiting TLR-dependent airway infl ammation. The net effect facilitates pathogen removal and returns the lungs to homeostasis. Future experiments are needed to provide additional support for this proposed negative feed-back loop model system.

TNF is the major pro-infl ammatory molecule during airway infection. Ulich et al. [ 114 ] demonstrated that intratracheal LPS-induced neutrophilic infl ammation in rats can be inhibited by intratracheal administration of soluble TNFR, suggesting that TNF/TNFR interaction plays a key role in LPS-induced airway neutrophilic infl ammation. Interestingly, TNFR1 defi cient mice not only failed to control either LPS or Pseudomonas aeruginosa -induced neutrophilic infl ammation [ 111 ] but showed greater neutrophilic infl ammation to the contrary. Recently Choi et al. [ 113 ] showed that Muc1 −/− mice behaves exactly the similar way as TNFR defi cient mice in response to airway Pseudomonas aeruginosa infection, i.e., an increased neutrophilc infl ammation, compared with their WT Muc1 +/+ mice. The relationship between TNFR and Muc1 can be explained by Koga et al. [ 107 ] who demonstrated that the levels of MUC1 are controlled by the TNF/TNFR signaling pathway. Thus, MUC1/Muc1 seems to be controlled mainly by TNF both in vivo [ 113 ] and in vitro [ 107 ] . This timely regulation of infl ammation and its resolution has also been demonstrated in vivo between TNF and IL-10, in which the former induces the latter, one of the major anti-infl ammatory molecule [ 115 ] . Thus, failure to induce sufficient levels of anti-infl ammatory molecules in a timely manner during the course of lung infl ammation will result in lung tissue damage, and the subsequent repair processes will result in lung remodeling, a major characteristic of infl ammatory lung diseases such as COPD and CF. Whether other anti-infl ammatory molecules are also controlled through the similar mechanism remains to be elucidated.

One of the interesting questions that arise from this study is why there are multiple anti-infl ammatory molecules and how they interact with each other during airway infection. For example, it has been shown that MUC1 induces IL-10, an anti-infl ammatory cytokine, in dendritic cells [ 116 ] and that the levels of IL-10 in the BALF of Muc1 KO mice were signifi cantly greater following Pseudomonas aeruginosa infection as compared with those of Muc1+/+ mice (unpublished data), suggesting the possible collaboration between the two during infl ammation. The same question may be applied to other anti-infl ammatory molecules in the lung that have been reported recently, including CD44 [ 117 ] , aryl hydrocarbon receptor [ 118 ] and various lipid mediators [ 119 , 120 ] . Further studies are required to understand the functional relationships between the known anti-infl ammatory molecules during the resolution of airway infl ammation.

In summary, airway epithelial cells play a critical role in the pathogenesis of chronic infl ammatory lung disease. Their primary role in the process of host defense becomes dysregulated, and the excess infl ammation causes increased mucus production and hypersecretion, resulting in mucostasis, airway obstruction, and tissue remodeling from several downstream events. Clinical consequences include an accelerated decline in lung function, greater respiratory symtoms, exacerbations of underlying lung disease, recurrent lower respiratory tract infection, and higher mortality. Multiple complex interactions between infl ammatory cytokines and epithelial cells exist, and the precise roles of each in the generation of mucins and the amplifi cation of lung infl ammation remain unclear. There is, however, emerging evidence that the role of MUC1 mucin is essential to the airway epithelium's response to environmental toxic agents, and therefore essential to the development of chronic and persistent infl ammation. Further studies are required to better understand the roles of this mucin as well as others in the pathogenesis of infl ammatory lung disease.

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Pseudomonas aeruginosa fl agella activate airway epithelial cells through asialoGM1 and toll-like receptor 2 as well as toll-like receptor 5

Toll-like receptors in normal and cystic fi brosis airway epithelial cells

Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin

Induction of macrophage nitric oxide production by Gram-negative fl agellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes

TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

Lack of adherence of clinical isolates of Pseudomonas aeruginosa to asialo-GM(1) on epithelial cells

The neutrophil: function and regulation in innate and humoral immunity

Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis

Neutrophil elastase: path clearer, pathogen killer, or just pathologic?

Effects of neutrophil elastase inhibitor (ONO-5046) on lung injury after intestinal ischemia-reperfusion

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells

Effects of activation of polymorphonuclear leukocytes on airway goblet cell mucin release in a co-culture system

Neutrophil elastase induces mucin production by liganddependent epidermal growth factor receptor activation

Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C{delta}-mediated mechanism

Peroxisome proliferator-activated receptors: potential therapeutic targets in lung disease?

Chronic infl ammation in the cystic fi brosis lung: alterations in inter-and intracellular signaling

IL-10 family of cytokines

Intracellular cytokine profi le of T lymphocytes in patients with chronic obstructive pulmonary disease

Regulatory T cells in many fl avors control asthma

Regulatory T cells in asthma

Naturally occurring lung CD4(+)CD25(+) T cell regulation of airway allergic responses depends on IL-10 induction of TGF-beta

Blockade of transforming growth factor beta/Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway infl ammation and airway reactivity

TGF-beta signaling in COPD: deciphering genetic and cellular susceptibilities for future therapeutic regimen

Isoprenoid-mediated control of SMAD3 expression in a cultured model of cystic fi brosis epithelial cells

The organization, promoter analysis, and expression of the human PPARgamma gene

Regulation of PPAR gamma gene expression by nutrition and obesity in rodents

Peroxisome proliferator-activated receptorgamma (PPAR-gamma) ligands as potential therapeutic agents to treat arthritis

Peroxisome proliferator-activated receptor gamma agonists as therapy for chronic airway infl ammation

Peroxisome proliferator activated receptor ligands as regulators of airway infl ammation and remodelling in chronic lung disease

Peroxisome proliferator-activated receptor-gamma in cystic fi brosis lung epithelium

A paradigm for gene regulation: infl ammation, NF-kappaB and PPAR

Decreased expression of peroxisome proliferator activated receptor gamma in cftr−/− mice

Peroxisome proliferator-activated receptor alpha (PPAR alpha) down-regulation in cystic fi brosis lymphocytes

Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin

Cloning and sequencing of a human pancreatic tumor mucin cDNA

Identifi cation of MUC1 proteolytic cleavage sites in vivo

Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins. Cytokine receptor-like molecules

Delayed mammary tumor progression in Muc-1 null mice

Bacterial conjunctivitis in Muc1 null mice

Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice

TNF-alpha induces MUC1 gene transcription in lung epithelial cells: its signaling pathway and biological implication

Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1-induced suppression of Toll-like receptor 5 signaling

Anti-infl ammatory effect of MUC1 during respiratory syncytial virus infection of lung epithelial cells in vitro

MUC1 mucin is a negative regulator of toll-like receptor signaling

Role of the type 1 TNF receptor in lung infl ammation after inhalation of endotoxin or Pseudomonas aeruginosa

The signaling pathway involved in neutrophil elastase stimulated MUC1 transcription

TNF-alpha is a key regulator of MUC1, an anti-infl ammatory molecule, during airway Pseudomonas aeruginosa infection

Intratracheal administration of endotoxin and cytokines. IV. The soluble tumor necrosis factor receptor type I inhibits acute infl ammation

IL-10 enhances resolution of pulmonary infl ammation in vivo by promoting apoptosis of neutrophils

Tumor-derived MUC1 mucins interact with differentiating monocytes and induce IL-10highIL-12low regulatory dendritic cell

CD44 is a negative regulator of acute pulmonary infl ammation and lipopolysaccharide-TLR signaling in mouse macrophages

Aryl hydrocarbon receptor-defi cient mice develop heightened infl ammatory responses to cigarette smoke and endotoxin associated with rapid loss of the nuclear factor-kappaB component RelB

Prostaglandin I2 analogs inhibit proinfl ammatory cytokine production and T cell stimulatory function of dendritic cells

Lipid mediators as agonists for the resolution of acute lung infl ammation and injury