Isolated abnormal FEF75% detects unsuspected bronchiolar obstruction in CF children.

BACKGROUND: Physiologic detection of bronchiolar obstruction in children with cystic fibrosis (CF) may be clinically unsuspected because of normal routine spirometry despite bronchiectasis on lung CT. METHODS: Children from two accredited CF facilities had spirometry obtained every 3 months when clinically stable. Pre-bronchodilator maximum expiratory flow volume curves were retrospectively analyzed over 16 years to detect an isolated abnormal FEF75%, despite normal routine spirometry. RESULTS: At Miller Children's and Women's Hospital (MCWH), an abnormal FEF75% was initially detected in 26 CF children at age 7.5 ± 4 (SD) years despite normal routine spirometry initially. FEF75% remained an isolated abnormality for 2.5 ± 1.5 years after it was initially detected in these 26 CF children. At Cohen Children's Medical Center (CCMC), despite normal routine spirometry initially, abnormal FEF75% occurred in 13 children at age 11.7 ± 4.5 years, and abnormal FEF25-75% in 10 children at age 11.8 ± 5.3 years. CONCLUSIONS: FEF75% was most sensitive spirometric test for diagnosing both early and isolated progressive bronchiolar obstruction. Data from CCMC in older children demonstrated the simultaneous detection of abnormal FEF75% and FEF25-75% values consistent with greater bronchiolar obstruction when serial spirometry was initiated at an older age. IMPACT: There is very little published spirometric data regarding diagnosis of isolated small airways obstruction in CF children. FEF75% can easily detect unsuspected small airways obstruction in CF children with normal routine spirometry and bronchiectasis on lung CT and optimize targeted modulatory therapies.

Normal Routine Spirometry Can Mask COPD/Emphysema in Symptomatic Smokers.

BACKGROUND: Recent studies have emphasized the difficulty of early detection of chronic obstructive pulmonary disease (COPD) in symptomatic smokers with normal routine spirometry. This includes post-bronchodilator normal forced expiratory volume in 1 second (FEV1)(L)≥80% predicted, forced vital capacity (FVC)(L)≥80% predicted, and FEV1/FVC ≥70% or greater than age corrected lower limit of normal (LLN). However, in COPD the pathologic site of small airway obstruction and emphysema begins in the small peripheral airways ≤2 mm id which normally contribute <20% of total airway resistance. METHODS: Expiratory airflow at high and low lung volumes post-bronchodilator were measured and correlated with lung computed tomography (CT) and lung pathology (6 patients) in 16 symptomatic, treated smokers, and all with normal routine spirometry. RESULTS: Despite normal routine spirometry, all16 patients had isolated, abnormal forced expiratory flow at 75% of FVC (FEF75) using data from Knudson et al, Hankinson et al NHAMES III, and Quanjer et al and the Global Lung Function Initiative. This reflects isolated detection of small airways obstruction and/or emphysema. Measuring airflow at FEF50 detected only 8 of 16 patients, maximal expiratory flow at 25%-75% of FVC (MEF25-75) only 4 of 16, residual volume (RV) 4 of 16, and RV to total lung capacity ratio only 2 of 16. There was excellent correlation between limited lung pathology and lung CT for absence of emphysema. CONCLUSION: This study confirms our earlier observations that detection of small airways obstruction and/or emphysema, in symptomatic smokers with normal routine spirometry, requires analysis of expiratory airflow at low lung volumes, including FEF75. Dependence upon normal routine spirometry may result in clinical and physiologic delay in the diagnosis and treatment in symptomatic smokers with emphysema and small airways obstruction.

Further Studies of Unsuspected Emphysema in Nonsmoking Patients With Asthma With Persistent Expiratory Airflow Obstruction.

BACKGROUND: Previously, we and other investigators have described reversible loss of lung elastic recoil in patients with acute and persistent, moderate-to-severe, chronic, treated asthma who never smoked, and its adverse effect on maximal expiratory airflow. In four consecutive autopsies, we reported the pathophysiologic mechanism(s) has been unsuspected mild, diffuse, middle and upper lobe centrilobular emphysema. METHODS: We performed prospective studies (5 to 22 years) in 25 patients (12 female) with chronic asthma, age 55 ± 15 years, who never smoked, with persistent moderate-to-severe expiratory obstruction. Studies included measuring blood eosinophils, IgE, total exhaled nitric oxide (NO), central airway NO flux, peripheral airway/alveolar NO concentration, impulse oscillometry, heliox curves, lung elastic recoil, and high-resolution thin-section (1 mm) lung CT imaging at full inspiration with voxel quantification. RESULTS: In 25 patients with stable asthma with varying type 2 phenotype, after 270 µg of aerosolized albuterol sulfate had been administered with a metered dose inhaler with space chamber, FVC was 3.1 ± 1.0 L (83% ± 13% predicted) (mean ± SD), FEV1 was 1.8 ± 0.6 L (59% ± 11%), the FEV1/FVC ratio was 59% ± 10%, and the ratio of single-breath diffusing capacity of the lung for carbon monoxide to alveolar volume was 4.8 ± 1.1 mL/min/mm Hg/L (120% ± 26%). All 25 patients with asthma had loss of static lung elastic recoil pressure, which contributed equally to decreased intrinsic airway conductance in limiting expiratory airflow. Lung CT scanning detected none or mild emphysema. In all four autopsied asthmatic lungs previously reported and one unreported explanted lung, microscopy revealed unsuspected mild, diffuse centrilobular emphysema in the upper and middle lung fields, and asthma-related remodeling in airways. In eight cases, during asthma remission, there were increases in measured static lung elastic recoil pressure-calculated intrinsic airway conductance, and measured maximal expiratory airflow at effort-independent lung volumes. CONCLUSIONS: As documented now in five cases, unsuspected microscopic mild centrilobular emphysema is the sentinel cause of loss of lung elastic recoil. This contributes significantly to expiratory airflow obstruction in never-smoking patients with asthma, with normal diffusing capacity and near-normal lung CT scan results. TRIAL REGISTRY: Protocol No. 20070934 and Study No. 1090472, Western Institutional Review Board, Olympia, WA; ClinicalTrials.gov; No. NCT00576069; URL: www.clinicaltrials.gov.

Understanding the pathophysiology of the asthma-chronic obstructive pulmonary disease overlap syndrome.

PURPOSE OF REVIEW: The review will provide an update on the pathophysiology and studies of inflammation associated with the asthma-chronic obstructive pulmonary disease (COPD) overlap syndrome (ACOS) and the mechanism(s) responsible for persistent expiratory airflow limitation in never-smoked asthma patients who develop loss of lung elastic recoil consistent with an asthma-COPD clinical phenotype (ACOS in nonsmokers). RECENT FINDINGS: Patients with a clinical diagnosis of ACOS have more frequent respiratory exacerbations and hospitalizations than COPD patients without ACOS. ACOS patients should be treated with inhaled corticosteroids, short and long-acting ß2-agonist, and long-acting muscarinic receptor antagonist. Biomarker work suggests that a molecular phenotype of ACOS (e.g., elevated markers of eosinophilic or type 2 inflammation) incompletely corresponds to clinical phenotypes. Recently, we reported sentinel observation of unsuspected mild diffuse centrilobular emphysema in never-smoked asthma patients at autopsy, despite mild changes in lung computed tomography and normal diffusing capacity. SUMMARY: Recent studies have shown that subgroups of COPD and asthma patients may have overlapping immune responses. Never-smoked asthma patients may have persistent expiratory airflow limitation because of loss of lung elastic recoil. This may be because of unsuspected centrilobular emphysema detected at autopsy, and not easily diagnosed on lung computed tomography and diffusing capacity.

Unraveling the Pathophysiology of the Asthma-COPD Overlap Syndrome: Unsuspected Mild Centrilobular Emphysema Is Responsible for Loss of Lung Elastic Recoil in Never Smokers With Asthma With Persistent Expiratory Airflow Limitation.

Investigators believe most patients with asthma have reversible airflow obstruction with treatment, despite airway remodeling and hyperresponsiveness. There are smokers with chronic expiratory airflow obstruction despite treatment who have features of both asthma and COPD. Some investigators refer to this conundrum as the asthma-COPD overlap syndrome (ACOS). Furthermore, a subset of treated nonsmokers with moderate to severe asthma have persistent expiratory airflow limitation, despite partial reversibility. This residuum has been assumed to be due to large and especially small airway remodeling. Alternatively, we and others have described reversible loss of lung elastic recoil in acute and persistent loss in patients with moderate to severe chronic asthma who never smoked and its adverse effect on maximal expiratory airflow. The mechanism(s) responsible for loss of lung elastic recoil and persistent expiratory airflow limitation in nonsmokers with chronic asthma consistent with ACOS remain unknown in the absence of structure-function studies. Recently we reported a new pathophysiologic observation in 10 treated never smokers with asthma with persistent expiratory airflow obstruction, despite partial reversibility: All 10 patients with asthma had a significant decrease in lung elastic recoil, and unsuspected, microscopic mild centrilobular emphysema was noted in all three autopsies obtained although it was not easily identified on lung CT scan. These sentinel pathophysiologic observations need to be confirmed to further unravel the epiphenomenon of ACOS. The proinflammatory and proteolytic mechanism(s) leading to lung tissue breakdown need to be further investigated.

EGFR activation suppresses respiratory virus-induced IRF1-dependent CXCL10 production.

Airway epithelial cells are the primary cell type involved in respiratory viral infection. Upon infection, airway epithelium plays a critical role in host defense against viral infection by contributing to innate and adaptive immune responses. Influenza A virus, rhinovirus, and respiratory syncytial virus (RSV) represent a broad range of human viral pathogens that cause viral pneumonia and induce exacerbations of asthma and chronic obstructive pulmonary disease. These respiratory viruses induce airway epithelial production of IL-8, which involves epidermal growth factor receptor (EGFR) activation. EGFR activation involves an integrated signaling pathway that includes NADPH oxidase activation of metalloproteinase, and EGFR proligand release that activates EGFR. Because respiratory viruses have been shown to activate EGFR via this signaling pathway in airway epithelium, we investigated the effect of virus-induced EGFR activation on airway epithelial antiviral responses. CXCL10, a chemokine produced by airway epithelial cells in response to respiratory viral infection, contributes to the recruitment of lymphocytes to target and kill virus-infected cells. While respiratory viruses activate EGFR, the interaction between CXCL10 and EGFR signaling pathways is unclear, and the potential for EGFR signaling to suppress CXCL10 has not been explored. Here, we report that respiratory virus-induced EGFR activation suppresses CXCL10 production. We found that influenza virus-, rhinovirus-, and RSV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1-dependent CXCL10 production. In addition, inhibition of EGFR during viral infection augmented IRF1 and CXCL10. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.

Respiratory virus-induced EGFR activation suppresses IRF1-dependent interferon λ and antiviral defense in airway epithelium.

Viruses suppress host responses to increase infection, and understanding these mechanisms has provided insights into cellular signaling and led to novel therapies. Many viruses (e.g., Influenza virus, Rhinovirus [RV], Cytomegalovirus, Epstein-Barr virus, and Hepatitis C virus) activate epithelial epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, but the role of EGFR in viral pathogenesis is not clear. Interferon (IFN) signaling is a critical innate antiviral host response and recent experiments have implicated IFN-λ, a type III IFN, as the most significant IFN for mucosal antiviral immune responses. Despite the importance of IFN-λ in epithelial antiviral responses, the role and mechanisms of epithelial IFN-λ signaling have not been fully elucidated. We report that respiratory virus-induced EGFR activation suppresses endogenous airway epithelial antiviral signaling. We found that Influenza virus- and RV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1-induced IFN-λ production and increased viral infection. In addition, inhibition of EGFR during viral infection augmented IRF1 and IFN-λ, which resulted in decreased viral titers in vitro and in vivo. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.

Normal CFTR inhibits epidermal growth factor receptor-dependent pro-inflammatory chemokine production in human airway epithelial cells.

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis, a disease characterized by exaggerated airway epithelial production of the neutrophil chemokine interleukin (IL)-8, which results in exuberant neutrophilic inflammation. Because activation of an epidermal growth factor receptor (EGFR) signaling cascade induces airway epithelial IL-8 production, we hypothesized that normal CFTR suppresses EGFR-dependent IL-8 production and that loss of CFTR at the surface exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade. We examined this hypothesis in human airway epithelial (NCI-H292) cells and in normal human bronchial epithelial (NHBE) cells containing normal CFTR treated with a CFTR-selective inhibitor (CFTR-172), and in human airway epithelial (IB3) cells containing mutant CFTR versus isogenic (C38) cells containing wild-type CFTR. In NCI-H292 cells, CFTR-172 induced IL-8 production EGFR-dependently. Pretreatment with an EGFR neutralizing antibody or the metalloprotease TACE inhibitor TAPI-1, or TACE siRNA knockdown prevented CFTR-172-induced EGFR phosphorylation (EGFR-P) and IL-8 production, implicating TACE-dependent EGFR pro-ligand cleavage in these responses. Pretreatment with neutralizing antibodies to IL-1R or to IL-1alpha, but not to IL-1beta, markedly suppressed CFTR-172-induced EGFR-P and IL-8 production, suggesting that binding of IL-1alpha to IL-1R stimulates a TACE-EGFR-IL-8 cascade. Similarly, in NHBE cells, CFTR-172 increased IL-8 production EGFR-, TACE-, and IL-1alpha/IL-1R-dependently. In IB3 cells, constitutive IL-8 production was markedly increased compared to C38 cells. EGFR-P was increased in IB3 cells compared to C38 cells, and exaggerated IL-8 production in the IB3 cells was EGFR-dependent. Activation of TACE and binding of IL-1alpha to IL-1R contributed to EGFR-P and IL-8 production in IB3 cells but not in C38 cells. Thus, we conclude that normal CFTR suppresses airway epithelial IL-8 production that occurs via a stimulatory EGFR cascade, and that loss of normal CFTR activity exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade.

John Widdicombe's contribution to respiratory physiology and cough: reminiscences.

John Widdicombe has made substantial contributions to respiratory physiology and to the field of cough particularly. He was one of the first to characterise Aδ-myelinated fibres in the airways that could mediate cough and increased breathing. Later on, he initiated the series of international London Cough Symposia that gathered researchers and clinicians on a two-yearly basis to discuss recent results and concepts regarding cough. John Widdicombe was interested in all aspects of cough from the definition to potential new antitussives. This article will focus on his contributions and on his generous personality through reminiscences from three friends.

Mucous hypersecretion and relationship to cough.

A variety of foreign "invaders" such as viruses, bacteria and other particulates e.g., cigarette smoke, are inhaled, deposit on the airway surface and invade the "host." Mucins produced by the surface airway epithelium and by the submucosal glands are secreted into the airway lumen. Deposited particulates adhere to the mucus and are cleared via mucociliary transport and via cough. Mucins are major constituents of mucus, which is important in the clearance of inhaled materials. Normally, secreted mucus is cleared without symptoms or interference with lung function. However, in obstructive airway diseases such as COPD, asthma, and cystic fibrosis, excessive mucus is produced. Because of the prominence of mucous hypersecretion as a cause of cough, this discussion focuses on mechanisms regulating normal production of mucins and the mechanisms underlying exaggerated mucin secretion in chronic obstructive airway diseases. Mucins are produced by airway epithelial cells via a cascade of signals (the Epidermal Growth Factor Cascade) and secreted on the luminal epithelial surface, often in response to the deposition of inhaled irritants. Normally, only minimal amounts of mucins are secreted, which assist in clearance of the inhaled particulates. However, in disease, additional pathways are induced via positive feedback systems, which lead to mucous hypersecretion. In the large conducting airways, where cough receptors are concentrated, mucous hypersecretion causes stimulation of neural receptors that result in cough. However, in small airways (e.g., bronchioles), because of their small diameters, mucous hypersecretion leads to plugging of the airways. Because there are so many small airways, their plugging is difficult to detect early, and this locus is known as a "silent zone." In chronic obstructive airway diseases, plugging of small airways may persist and increase over time, finally resulting in severe airway obstruction. Different obstructive airway diseases induce inflammatory signaling (including mucous hypersecretion) via different stimuli, but often via similar signaling pathways. Application of present knowledge of signaling that occurs with mucous hypersecretion can lead to novel therapies for hypersecretion and cough induced in conducting airways and could prevent plugging in small airways that can lead to clinical deterioration and death.

Epidermal growth factor receptor reactivation induced by E-prostanoid-3 receptor- and tumor necrosis factor-alpha-converting enzyme-dependent feedback exaggerates interleukin-8 production in airway cancer (NCI-H292) cells.

Airway epithelial cancer cells produce increased amounts of the chemokine interleukin-8 (IL-8), inducing pro-tumor responses. Multiple stimuli induce airway epithelial IL-8 production epidermal growth factor receptor (EGFR) dependently, but the mechanisms that exaggerate IL-8 production in airway cancers remain unknown. Here we show that direct activation of EGFR (EGFR-P) by its ligand transforming growth factor (TGF)-alpha induces a second EGFR-P in human airway (NCI-H292) cancer cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating IL-8 production in these cancer cells. The second EGFR-P in NCI-H292 cells was caused by metalloprotease TNF-alpha-converting enzyme (TACE)-dependent cleavage of EGFR pro-ligands and was responsible for most of the total IL-8 induced by TGF-alpha. In NCI-H292 cells, TGF-alpha induced cyclooxygenase (COX)-2-dependent prostaglandin (PG)E2 production and release. PGE2 increased the second EGFR-P and IL-8 production via binding to its Gi-protein-coupled E-prostanoid (EP)3 receptor. In NHBE cells, TGF-alpha-induced EGFR-P did not lead to PGE2 production or to a second EGFR-P, and less IL-8 was produced. Thus, we conclude that a positive feedback pathway involving COX-2/PGE2/EP3 receptor-dependent EGFR reactivation exaggerates IL-8 production in NCI-H292 cancer cells but not in NHBE (normal) cells.

CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells.

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.

Twenty-five years of airway research: personal thoughts.

The airway research presented for 25 years annually at the Transatlantic Airway Conferences is impressive! The imaginative Conference design, the quality of speakers, and the generosity of the sponsor are outstanding. The breadth of the Conferences is too broad for an objective review here. Instead, I offer some subjective thoughts by one who has observed the evolution of modern pulmonology. Pulmonology came late as a specialty. The National Institutes of Health deserves credit for recognizing the increasing importance of lung disease through its funded grants and changing the designation to the Heart and Lung Institute. The combined effect of enthusiastic young investigators and an increasing commitment from what was to become the National Heart, Lung, and Blood Institute provided us pulmonologists with a jump start. Here, I note the importance of the emergence of modern biology in shaping evolving airway research, and our special opportunities because of accessible anatomic features of airways. My presentation of opportunities here in immunology and in signaling may be the result of individual prejudice! Cystic fibrosis was chosen for discussion because of the importance of cloning the cystic fibrosis transmembrane regulator gene and the limited novel therapies thus far, with a plea for new ideas. The gene was (brilliantly) cloned 2 decades ago, but insights that give rise to novel life-saving therapies are still lacking. I finish with accolades to those who seek to learn from a variety of sources, including artists; I am proud and grateful that I became an early convert from cardiology to pulmonology. It continues to be an exciting experience.

Fibrinogen binding to ICAM-1 promotes EGFR-dependent mucin production in human airway epithelial cells.

Mucous hypersecretion is a serious feature of chronic airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Although mucins are produced via activation of an EGF receptor (EGFR) signaling cascade, the mechanisms leading to exaggerated mucin production in mucous hypersecretory diseases are unknown. Because expression of ICAM-1 and of the ICAM-1 ligand fibrinogen is increased in the airways of subjects with mucous hypersecretory diseases, we hypothesized that fibrinogen binding to ICAM-1 could increase EGFR-dependent mucin production in human airway (NCI-H292) epithelial cells. Consistent with this hypothesis, we found that an ICAM-1 neutralizing antibody and an ICAM-1(8-22) peptide that binds fibrinogen decreased mucin production induced by the EGFR ligand transforming growth factor (TGF)-alpha dose-dependently. Exogenous fibrinogen and a fibrinogen(117-133) peptide that binds ICAM-1 rescued mucin production in cells treated with the ICAM-1(8-22) peptide. Surprisingly, the ICAM-1(8-22) peptide increased EGFR phosphotyrosine and phospho-ERK1/2 in cells treated with TGF-alpha. The ICAM-1(8-22) peptide-induced increases in EGFR phosphotyrosine and phospho-ERK1/2 were prevented by exogenous fibrinogen, by the fibrinogen(117-133) peptide, and by selective inhibitors of phospholipase C (PLC), protein kinase C (PKC)-alpha/beta, and metalloproteases. These results suggest that fibrinogen binding to ICAM-1 promotes mucin production by decreasing TGF-alpha-induced EGFR and ERK1/2 activation and that the fibrinogen-ICAM-1-dependent decrease in EGFR and ERK1/2 activation occurs via inhibition of an early positive feedback pathway involving PLC- and PKC-alpha/beta-dependent metalloprotease activation and subsequent metalloprotease-dependent EGFR reactivation.

Lipopolysaccharide initiates a positive feedback of epidermal growth factor receptor signaling by prostaglandin E2 in human biliary carcinoma cells.

Bacterial products (e.g., LPS) are viewed as critical stimuli in inflammation-associated cancer. Cyclooxygenase 2 (COX-2), a major effector of LPS, and EGFR, are key to carcinogenesis, notably in the hepatobiliary tract. In this study, we tested the hypothesis that LPS can initiate an interaction between the epidermal growth factor receptor (EGFR) and COX-2 pathways. We examined the effect of LPS in biliary carcinoma cells that displayed constitutive COX-2 expression and PGE(2) production and in normal human biliary epithelial cells in which COX-2/PGE(2) expression was virtually absent. LPS induced early phosphorylation of EGFR and ERK1/2 in both types of cells, which reached maximum levels within 30 min (first phase). However, only the carcinoma cells showed a second significant rise in both EGFR and ERK phosphorylation 6 h after exposure to LPS (second phase). Inhibition of COX-2/PGE(2) production prevented the second, but not the first, phase of EGFR and ERK1/2 phosphorylation, implicating COX-2/PGE(2) in the second phase of phosphorylation. LPS induced COX-2-derived PGE2 production at 4 h, which was before the rise in the second phosphorylation that occurred at 6 h. Exogenous PGE(2) also caused EGFR activation via a signaling pathway involving TACE-dependent TGF-alpha release. Inhibition of the second phase of EGFR phosphorylation with EGFR or COX-2 inhibitor prevented LPS-induced cell invasion in vitro, demonstrating the biological importance of this COX-2 feedback signaling in cancer cells. We conclude that LPS triggers a positive feedback loop involving COX-2/PGE(2) in biliary carcinoma cells and that this second phase of EGFR phosphorylation is implicated in cell invasion by LPS.