Thomas O. Moninger

Motile Cilia of Human Airway Epithelia Are Chemosensory

Beat It Primary cilia are specialized organelles that serve important sensory functions in many different tissues and cells, and defects in their structure and function underlie a variety of genetic diseases. In contrast to primary cilia, motile cilia serve a mechanical function. For example, the cilia on airway epithelia remove inhaled material from the lung. Shah et al. (p. 1131, published online 23 July; see the cover; see the Perspective by Kinnamon and Reynolds) now show that these classic motile cilia are also chemosensory. The motile cilia on airway epithelia contain bitter-taste receptors and their associated signaling machinery. Moreover, application of bitter substances triggers an elevation of intracellular Ca2+ levels and increases cilia beat frequency. Thus, in airway epithelia, bitter-taste receptors may be able to detect noxious substances entering the airways and initiate an autonomous defensive mechanism designed to accelerate elimination of the offending compound. Airway epithelia directly sense and respond to noxious substances. Cilia are microscopic projections that extend from eukaryotic cells. There are two general types of cilia; primary cilia serve as sensory organelles, whereas motile cilia exert mechanical force. The motile cilia emerging from human airway epithelial cells propel harmful inhaled material out of the lung. We found that these cells express sensory bitter taste receptors, which localized on motile cilia. Bitter compounds increased the intracellular calcium ion concentration and stimulated ciliary beat frequency. Thus, airway epithelia contain a cell-autonomous system in which motile cilia both sense noxious substances entering airways and initiate a defensive mechanical mechanism to eliminate the offending compound. Hence, like primary cilia, classical motile cilia also contain sensors to detect the external environment.

Reduced Airway Surface pH Impairs Bacterial Killing in the Porcine Cystic Fibrosis Lung

Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO3− transport. Without CFTR, airway epithelial HCO3− secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.

Adenovirus Fiber Disrupts CAR-Mediated Intercellular Adhesion Allowing Virus Escape

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

Segregation of receptor and ligand regulates activation of epithelial growth factor receptor.

Interactions between ligands and receptors are central to communication between cells and tissues. Human airway epithelia constitutively produce both a ligand, the growth factor heregulin, and its receptors—erbB2, erbB3 and erbB4 (refs 1–3). Although heregulin binding initiates cellular proliferation and differentiation, airway epithelia have a low rate of cell division. This raises the question of how ligand–receptor interactions are controlled in epithelia. Here we show that in differentiated human airway epithelia, heregulin-α is present exclusively in the apical membrane and the overlying airway surface liquid, physically separated from erbB2–4, which segregate to the basolateral membrane. This physical arrangement creates a ligand–receptor pair poised for activation whenever epithelial integrity is disrupted. Indeed, immediately following a mechanical injury, heregulin-α activates erbB2 in cells at the edge of the wound, and this process hastens restoration of epithelial integrity. Likewise, when epithelial cells are not separated into apical and basolateral membranes (‘polarized’), or when tight junctions between adjacent cells are opened, heregulin-α activates its receptor. This mechanism of ligand–receptor segregation on either side of epithelial tight junctions may be vital for rapid restoration of integrity following injury, and hence critical for survival. This model also suggests a mechanism for abnormal receptor activation in diseases with increased epithelial permeability.

Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo.

Improving the efficiency of gene transfer remains an important goal in developing new treatments for cystic fibrosis and other diseases. Adenovirus vectors and nonviral vectors each have specific advantages, but they also have limitations. Adenovirus vectors efficiently escape from the endosome and enter the nucleus, but the virus shows limited binding to airway epithelia. Nonviral cationic vectors bind efficiently to the negatively charged cell surface, but they do not catalyze subsequent steps in gene transfer. To take advantage of the unique features of the two different vector systems, we noncovalently complexed cationic molecules with recombinant adenovirus encoding a transgene. Complexes of cationic polymers and cationic lipids with adenovirus increased adenovirus uptake and transgene expression in cells that were inefficiently infected by adenovirus alone. Infection by both complexes was independent of adenovirus fiber and its receptor and occurred via a different cellular pathway than adenovirus alone. Complexes of cationic molecules and adenovirus also enhanced gene transfer to differentiated human airway epithelia in vitro and to the nasal epithelium of cystic fibrosis mice in vivo These data show that complexes of adenovirus and cationic molecules increase the efficiency of gene transfer, which may enhance the development of gene therapy.

The morphologic characteristics of tumor blood vessels as a marker of tumor progression in primary human uveal melanoma: A matched case-control study☆

Nine morphologic patterns of tumor vessels were identified in eyes removed for ciliary body or choroidal melanoma by the examination of tissue sections stained with fluorescein-conjugated Ulex europaeus I using laser scanning confocal microscopy. This technique also highlights intravascular tumor invasion. Each of these nine morphologic patterns of tumor vessels also may be demonstrated by a modification of the periodic acid-Schiff reaction, viewed with a green narrow band pass filter, but this modified histochemical technique does not accurately identify intravascular tumor invasion. Most tumors have a heterogeneous distribution of vascular patterns. Melanomas in two groups of 20 tumors each were matched by tumor size and location (one group of tumors from patients who survived at least 15 years free of metastatic melanoma after enucleation and one group of tumors from patients who died of metastatic melanoma). A matched case-control analysis indicates that the presence of at least one closed vascular loop in a uveal melanoma is the most significant vascular pattern associated with death from metastatic melanoma after enucleation. Closed loops are associated with other histologic features that are predictive of an unfavorable outcome after enucleation: epithelioid cells and mitotic figures. In this preliminary study the formation of closed vascular loops is a marker of tumor progression in ciliary body and choroidal melanomas.

Product ion of the cytokines interleukin 1 and 6 by murine brain microvessel endothelium and smooth muscle pericytes

Murine brain microvessel endothelial cells and smooth muscle/pericytes (SM/P) cells were cultured from newborn BALB/c (normal strain) and SJL/j (autoimmune-prone strain) mice. These cells were evaluated for their ability to produce interleukin (IL)-1 and IL-6 cytokines. The expression of mRNA for IL-1 and IL-6 was shown in highly purified BALB/c endothelial cells and SM/P cells using polymerase chain reaction with specific primers for IL-1 alpha, IL-1 beta and IL-6. IL-6 but not IL-1 mRNA was detected in unstimulated SJL/j brain microvessel cells. The presence of IL-1 and IL-6 mRNA in the BALB/c brain microvessel endothelial cells and SM/P was confirmed by in situ hybridization. By D10.G4.1 assay, unstimulated BALB/c endothelial cells were shown to produce active IL-1 to a higher degree than SM/P. By B9 bioassay, a low amount of active IL-6 was detected in the supernatant of endothelial cells and SM/P. The production of IL-1 and IL-6 in the bioassays was upregulated by lipopolysaccharide (LPS) activation of the cells in a time- and dose-dependent way. IL-6 production was also shown to be upregulated by IL-1 beta activation of the cells. Brain microvessel endothelial cells of SJL/j origin released equivalent amounts of IL-6 compared to endothelial cells of BALB/c origin. However, the production of IL-6 was markedly higher in SM/P of SJL/j origin than in those of BALB/c origin. These observations, together with our previous data showing that brain microvessel SM/P cells produce GM-CSF, emphasize the possibility for active participation of brain microvasculature SM/P as well as endothelium in inflammatory reactions of the central nervous system.

Loss of Anion Transport without Increased Sodium Absorption Characterizes Newborn Porcine Cystic Fibrosis Airway Epithelia

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR⁻(/)⁻ pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR⁻(/)⁻ epithelia showed markedly reduced Cl⁻ and HCO₃⁻ transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR⁻(/)⁻ pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl⁻ conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl⁻ and HCO₃⁻ in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.

Impaired Mucus Detachment Disrupts Mucociliary Transport in a Piglet Model of Cystic Fibrosis

A breathtaking tale of sticky mucus Patients with cystic fibrosis have difficulty breathing because their airways are clogged with thick mucus. Does this mucus accumulate because there is a defect in the way it is produced? Or does it accumulate because of other disease features, such as dehydration or airway wall remodeling? Distinguishing between these possibilities is important for future drug development. In a study of piglets with cystic fibrosis, Hoegger et al. identify mucus production as the primary defect (see the Perspective by Wine). The airway glands of the piglets synthesized strands of mucus normally, but the strands were never released and stayed tethered to the gland ducts. Science, this issue p. 818; see also p. 730 Lung disease in pigs with cystic fibrosis is caused by aberrant tethering of mucus to the airway glands that produce it. [Also see Perspective by Wine] Lung disease in people with cystic fibrosis (CF) is initiated by defective host defense that predisposes airways to bacterial infection. Advanced CF is characterized by a deficit in mucociliary transport (MCT), a process that traps and propels bacteria out of the lungs, but whether this deficit occurs first or is secondary to airway remodeling has been unclear. To assess MCT, we tracked movement of radiodense microdisks in airways of newborn piglets with CF. Cholinergic stimulation, which elicits mucus secretion, substantially reduced microdisk movement. Impaired MCT was not due to periciliary liquid depletion; rather, CF submucosal glands secreted mucus strands that remained tethered to gland ducts. Inhibiting anion secretion in non-CF airways replicated CF abnormalities. Thus, impaired MCT is a primary defect in CF, suggesting that submucosal glands and tethered mucus may be targets for early CF treatment.

Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo.