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.