Adenosine promotion of cellular migration in bronchial epithelial cells is mediated by the activation of cyclic adenosine monophosphate-dependent protein kinase A.

Migration of neighboring cells into the injury is important for rapid repair of damaged airway epithelium. We previously reported that activation of the A(2A )receptors (A(2A)ARs) mediates adenosine-stimulated epithelial wound healing, suggesting a role for adenosine in migration. Because A(2A)AR increases cyclic adenosine monophosphate (cAMP) levels in many cells, we hypothesized that cAMP-dependent protein kinase A (PKA) is involved in adenosine-mediated cellular migration. To test this hypothesis, we stimulated a human bronchial epithelial cell line with adenosine and/or A(2A)AR agonist (5'-(N-cyclopropyl)-carboxamido-adenosine [CPCA]) in the presence or absence of adenosine deaminase inhibitor (erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride [EHNA]). Cells treated with adenosine or CPCA demonstrated a concentration-dependent increase in migration. Similar results were observed in the presence and absence of EHNA. To confirm A(2A) involvement, we pretreated the cells for 1 hour with the A(2A) receptor antagonist ZM241385 and then stimulated them with either adenosine or CPCA. To elucidate PKA's role, cells were pretreated for 1 hour with either a PKA inhibitor (KT5720) or a cAMP antagonist analogue (Rp-cAMPS) and then stimulated with adenosine and/or CPCA. Pretreatment with KT5720 or Rp-cAMPS resulted in a significant decrease in adenosine-mediated cellular migration. PKA activity confirmed that bronchial epithelial migration requires cAMP and PKA activity. When cells were wounded and stimulated with CPCA, an increase in PKA activity occurred. Pretreatment for 1 hour with either KT5720 or Rp-cAMPS resulted in a significant decrease in adenosine-mediated PKA activation. These data suggest that adenosine activation of A(2A)AR augments epithelial repair by increasing airway cellular migration by PKA-dependent mechanisms.

TGF-beta1 and serum both stimulate contraction but differentially affect apoptosis in 3D collagen gels.

Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-beta1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-beta1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-beta1 and serum significantly augmented collagen gel contraction. TGF-beta1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-beta1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-beta induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.

Malondialdehyde-acetaldehyde adducts decrease bronchial epithelial wound repair.

Most people who abuse alcohol are cigarette smokers. Previously, we have shown that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, a component of both ethanol metabolism and cigarette smoke, form protein adducts that stimulate protein kinase C (PKC) activation in bronchial epithelial cells. We have also shown that PKC can regulate bronchial epithelial cell wound repair. We hypothesize that bovine serum albumin adducted with malondialdehyde and acetaldehyde (BSA-MAA) decreases bronchial epithelial cell wound repair via binding to scavenger receptors on bronchial epithelial cells. To test this, confluent monolayers of bovine bronchial epithelial cells were grown in serum-free media prior to wounding the cells. Bronchial epithelial cell wound closure was inhibited in a dose-dependent manner (up to 60%) in the presence of BSA-MAA than in media treated cells (Laboratory of Human Carcinogenesis [LHC]-9-Roswell Park Memorial Institute [RPMI]). The specific scavenger receptor ligand, fucoidan, also stimulated PKC activation and decreased wound repair. Pretreatment with fucoidan blocked malondialdehyde-acetaldehyde binding to bronchial epithelial cells. When bronchial epithelial cells were preincubated with a PKC alpha inhibitor, Gö 6976, the inhibition of wound closure by fucoidan and BSA-MAA was blocked. Western blot demonstrated the presence of several scavenger receptors on bronchial epithelial cell membranes, including SRA, SRBI, SRBII, and CD36. Scavenger receptor-mediated activation of PKC alpha may function to reduce wound healing under conditions of alcohol and cigarette smoke exposure where malondialdehyde-acetaldehyde adducts may be present.

Alcohol intake is associated with altered pulmonary function.

Little is known about the effect of moderate alcohol intake on lung function in the general population. Because moderate alcohol intake appears to reduce cardiovascular disease risk, we hypothesized that moderate alcohol intake is associated with better pulmonary function. To test this hypothesis, we examined the association between alcohol intake and pulmonary function, measured by spirometry, in a representative sample of U.S. adults who participated in the Third National Health and Nutrition Examination Survey. A stratified multistage clustered probability design was used to select a population-based sample. Data analyzed included alcohol intake, smoking status, education, body mass, sex, age, race, diabetes status, and CHF status. The Third National Health and Nutrition Examination Survey was conducted from 1988 to 1994 by the National Center for Health Statistics of the Centers for Disease Control and Prevention, Atlanta, GA. We analyzed data from 15,294 study participants who completed extensive questionnaires in the household and a comprehensive physical examination, including pulmonary function testing, either in the household or at a specially equipped mobile examination center. Low-to-moderate alcohol intake was not associated with reduced odds of obstructive lung function. In fact, increased odds for obstructive lung pattern were observed only in former heavy drinkers. In contrast, low-to-moderate alcohol intake was associated with better forced vital capacity and forced exhaled volume in 1s in the absence of obstruction, consistent with reduced odds for lung restriction. Using a logistic regression model, we found that individuals reporting alcohol consumption had a lower risk of lung restriction both before and after adjusting for confounding factors including smoking (P< or =.001). Alcohol intake-related reduced risk for restriction was associated with lower risk of CHF, diabetes, obesity, and lower markers of inflammation (white blood cell, fibrinogen, and C-reactive protein) consistent with less lung congestion, external restriction, and/or lung inflammation. Our analyses indicate that alcohol consumption, even at very modest intake levels, is associated with less lung restriction.

Agricultural lung disease.

Agricultural work is associated with high rates of injury, disability, and illness. Agricultural workers are at increased risk for a variety of illnesses including respiratory disorders, dermatologic conditions, and cancer. The recognition of ODTS led to increased understanding of acute illness in farmers and grain workers. Previously, many cases of acute illness were probably erroneously called farmer's lung. The same agents that are responsible for ODTS are responsible for the high prevalence of bronchitis in certain agricultural workers. The recent description of the innate immune system is very exciting because it will lead to increased understanding of the pathogenesis of organic dust induced disorders.

Ethanol increases phosphodiesterase 4 activity in bovine bronchial epithelial cells.

Ethanol exposure in airway epithelium increases cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. Activation of PKA and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) has been shown to increase ciliary beat frequency (CBF) in bovine bronchial epithelial cells (BBECs). We have shown that biologically relevant concentrations of ethanol stimulate increases in CBF in a nitric oxide-dependent manner, mediated through elevated cAMP levels and subsequent PKA activation. This ethanol-driven rapid and transient increase in CBF occurs 15 to 30 min after exposure to 100 mM ethanol. However, after prolonged exposure to 100 mM ethanol (>/=6 h), CBF and the catalytic activity of PKA return to baseline levels. We hypothesize that cyclic nucleotide-dependent phosphodiesterase (PDE) activity attenuates the duration of ethanol-stimulated ciliary motility. The effect of ethanol on the PDE activity in BBECs was determined through direct assay of catalytic activity. When BBECs were incubated with 100 mM ethanol, significant increases in cAMP levels occurred within 1 h, with corresponding increases in PKA activity. Treatment of BBECs with 100 mM ethanol increased cAMP-PDE activity significantly by 4 h. 3-Isobutyl-1-methylxanthine, Ro 20-1724, and rolipram inhibited ethanol-stimulated cAMP-PDE activity. These agents inhibited ethanol-stimulated cAMP-PDE activity and increased the magnitude of ethanol-stimulated PKA activity observed under the same conditions. These findings support the idea that acute exposure (<6 h) to ethanol increases cAMP levels, and the associated increase in PKA activation is regulated by cAMP-dependent PDE, specifically PDE4. Other compensatory mechanisms however, may be responsible for the down-regulation of PKA, which occurs after chronic epithelial exposure (>/=6 h) to ethanol.

Outpatient management of patients following pulmonary embolism.

Pulmonary embolus is a common reason for hospitalization and requires close follow-up and management in the office setting. The main issues facing the clinician include determination of the appropriate anticoagulation regimen, how long to anticoagulate, and whether an evaluation for hypercoagulable states is indicated. The decisions will depend on individual patient factors and assessment of the risks and benefits for that patient.

Pathogenesis of COPD.

Chronic obstructive pulmonary disease (COPD) is characterized and defined by limitation of expiratory airflow. This can result from several types of anatomical lesions, including loss of lung elastic recoil and fibrosis and narrowing of small airways. Inflammation, edema, and secretions also contribute variably to airflow limitation. Smoking can cause COPD through several mechanisms. First, smoke is a powerful inducer of an inflammatory response. Inflammatory mediators, including oxidants and proteases, are believed to play a major role in causing lung damage. Smoke can also alter lung repair responses in several ways. Inhibition of repair may lead to tissue destruction that characterizes emphysema, whereas abnormal repair can lead to the peribronchiolar fibrosis that causes airflow limitation in small airways. Genetic factors likely play a major role and probably account for much of the heterogeneity susceptibility to smoke and other factors. Many factors may play a role, but to date, only alpha-1 protease inhibitor deficiency has been unambiguously identified. Exposures other than cigarette smoke can contribute to the development of COPD. Inflammation of the lower respiratory tract that results from asthma or other chronic disorders may also contribute to the development of fixed airway obstruction. COPD is not only a disease of the lungs but is also a systemic inflammatory disorder. Muscular weakness, increased risk for atherosclerotic vascular disease, depression, osteoporosis, and abnormalities in fluids and electrolyte balance may all be consequences of COPD. Advances in understanding the pathogenesis of COPD have the potential for identifying new therapeutic targets that could alter the natural history of this devastating disorder.

Ethanol treatment reduces bovine bronchial epithelial cell migration.

BACKGROUND: Chronic ethanol abuse is associated with significant lung disease. Excessive alcohol intake increases risk for a variety of respiratory tract diseases, including pneumonia and bronchitis. Damage to airway epithelium is critical to the pathogenesis of airway disorders such as chronic bronchitis and chronic obstructive pulmonary disease. The ability of the airway epithelium to repair itself is an important step in the resolution of airway inflammation and disease. Ethanol exposure is known to modulate signaling systems in bronchial epithelial cells. We hypothesize that chronic ethanol exposure down-regulates the adenosine 3':5'-cyclic monophosphate signaling cascade in airway epithelial cells, resulting in decreased epithelial cell migration and repair. METHODS: We evaluated the effect of ethanol on primary cultures of bovine bronchial epithelial cells in in vitro models of cell migration, wound repair, cell attachment, and cell spreading. RESULTS: Ethanol causes a concentration-dependent effect on closure of mechanical wounds in cell monolayers. Pretreatment of cells with 100 mm ethanol for 24 hr further slows wound closure. Ethanol pretreatment also reduced the protein kinase A response to wounding and made the cells unresponsive to stimuli of protein kinase A that accelerate wound closure. The effects of ethanol on cell migration in wound closure were confirmed in another assay of migration, the Boyden chamber cell migration assay. Prolonged treatment with ethanol also reduced other cell functions, such as spreading and attachment, which are necessary for epithelial repair. CONCLUSIONS: Ethanol modulates signaling systems that are relevant to airway injury and repair, suggesting that chronic, heavy ethanol ingestion has a detrimental impact on airway repair. Impaired response to inflammation and injury may contribute to chronic airway disease.

Tumor necrosis factor-alpha hyper-responsiveness to endotoxin in whole blood is associated with chronic bronchitis in farmers.

Many farmers experience chronic bronchitis, airflow obstruction, and asthma. It is thought that these respiratory problems may be related to workplace inhalation of organic dust containing endotoxin. The purpose of this study was to determine whether whole blood cytokine responsiveness to endotoxin is associated with airflow disorders (i.e., airflow obstruction, chronic bronchitis, and doctor-diagnosed asthma). Farmers (N = 95) were recruited from a rural cohort study and completed a respiratory symptom and history questionnaire, spirometry, and blood sampling. Blood was incubated 24 hours in the presence and absence of endotoxin and supernatants were analyzed for TNF-alpha, IL-1beta, IL-6, and IL-8. Hypo- or hyper-responsiveness to endotoxin was based on whether cytokine values were in the lower or upper 10% of the group range, respectively. A significant association existed between TNF-alpha hyper-responsiveness and chronic bronchitis. These results indicate that the whole blood cytokine assay may be useful to identify individual responsiveness to endotoxin, and may provide an additional diagnostic tool to evaluate persons potentially at risk for developing chronic bronchitis following exposure to organic dust in the workplace.

Activation of protein kinase A accelerates bovine bronchial epithelial cell migration.

Bronchial epithelial cell migration is required for the repair of damaged airway epithelium. We hypothesized that bronchial epithelial cell migration during wound repair is influenced by cAMP and the activity of its cyclic nucleotide-dependent protein kinase, protein kinase A (PKA). We found that, when confluent monolayers of bronchial epithelial cells are wounded, an increase in PKA activity occurs. Augmentation of PKA activity with a cell-permeable analog of cAMP, dibutyryl adenosine 3',5'-cyclic monophosphate, isoproterenol, or a phosphodiesterase inhibitor accelerated migration of normal bronchial epithelial cells in in vitro wound closure assays and Boyden chamber migration assays. A role for PKA activity was also confirmed with a PKA inhibitor, KT-5720, which reduced stimulated migration. Augmentation of PKA activity reduced the levels of active Rho and the formation of focal adhesions. These studies suggest that PKA activation modulates Rho activity, migration mechanisms, and thus bronchial epithelial repair mechanisms.

Interleukin-4- and interleukin-13-enhanced transforming growth factor-beta2 production in cultured human bronchial epithelial cells is attenuated by interferon-gamma.

Cytokines derived from lymphocytes are believed to play key roles in a variety of diseases, including airway diseases such as asthma. The current study was designed to evaluate the hypothesis that cytokines derived from Th2 cells, interleukin (IL)-4 and IL-13, might contribute to tissue remodeling by modulating the production of transforming growth factor (TGF)-beta. In addition, the ability of interferon (IFN)-gamma, a cytokine derived from Th1 cells that can antagonize many effects of IL-4 and IL-13, was also assessed for its effects on TGF-beta production. IL-4 and IL-13 both stimulated production of TGF-beta2 release from human bronchial epithelial cells in a time- and concentration-dependent manner. Both with and without acidification, TGF-beta2 were detected. Neither TGF-beta1 nor TGF-beta3 was released. In contrast to the stimulatory effect on human bronchial epithelial cells, neither IL-4 nor IL-13 stimulated release of any TGF-beta isoform from human lung fibroblasts. IFN-gamma reduced both basal, IL-4-, and IL-13-stimulated release of TGF-beta2 in human bronchial epithelial cells. The stimulatory effects of IL-4 and IL-13 and the inhibitory effect of IFN-gamma on TGF-beta2 release were paralleled by mRNA levels, as assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In summary, the Th2-derived cytokines, IL-4 and IL-13, can stimulate production of TGF-beta from airway epithelial cells but not from lung fibroblasts. IFN-gamma, in contrast, can inhibit TGF-beta2 release both under basal conditions and following IL-4 or IL-13 stimulation. The ability of these cytokines to modulate TGF-beta release may contribute to both normal airway repair and to the development of subepithelial fibrosis in asthma.

Effect of cigarette smoke on fibroblast-mediated gel contraction is dependent on cell density.

Cigarette smoke exposure has been associated with a variety of diseases, including emphysema. The current study evaluated the interaction of cell density and cigarette smoke extract (CSE) on fibroblast contraction of collagen gels. Protein levels of transforming growth factor (TGF)-beta1, fibronectin, PGE(2), and TGF-beta1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 x 10(5) cell/ml), only 5% CSE augmented contraction in higher-density cultures (3-5 x 10(5) cells/ml). CSE also inhibited fibronectin and TGF-beta1 production in low-density cultures but stimulated fibronectin production in high-density cultures. Active TGF-beta1 was readily detectable only in higher-density cultures and was markedly augmented by 5% CSE. In contrast, although TGF-beta1 mRNA expression was inhibited in high-density cultures by 10% CSE, expression was increased in the presence of 5% CSE. These results suggest that CSE-induced inhibition of low-density fibroblast contraction is due to inhibition of fibronectin production, whereas CSE's stimulatory effect on high-density cells is the result of increased release of TGF-beta1. These effects may help explain the varied pathologies associated with exposure to cigarette smoke.