Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology.

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease.

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

Effects of inhibitors of phosphodiesterase, on antigen-induced bronchial hyperreactivity in conscious sensitized guinea-pigs and airway leukocyte infiltration.

1. The aim of this study was to determine the effects of inhibitors of phosphodiesterase (PDE) on the early and late phase bronchoconstriction in sensitized, conscious guinea-pigs and the subsequent development of acute airway hyperreactivity to the inhaled thromboxane mimetic, U46619, and leukocyte infiltration following ovalbumin (OvA) challenge. 2. Following an inhalation challenge with OvA, there was an early bronchoconstriction which peaked at 15 min with recovery after 3-4 h. A late phase bronchoconstriction occurred between 17 and 24 h after challenge. The PDE 4 inhibitors, Ro 20-1724 (3 mg kg-1, i.p.) and rolipram (1 mg kg-1, i.p.) administered 30 min before and 6 h after antigen challenge (double dosing regimen), did not affect the development of the early or late phase responses. 3. Seventeen to twenty four hours following an acute OvA or saline challenge, a consistently greater bronchoconstrictor response to inhaled U46619 was observed in the OvA challenged group. This increase in responsiveness was significantly attenuated by the administration of Ro 20-1724 and rolipram 30 min before and 6 h after antigen challenge (P < 0.05); this was not attributable to a residual bronchodilator effect of these compounds. There was a trend towards inhibition of the hyperreactivity to U46619 by aminophylline but not by the PDE3 inhibitors, siguazodan or SKF 95654. 4. Aminophylline, rolipram and Ro 20-1724 when administered as the double dose regimen attenuated the rise in macrophages, eosinophils and neutrophils recovered in bronchial lavage fluid 17 to 24 h after antigen challenge. 5. The dose of Ro 20-1724 given at 6 h post challenge was essential for attenuation of airway hyperreactivity and to protect against leukocyte influx. 6. In summary, aminophylline, rolipram and Ro 20-1724 have anti-inflammatory effects against antigen-induced airway leukocyte infiltration. Rolipram and Ro 20-1724 additionally attenuated the development of acute airway hyperreactivity, effects which are probably mediated through inhibition of PDE type 4. A dose of PDE inhibitor 6 h after the antigen challenge appears to be essential to achieve this protection. Inhibitors of PDE type 3 were generally without effect. However, there was no effect of rolipram or Ro 20-1724 on the development of either the early or late phase type responses.

Analysis of the bronchoconstrictor responses to adenosine receptor agonists in sensitized guinea-pig lungs and trachea.

Airway perfused lungs and half-lungs and superfused tracheal spirals from ovalbumin-sensitized guinea pigs were set up. Adenosine and the analogues, 5'-(N-ethylcarboxamido)adenosine (NECA), R-N6-phenylisopropyladenosine (R-PIA), 2-chloroadenosine, N6-2-(4-aminophenyl)ethyladenosine (APNEA) and 5'-AMP yielded bronchoconstrictor responses as increases in perfusion pressure or of tension, respectively, of these two preparations. These responses were greater in tissues from sensitized compared with un-sensitized guinea pigs. Cross-tachyphylaxis occurred between the constrictor responses to adenosine and the other constrictor adenosine agonists which indicated a common site of action. The adenosine transport inhibitors, dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), inhibited the constrictor responses to adenosine and the analogues, except 2-chloroadenosine. This was attributed to a potentiation of the opposing relaxant effects which generally occurred at higher concentrations of the agonists. The P1 purinoceptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline (A1 receptor selective) failed to remove the constrictor responses to adenosine either alone or in the presence of dipyridamole. This suggests that the bronchoconstrictor response of sensitized airways tissues is mediated via the novel xanthine-resistant A3 receptor.

A simple, rapid, and sensitive scintillation proximity assay for the determination of levels of guinea-pig interleukin-5 in bronchoalveolar lavage samples.

This article describes the development and validation of a scintillation proximity assay (SPA) sensitive for guinea-pig interleukin-5 (IL-5). SPA beads were coated with TRFK-5, a monoclonal antibody directed against mouse IL-5, which is known also to bind guinea-pig IL-5. The assay is a simple competitive binding assay between [125I]-rh-IL-5 and the IL-5, in a sample of guinea-pig bronchoalveolar lavage fluid (BALF), for the binding site on the TRFK-5-coated beads. IL-5 levels in BALF ([IL-5]BALF) were shown to increase in guinea-pigs sensitized to ovalbumin (OvA) and challenged with an OvA inhalation. This occurred at a time (24 h) after challenge when there was also a marked eosinophilia. The assay was validated by treating guinea-pigs with a second antibody, Genzyme 2374-01, directed against IL-5. Treatment with this antibody resulted in a significant reduction of the antigen-induced eosinophilia and concentration of [IL-5]BALF. This observation confirms that the IL-5 identified in BALF also cross-reacts with the antibody Genzyme 2374-01. Interestingly, plasma from sensitized, but unchallenged, guinea-pigs also contained detectable levels of IL-5, and the stimulation of plasma protein extravasation (PPE) within the airways with inhaled histamine also induced a rise in [IL-5]BALF. These observations suggest that the plasma may be an additional source of the IL-5 present in the airways of antigen-challenged guinea-pigs.

ENaC-mediated effects assessed by MRI in a rat model of hypertonic saline-induced lung hydration.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) regulates airway mucosal hydration and mucus clearance. The lack of such regulation in cystic fibrosis patients leads to desiccation of the airway lumen, resulting in mucostasis that establishes the environment for infections. Osmotic agents and negative ENaC regulators can be used to restore mucosal hydration. We aimed to assess whether: (i) osmotically driven fluid flux into the rat lung could be quantified in vivo by magnetic resonance imaging (MRI); and (ii) the MRI signals could be modulated through the regulation of ENaC function. EXPERIMENTAL APPROACH: Lung images from spontaneously breathing rats were acquired following intra-tracheal (i.t.) administration of physiological or hypertonic saline (HS). Compounds known to modulate the ENaC function were given i.t. prior to saline. Volumes of fluid signals were quantified on the images. KEY RESULTS: A tonicity-dependent increase in lung fluid was demonstrated following HS administration. Pretreatment with the ENaC blockers, amiloride or 552-02, resulted in an enhancement of HS-induced lung fluid signals, which were detectable for up to 4 h, consistent with a role for ENaC in fluid clearance. Aprotinin, a serine protease inhibitor that attenuates ENaC function, likewise enhanced the HS-induced increase in lung fluid signal, while alpha(1)-anti-trypsin was without significant effect. CONCLUSIONS AND IMPLICATIONS: Proton MRI provides a non-invasive technique for studying modulators of lung fluid hydration in rat lung in vivo. The pharmacological sensitivity of MRI-detected fluid signals is consistent with ENaC-mediated fluid reabsorption after HS. This target-related readout may be used to characterize new ENaC modulators.

The guinea-pig tracheal potential difference as an in vivo model for the study of epithelial sodium channel function in the airways.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) is a key regulator of airway mucosal hydration and mucus clearance. Negative regulation of airway ENaC function is predicted to be of clinical benefit in the cystic fibrosis lung. The aim of this study was to develop a small animal model to enable the direct assessment of airway ENaC function in vivo. EXPERIMENTAL APPROACH: Tracheal potential difference (TPD) was utilized as a measure of airway epithelial ion transport in the guinea-pig. ENaC activity in the trachea was established with a dose-response assessment to a panel of well-characterized direct and indirect pharmacological modulators of ENaC function, delivered by intra-tracheal (i.t.) instillation. KEY RESULTS: The TPD in anaesthetized guinea-pigs was attenuated by the direct ENaC blockers: amiloride, benzamil and CF552 with ED(50) values of 16, 14 and 0.2 microg kg(-1) (i.t.), respectively. 5-(N-Ethyl-N-isopropyl) amiloride, a structurally related compound but devoid of activity on ENaC, was without effect on the TPD. Intra-tracheal dosing of the Kunitz-type serine protease inhibitors aprotinin and placental bikunin, which have previously been demonstrated to inhibit proteolytic activation of ENaC, likewise potently attenuated TPD in guinea-pigs, whereas alpha(1)-antitrypsin and soya bean trypsin inhibitor were without effect. CONCLUSIONS AND IMPLICATIONS: The pharmacological sensitivity of the TPD to amiloride analogues and also to serine protease inhibitors are both consistent with that of ENaC activity in the guinea-pig trachea. The guinea-pig TPD therefore represents a suitable in vivo model of human airway epithelial ion transport.

PDE4 inhibition and a corticosteroid in chronically antigen exposed conscious guinea-pigs.

BACKGROUND: The physiological and pharmacological consequences of repeated aero-allergen challenge have not been previously characterized in conscious, sensitized guinea-pigs. OBJECTIVES: This study was undertaken to compare the effects of two anti-inflammatory compounds, dexamethasone and Ro 20- 1724, on an acute and chronic airway inflammation, in terms of airway function, reactivity and leucocyte infiltration. METHODS: Sensitized guinea-pigs received eight saline or ovalbumin (OvA) inhalation exposures over 4 weeks and either vehicle, the type 4 PDE inhibitor, Ro 20-1724 (3 mgkg(-1)), or dexamethasone (1.5 mg/kg(-1)), 30 min before and 6 h after each challenge. Airway function of the conscious animal (sGaw) was monitored over the duration of the first and final OvA challenge. Airway reactivity to the thromboxane mimetic, U46619, was also determined following the final OvA exposure as was the leucocyte infiltration. RESULTS: The first antigen challenge induced a large early (0-3h) and smaller late (17-24h) bronchoconstrictor response. Neither phase was affected by the drug treatments. The final OvA challenge induced early and late phase bronchoconstrictor responses but of similar magnitude. The late phase was also significantly prolonged. Ro 20-1724 and dexamethasone significantly attenuated both phases. Airway reactivity to the inhaled thromboxane mimetic, U46619, was also significantly enhanced at 120h after the final OvA exposure in contrast to the saline challenged group. This hyperreactivity was attenuated by Ro 20-1724 and dexamethasone. Bronchoalveolar lavage after repeated OvA exposures revealed eosinophilia which was attenuated by Ro 20-1724 and dexamethasone. CONCLUSIONS: This model demonstrates differential airway responses to acute and chronic antigen challenge. Repeated administration of dexamethasone and Ro 20-1724 with each OvA exposure attenuated all of the chronic inflammatory responses: early and late phase responses, hyperreactivity and eosinophilia.

The in vitro and in vivo pharmacology of antisense oligonucleotides targeted to murine Stat6.

OBJECTIVE AND DESIGN: This study was designed to establish whether phosphorothioate (PS) antisense oligonucleotides (AS-ODN) targeted to Stat6 were active in vivo in a mouse model of active sensitisation. MATERIALS: Female Balb/c mice (6-8) per group were used for in vivo study. TREATMENT: Mice were treated with active PS AS-ODNs determined in initial in vitro studies. Compounds were dosed daily (3-30mg/kg i.v.) over the course of sensitisation to ovalbumin. METHODS: Stat6 mRNA and protein levels were determined in the spleen after treatment (quantitative northern and western analysis respectively), in addition to serum IgE (ELISA). ANOVA was used to determine any significant differences between groups. RESULTS: Both of the AS-ODNs tested in vivo, down regulated Stat6 mRNA and protein levels in the spleen by 40-50% although there was no effect on serum IgE. These treatments also induced splenomegaly in vivo and caused splenocyte proliferation in vitro. CONCLUSIONS: The AS-ODNs used can down regulate Stat6 mRNA and protein although not sufficiently to influence IgE-levels. These effects are likely to be complicated in vivo by the immune-stimulation evident as splenomegaly.

The potential roles of cytokines, IL-5 and IL-8, and plasma cortisol in the anti-inflammatory actions of phosphodiesterase inhibitors in sensitized guinea-pig airways.

Ovalbumin (OvA) inhalation by sensitized guinea-pigs caused a pronounced rise in interleukin (IL)-5 in bronchoalveolar lavage (BAL) fluid at both 3 and 24 h after antigen exposure. The increased levels at 24 h were attenuated by the phosphodiesterase inhibitors Ro 20-1724 and aminophylline and by dexamethasone, all of which also attenuated the concurrent lung eosinophilia. The rise in IL-5 at 3 h was additionally attenuated by the PDE3 inhibitor, siguazodan, which failed to attenuate the eosinophilia at 24 h. These results suggest a pivotal action of these compounds on the later rise in IL-5. Ro 20-1724, aminophylline, siguazodan and dexamethasone attenuated a rise in IL-8 levels in BAL fluid at 3 h and the subsequent neutrophilia at 24 h. There was no increase in plasma ACTH at 3 and 24 h after OvA challenge but cortisol levels were elevated at 3 h. This was inhibited by Ro 20-1724, siguazodan and dexamethasone. Thus, elevation of plasma cortisol does not explain the anti-inflammatory actions of these compounds. Aminophylline, however, did raise plasma cortisol at both 3 and 24 h after antigen challenge which may be an important further mechanism of action for this compound.

Temporal relationships between leukocytes, IL-5 and IL-8 in guinea pig lungs, plasma cortisol and airway function after antigen challenge.

OBJECTIVE AND DESIGN: The aim was to determine the time courses for the changes in airway function, airway reactivity, influx of inflammatory cells and levels of the pro-inflammatory cytokines, interleukin (IL)-5 and IL-8 in bronchoalveolar lavage fluid (BALF), and the plasma levels of cortisol and ACTH after antigen challenge to determine whether a temporal link could be established between these events. METHODS: Airway function was measured as specific airway conductance (sGsw) in conscious ovalbumin (OvA)-sensitized guinea pigs using whole body plethysmography at intervals after an inhalation challenge with ovalbumin (0.5% for 10 min). Airway responses to the inhaled spasmogen, U46619 (30 ng/ml, 60 s), were measured at 3, 6 and 24 h after challenge. In separate animals, bronchoalveolar lavage fluid (BALF) was obtained after anaesthetic overdose either before challenge or at 1, 3, 6, 12, or 24 h after OvA challenge. Total and differential cell counts of eosinophils and neutrophils were performed on BALF and levels of IL-5 and IL-8 determined by scintillation proximity assays and ELISA, respectively. Plasma cortisol and ACTH levels were determined by RIA kits in blood removed by cardiac puncture at intervals after challenge. RESULTS: An early phase bronchoconstriction occurred which resolved by 3 h and was followed by a late phase between 17 and 24 h. Airway hyperresponsiveness to inhaled U46619, was evident at 3, 6 and 24 h after antigen challenge. Increased IL-5[BALF] was observed by 60 min post challenge implicating a performed storage site. In contrast, IL-8[BALF] was not raised until 3 h post challenge. There was a significant infiltration of neutrophils and eosinophils by 3 and 6 h, respectively. IL-5[BALF] further increased up to 24 h, during the appearance of the late phase of bronchoconstriction and whilst eosinophilia was maximal. Plasma cortisol levels were increased 1 and 3 hours after antigen challenge, thereafter returning to baseline levels. CONCLUSIONS: The hyperresponsiveness appears to be dissociated from the appearance of eosinophils in lavage fluid. The early appearance of IL-5, however, could be a trigger for the migration of eosinophils and development of hyper-responsiveness. The increased plasma cortisol levels occurring after antigen challenge were presumably due to the stress involved and these would be expected to exert an endogenous anti-inflammatory effect.

Distribution of a 20-mer phosphorothioate oligonucleotide, CGP69846A (ISIS 5132), into airway leukocytes and epithelial cells following intratracheal delivery to brown-norway rats.

PURPOSE: To evaluate the pulmonary distribution of CGP69846A (ISIS 5132), a phosphorothioate oligonucleotide, following intra-tracheal (i.t.) instillation into Brown-Norway rats. METHODS: The pharmacokinetic profile of [3H]-CGP69846A was investigated following i.t. instillation into both naive and inflamed airways of Brown-Norway rats. The cellular distribution was determined using autoradiography, immunohistochemistry and flow cytometry/fluorescence microscopy, in inflamed airways. RESULTS: CGP69846A displayed a dose-dependent lung retention following i.t. administration which was unaffected by local inflammation. Autoradiography and immunohistochemistry showed distribution to alveolar macrophages, eosinophils, bronchial and tracheal epithelium and alveolar cells. Studies with [FITC]-CGP69846A demonstrated a preferential association of oligonucleotide with leukocytes in bronchial lavage fluid of: macrophages > eosinophils = neutrophils >> lymphocytes. CONCLUSIONS: The dose-dependency of lung retention together with cell-specific uptake suggests that the lung can be used as a local target for antisense molecules with potentially minimal systemic effects. Furthermore, the preferential targeting of macrophages and the airway epithelium by oligonucleotides may represent rational cellular targets for antisense therapeutics.

Protease-activated receptor-2-mediated inhibition of ion transport in human bronchial epithelial cells.

A cytoprotective role for protease-activated receptor-2 (PAR2) has been suggested in a number of systems including the airway, and to this end, we have studied the role that PARs play in the regulation of airway ion transport, using cultures of normal human bronchial epithelial cells. PAR2 activators, added to the basolateral membrane, caused a transient, Ca2+-dependent increase in short-circuit current (I(sc)), followed by a sustained inhibition of amiloride-sensitive I(sc). These phases corresponded with a transient increase in intracellular Ca2+ concentration and then a transient increase, followed by decrease, in basolateral K+ permeability. After PAR2 activation and the addition of amiloride, the forskolin-stimulated increase in I(sc) was also attenuated. By contrast, PAR2 activators added to the apical surface of the epithelia or PAR1 activators added to both the apical and basolateral surfaces were without effect. PAR2 may, therefore, play a role in the airway, regulating Na+ absorption and anion secretion, processes that are central to the control of airway surface liquid volume and composition.

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line.

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.