Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

Mycoplasma pneumoniae is an important cause of respiratory disease, especially in school-age children and young adults. We employed normal human bronchial epithelial (NHBE) cells in air-liquid interface culture to study the interaction of M. pneumoniae with differentiated airway epithelium. These airway cells, when grown in air-liquid interface culture, polarize, form tight junctions, produce mucus, and develop ciliary function. We examined both qualitatively and quantitatively the role of mycoplasma gliding motility in the colonization pattern of developing airway cells, comparing wild-type M. pneumoniae and mutants thereof with moderate to severe defects in gliding motility. Adherence assays with radiolabeled mycoplasmas demonstrated a dramatic reduction in binding for all strains with airway cell polarization, independent of acquisition of mucociliary function. Adherence levels dropped further once NHBE cells achieved terminal differentiation, with mucociliary activity strongly selecting for full gliding competence. Analysis over time by confocal microscopy demonstrated a distinct colonization pattern that appeared to originate primarily with ciliated cells, but lateral spread from the base of the cilia was slower than expected. The data support a model in which the mucociliary apparatus impairs colonization yet cilia provide a conduit for mycoplasma access to the host cell surface and suggest acquisition of a barrier function, perhaps associated with tethered mucin levels, with NHBE cell polarization.M ycoplasma pneumoniae is a human respiratory tract pathogen primarily associated with tracheobronchitis and pneumonia. Infections are typically not life threatening but can be life altering due to the long-term lung damage that can result, including asthma and chronic obstructive pulmonary disease (1). M. pneumoniae initiates colonization of the airway mucosal epithelium via its terminal organelle (2-4). This highly differentiated polar structure functions in adhesion to host cell receptors, gliding motility, and cell division (5-8). Adhesin proteins P1 and P30 localize to the terminal organelle surface, where they participate directly in adherence to host cells and gliding motility (5, 6, 9, 10).Colonization of the human airways requires circumvention of mucociliary defenses, which effectively obstruct, capture, and remove inhaled substances, limiting access to the epithelium (11-13). Previous M. pneumoniae colonization models employed submerged organ and tissue culture systems and have contributed to our current understanding of pathogen-host cell interactions, but they are limited in their ability to accurately reflect the environment of the airway mucosa (3, 4, 14-17). Mycoplasma-host interactions in vivo typically begin at mucosal barriers (11-13), which we define here as including ciliary motion, mucus production, and tight-junction formation (11, 18). Gliding motility is required for lung colonization in experimentally infected hamsters and mice (19,20), and we speculate that this requirement begins with...

Respiratory syncytial virus (RSV) is a common cause of repeat infections throughout life and potentially severe lower respiratory tract illness in infants, young children, and the elderly. RSV proteins have been shown to contribute to immune evasion by several means, including modification of cytokine and chemokine responses whose expression is negatively regulated by suppressor of cytokine signaling (SOCS) proteins. In this study, we examine the role of SOCS1 and SOCS3 regulation of the type I interferon (IFN) response in normal fully-differentiated human bronchial epithelial cells infected with RSV or with an RSV mutant virus lacking the G gene. The results show that RSV G protein modulates SOCS expression to inhibit type I IFN and interferon-stimulated gene (ISG)-15 expression very early as well as late in infection, and that SOCS induction is linked to toll-like receptor (TLR) signaling by RSV F protein, as indicated by interferon-regulatory factor (IRF)-3 activation and nuclear translocation. These findings indicate that RSV surface proteins signal through the TLR pathway, suggesting that this may be an important mechanism to reduce type I IFN expression to aid virus replication.

No abstract

Mycoplasma pneumoniae causes acute and chronic respiratory infections, including tracheobronchitis and community acquired pneumonia, and is linked to asthma and an array of extra-pulmonary disorders. Recently, we identified an ADP-ribosylating and vacuolating toxin of M. pneumoniae, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we analysed CARDS toxin gene (annotated mpn372) transcription and identified its promoter. We also compared CARDS toxin mRNA and protein profiles in M. pneumoniae during distinct in vitro growth phases. CARDS toxin mRNA expression was maximal, but at low levels, during early exponential growth and declined sharply during mid-to-late log growth phases, which was in direct contrast to other mycoplasma genes examined. Between 7% and 10% of CARDS toxin was localized to the mycoplasma membrane at mid-exponential growth, which was reinforced by immunogold electron microscopy. No CARDS toxin was released into the medium. Upon M. pneumoniae infection of mammalian cells, increased expression of CARDS toxin mRNA was observed when compared with SP-4 broth-grown cultures. Further, confocal immunofluorescence microscopy revealed that M. pneumoniae readily expressed CARDS toxin during infection of differentiated normal human bronchial epithelial cells. Analysis of M. pneumoniae-infected mouse lung tissue revealed high expression of CARDS toxin per mycoplasma cell when compared with M. pneumoniae cells grown in SP-4 medium alone. Taken together, these studies indicate that CARDS toxin expression is carefully controlled by environmental cues that influence its transcription and translation. Further, the acceleration of CARDS toxin synthesis and accumulation in vivo is consistent with its role as a bona fide virulence determinant.

Mycoplasma pneumoniae protein P200 was localized to the terminal organelle, which functions in cytadherence and gliding motility. The loss of P200 had no impact on binding to erythrocytes and A549 cells but resulted in impaired gliding motility and colonization of differentiated bronchial epithelium. Thus, gliding may be necessary to overcome mucociliary clearance.Mycoplasma pneumoniae causes tracheobronchitis, bronchopneumonia, and a variety of extrapulmonary manifestations in humans (35). A distinct polar structure (5, 18) mediates host cell attachment (cytadherence) by this novel cell wall-less prokaryote. Electron micrographs of experimentally infected human and animal cells, as well as cells collected from natural M. pneumoniae infections, reveal an intimate relationship between this terminal organelle and the epithelial cell surface (7). The terminal organelle also mediates gliding motility (16,29), which may facilitate mycoplasma traversal of the airway surface liquid to allow access to host receptors on the apical surface of respiratory epithelium and refuge from the turbulent mucociliary escalator.

In horses, BWHE administration causes increases in MPO concentration and activity in laminar tissue and skin and the time of increased MPO concentration correlates with emigration of WBCs from the vasculature. These findings support the hypothesis that activation of peripheral WBCs is an early step in the pathogenesis of acute laminitis.