Bernard M. Fischer

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

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Pathogenic triad in COPD: oxidative stress, protease–antiprotease imbalance, and inflammation

Patients with chronic obstructive pulmonary disease (COPD) exhibit dominant features of chronic bronchitis, emphysema, and/or asthma, with a common phenotype of airflow obstruction. COPD pulmonary physiology reflects the sum of pathological changes in COPD, which can occur in large central airways, small peripheral airways, and the lung parenchyma. Quantitative or high-resolution computed tomography is used as a surrogate measure for assessment of disease progression. Different biological or molecular markers have been reported that reflect the mechanistic or pathogenic triad of inflammation, proteases, and oxidants and correspond to the different aspects of COPD histopathology. Similar to the pathogenic triad markers, genetic variations or polymorphisms have also been linked to COPD-associated inflammation, protease–antiprotease imbalance, and oxidative stress. Furthermore, in recent years, there have been reports identifying aging-associated mechanistic markers as downstream consequences of the pathogenic triad in the lungs from COPD patients. For this review, the authors have limited their discussion to a review of mechanistic markers and genetic variations and their association with COPD histopathology and disease status.

Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators

Deleterious environmental stimuli cause the airway epithelium to respond with increased secretions of mucus, reaction of oxygen/nitrogen species, changes in ciliary beating, and the influx of inflammatory cells. The epithelium is a target for factors released by infiltrating inflammatory cells, and has recently been shown to serve as an effector of such inflammation. Molecular mechanisms regulating production of secondary inflammatory mediators (cytokines, lipid mediators, and reactive oxygen/nitrogen species) have yet to be fully described. This report reviews the production of secondary mediators by epithelial cells and by airway epithelium. Lipid mediators are enzymatically produced by the airway epithelium in response to primary mediators. Molecular mechanisms regulating the production of cyclo-oxygenase, lipoxygenase and prostaglandin synthase are discussed, along with the potential of lipid mediators to produce inflammation. The molecular regulation of nitric oxide production is also described in the context of its role as a signalling molecule in pathways regulating secretion of mucus, ciliary motion, and intercellular adhesion molecule-1 (ICAM-1) expression. The production of cytokines by the airway epithelium is shown to play a role in causing inflammation associated with respiratory diseases. Particular attention is paid to molecular mechanisms governing the expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8).

Proteases and cystic fibrosis

Cystic fibrosis is the most common, inherited fatal disease in Caucasians. The major cause of morbidity and mortality is chronic lung disease due to infection and inflammation in the airways leading to bronchiectasis and respiratory failure. The signature pathologic features of CF lung disease including abnormal mucus obstructing airways, chronic infection with Staphylococcus aureus, Pseudomonas aeruginosa and other gram negative bacteria, and a robust neutrophil-dominant airway inflammation, are exacerbated by unopposed proteases present at high concentrations in the ASL. There is strong evidence that proteases, particularly neutrophil elastase, contribute to the pathology of CF by impairing mucociliary clearance, interfering with innate immune functions, and perpetuating neutrophilic inflammation. The mechanisms employed by proteases to impact airway function in CF will be reviewed.

CONCURRENT PRODUCTION OF REACTIVE OXYGEN AND NITROGEN SPECIES BY AIRWAY EPITHELIAL CELLS IN VITRO

Intracellularly generated reactive species of both oxygen (ROS) and nitrogen (RNS) have been implicated in signaling responses in airway epithelial cells, but these radicals have not been measured directly in such cells. In this study, intracellular production of both ROS and RNS were measured in the same cell lysates of guinea pig tracheal epithelial (GPTE) cells maintained in primary culture. ROS and RNS were quantified under basal (constitutive) conditions and in response to different stimuli: LPS and TNFalpha [activators of inducible nitric oxide synthase (iNOS)]; several activators of calcium-dependent cNOS (ATP, bradykinin, ionophore A23187, and thapsigargin); and exogenous oxidant stress generated by addition of xanthine oxidase to purine (p + XO). Studies with LPS and TNFalpha also were performed using the murine macrophage cell line, RAW 264.7, as a positive control. Intracellular oxidant production was detected from oxidation of dihydrorhodamine to rhodamine. NOx was quantified by either chemiluminescent or fluorescent detection. NOS activity was measured as citrulline production from arginine. Basal production of oxidants by GPTE cells (0.08 + 0.00 nmol rhodamine) was less than 10% that of RAW.267 cells (0.91 + 0.03 nmol rhodamine). TNFalpha and LPS significantly increased intracellular oxidant production in GPTE cells, as did p + XO, but none of the cNOS activators affected production of oxidants in these cells. Concentrations of NO2 after 4 h in unstimulated RAW 264.7 and GPTE cells were similar and comprised 63% of total NOx in GPTE and 62% in RAW cells. TNFalpha and LPS both increased NO2 in GPTE cells, but none of the Ca++-mobilizing agents nor p + XO significantly affected intracellular RNS. The results suggest both ROS and RNS can be measured in the same lysates from airway epithelial cells, and that both ROS and RNS are produced in these cells in response to different stimuli.

COPD: balancing oxidants and antioxidants

Chronic obstructive pulmonary disease (COPD) is one of the most common chronic illnesses in the world. The disease encompasses emphysema, chronic bronchitis, and small airway obstruction and can be caused by environmental exposures, primarily cigarette smoking. Since only a small subset of smokers develop COPD, it is believed that host factors interact with the environment to increase the propensity to develop disease. The major pathogenic factors causing disease include infection and inflammation, protease and antiprotease imbalance, and oxidative stress overwhelming antioxidant defenses. In this review, we will discuss the major environmental and host sources for oxidative stress; discuss how oxidative stress regulates chronic bronchitis; review the latest information on genetic predisposition to COPD, specifically focusing on oxidant/antioxidant imbalance; and review future antioxidant therapeutic options for COPD. The complexity of COPD will necessitate a multi-target therapeutic approach. It is likely that antioxidant supplementation and dietary antioxidants will have a place in these future combination therapies.

Maternal Exposure to Particulate Matter Increases Postnatal Ozone-induced Airway Hyperreactivity in Juvenile Mice

RATIONALE Epidemiologic studies implicate air pollutant exposure during pregnancy as a risk factor for wheezing in offspring. Ozone exposure is linked to exacerbations of wheezing in children. OBJECTIVES To determine if maternal pulmonary exposure to traffic-related particles during pregnancy augments ozone-induced airway hyperresponsiveness in offspring. METHODS C57BL6 time-mated mice were given NIST SRM#1648 (particulate matter [PM]) 0.48 mg, saline vehicle, or no treatment by tracheal insufflation twice weekly for 3 weeks. PM exposure augmented maternal lung inflammation and placental TNF-alpha, Keratinocyte-derived cytokine (KC), and IL-6 (measured at gestation Day 18). After parturition, dams and litters were exposed to air or ozone 1 ppm 3 h/d, every other day, thrice weekly for 4 weeks. Respiratory system resistance in pups was measured at baseline and after administration of nebulized methacholine. MEASUREMENTS AND MAIN RESULTS Ozone increased airway hyperresponsiveness, but the increase was greatest in pups born to PM-treated dams. Whole-lung TNF-alpha, IL-1beta, KC, IL-6, and MCP-1 were increased in ozone-treated pups, with the greatest increase in pups born to dams given PM. Airway epithelial mucous metaplasia estimated by periodic acid-Schiff Alcian blue staining was increased in ozone-exposed pups born to PM-treated dams. Alveolar development, determined by morphometry, and airway smooth muscle bulk, estimated using alpha-actin histochemistry, were unaffected by prenatal or postnatal treatment. CONCLUSIONS Maternal pulmonary exposure to PM during pregnancy augments placental cytokine expression and postnatal ozone-induced pulmonary inflammatory cytokine responses and ozone-induced airway hyperresponsiveness without altering airway structure.

Effects of TNFα on expression of ICAM-1 in human airway epithelial cells in vitro: Oxidant-mediated pathways and transcription factors

Abstract We demonstrate that two different cell-permeable antioxidants, pyrrolidine dithiocarbamate (PDTC) and dimethylthiourea (DMTU), inhibit TNFα-induced ICAM-1 surface and gene expression in primary cultures of differentiated normal human bronchial epithelial (NHBE) cells. In addition, TNFα stimulates binding of nuclear proteins to the nuclear factor kappa beta (NFκB) and the CAAT/enhancer binding protein (C/EBP) consensus sites in the ICAM-1 promoter in these cells. Because these transcription factors have been suggested to be oxidant-sensitive and important in ICAM-1 expression, the potential involvement of reactive oxygen species (ROS) in the response to TNFα was investigated. Interestingly, neither PDTC nor DMTU altered binding of NFκB complexes. In contrast, either the proteasome inhibitor carbobenzoxy-L-leucy-L-leucy-L-leucinal (MG 132) or the IκBα inhibitor BAY 11-7082 ablated TNFα-induced ICAM-1 gene expression and MG132 inhibited TNFα-induced NFκB complexes. Surprisingly, either PDTC or DMTU inhibited the binding of TNFα-enhanced C/EBP complexes to the consensus site directly adjacent to the NFκB site. These results suggest that although TNFα enhances binding of C/EBP and NFκB complexes in NHBE cells, C/EBP binding seems to involve an oxidant-dependent mechanism, whereas activation of NFκB complexes utilizes the ubiquitin-proteasome pathway, a mechanism that seems to be unaltered by the presence of antioxidants. Because interference with either signaling pathway abrogates TNFα-induced ICAM-1 expression, activation of both complexes seems to be involved in this response to TNFα, but this activation occurs via different intracellular pathways.

NAD(P)H Quinone Oxidoreductase 1 Is Essential for Ozone-Induced Oxidative Stress in Mice and Humans

One host susceptibility factor for ozone identified in epidemiologic studies is NAD(P)H quinone oxidoreductase 1 (NQO1). We hypothesized that after ozone exposure, NQO1 is required to increase 8-isoprostane (also known as F(2)-isoprostane) production, a recognized marker of ozone-induced oxidative stress, and to enhance airway inflammation and hyperresponsiveness. In this report, we demonstrate that in contrast to wild-type mice, NQO1-null mice are resistant to ozone and have blunted responses, including decreased production of F(2)-isoprostane and keratinocyte chemokine, decreased airway inflammation, and diminished airway hyperresponsiveness. Importantly, these results in mice correlate with in vitro findings in humans. In primary human airway epithelial cells, inhibition of NQO1 by dicumarol blocks ozone-induced F(2)-isoprostane production and IL-8 gene expression. Together, these results demonstrate that NQO1 modulates cellular redox status and influences the biologic and physiologic effects of ozone.

Tumor Necrosis Factor Alpha (TNFα)‐Induced ICAM‐1 Surface Expression in Airway Epithelial Cells in vitro: Possible Signal Transduction Mechanismsa

Within the past several years research on the interaction of cytokines and adhesion molecules with airway epithelium in diseases has allowed us to develop a better understanding of the disease process. The cytokine, TNF alpha and the adhesion molecule ICAM-1 are important mediators in the pathogenesis of airway diseases such as asthma, chronic bronchitis, and adult respiratory distress syndrome. Effects of TNF alpha on ICAM-1 surface expression was investigated in both primary cultures of normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cell line BEAS-2B. TNF alpha (0.015-150 ng/mL) significantly enhanced ICAM-1 surface expression (measured by flow cytometry) in a dose and time-dependent manner, with peak expression seen at 24 hours. This response was negated by heat inactivation of the TNF alpha prior to incubation. TNF alpha-induced ICAM-1 expression also was inhibited by pre- and coincubation of TNF alpha with 3 micrograms/mL soluble TNF-R1 or by the PKC inhibitor, Calphostin C (0.1 and 0.5 microM). The ROI scavengers, dimethylthiourea (4 mM), and dimethyl sulfoxide (0.001%), enhanced TNF alpha-induced ICAM-1 expression. Collectively, these results indicate that TNF alpha-induced ICAM-1 surface expression is a specific receptor-mediated response (TNF-R1), which is mediated by mechanisms dependent on PKC and intracellular reactive oxygen species.