Azithromycin inhibits mucin secretion, mucous metaplasia, airway inflammation, and airways hyperresponsiveness in mice exposed to house dust mite extract.

Excessive production, secretion, and retention of abnormal mucus is a pathological feature of many obstructive airways diseases including asthma. Azithromycin is an antibiotic that also possesses immunomodulatory and mucoregulatory activities, which may contribute to the clinical effectiveness of azithromycin in asthma. The current study investigated these nonantibiotic activities of azithromycin in mice exposed daily to intranasal house dust mite (HDM) extract for 10 days. HDM-exposed mice exhibited airways hyperresponsiveness to aerosolized methacholine, a pronounced mixed eosinophilic and neutrophilic inflammatory response, increased airway smooth muscle (ASM) thickness, and elevated levels of epithelial mucin staining. Azithromycin (50 mg/kg sc, 2 h before each HDM exposure) attenuated HDM-induced airways hyperresponsiveness to methacholine, airways inflammation (bronchoalveolar lavage eosinophil and neutrophils numbers, and IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and RANTES levels), and epithelial mucin staining (mucous metaplasia) by at least 50% (compared with HDM-exposed mice, P < 0.05). Isolated tracheal segments of HDM-exposed mice secreted Muc5ac and Muc5b (above baseline levels) in response to exogenous ATP. Moreover, ATP-induced secretion of mucins was attenuated in segments obtained from azithromycin-treated, HDM-exposed mice (P < 0.05). In additional ex vivo studies, ATP-induced secretion of Muc5ac (but not muc5b) from HDM-exposed tracheal segments was inhibited by in vitro exposure to azithromycin. In vitro azithromycin also inhibited ATP-induced secretion of Muc5ac and Muc5b in tracheal segments from IL-13-exposed mice. In summary, azithromycin inhibited ATP-induced mucin secretion and airways inflammation in HDM-exposed mice, both of which are likely to contribute to suppression of airways hyperresponsiveness.

Functional characterisation and application of an ex vivo perfusion-superfusion system in murine airways.

INTRODUCTION: The aim of this study was to develop two dynamic ex vivo airway explant systems, a perfusion-superfusion system and a ventilation-superfusion system, for the study of toxic airborne substances, such as the prevalent smoke constituent acrolein. METHODS: Mouse isolated tracheal segments were perfused with physiological media or ventilated with humidified air at 37°C to mimic dynamic flow conditions, and superfused with media over the exterior surface. At selected time points, the histological and functional integrity of segments was evaluated. The perfusion-superfusion system was subsequently used to examine mucin secretory responses elicited by acrolein in airways in which mucous metaplasia had been induced with lipopolysaccharide (LPS; 1µgml-1) prior to 24h of media perfusion, followed by stimulation with acrolein or ATP for 15min. Epithelial mucin levels were determined by quantitative analysis of periodic acid-Schiff's reagent (PAS)-stained sections. RESULTS: Epithelial morphology was successfully preserved in the perfusion-superfusion and ventilation-superfusion systems for at least 24h and up to 18h, respectively. At these time points, the contractile and relaxation responses of perfused and ventilated tracheal segments to carbachol, the neuropeptide substance P, and the prostanoid PGE2 were also preserved. Using the perfusion-superfusion system, acute exposure to acrolein caused a dose-dependent reduction in the levels of PAS-positive mucin stores induced by LPS, consistent with mucin secretion. DISCUSSION: Both the perfusion-superfusion and ventilation-superfusion systems successfully preserved the viability of mouse isolated tracheal segments on a histological and functional level, and the perfusion-superfusion system was used to characterise the mucin secretory responses elicited by acrolein. Thus, this system may be a useful model through which to conduct further toxicological studies in mammalian airways.

Investigating the role of MRGPRC11 and capsaicin-sensitive afferent nerves in the anti-influenza effects exerted by SLIGRL-amide in murine airways.

BACKGROUND: The hexapeptide SLIGRL-amide activates protease-activated receptor-2 (PAR-2) and mas-related G protein-coupled receptor C11 (MRGPRC11), both of which are known to be expressed on populations of sensory nerves. SLIGRL-amide has recently been reported to inhibit influenza A (IAV) infection in mice independently of PAR-2 activation, however the explicit roles of MRGPRC11 and sensory nerves in this process are unknown. Thus, the principal aim of this study was to determine whether SLIGRL-amide-induced inhibition of influenza infection is mediated by MRGPRC11 and/or by capsaicin-sensitive sensory nerves. METHODS: The inhibitory effect of SLIGRL-amide on IAV infection observed in control mice in vivo was compared to effects produced in mice that did not express MRGPRC11 (mrgpr-cluster∆ (-/-) mice) or had impaired sensory nerve function (induced by chronic pre-treatment with capsaicin). Complementary mechanistic studies using both in vivo and ex vivo approaches investigated whether the anti-IAV activity of SLIGRL-amide was (1) mimicked by either activators of MRGPRC11 (BAM8-22) or by activators (acute capsaicin) or selected mediators (substance P, CGRP) of sensory nerve function, or (2) suppressed by inhibitors of sensory nerve function (e.g. NK1 receptor antagonists). RESULTS: SLIGRL-amide and BAM8-22 dose-dependently inhibited IAV infection in mrgpr-cluster∆ (-/-) mice that do not express MRGPRC11. In addition, SLIGRL-amide and BAM8-22 each inhibited IAV infection in capsaicin-pre-treated mice that lack functional sensory nerves. Furthermore, the anti-IAV activity of SLIGRL-amide was not mimicked by the sensory neuropeptides substance P or CGRP, nor blocked by either NK1 (L-703,606, RP67580) and CGRP receptor (CGRP8-37) antagonists. Direct stimulation of airway sensory nerves through acute exposure to the TRPV1 activator capsaicin also failed to mimic SLIGRL-amide-induced inhibition of IAV infectivity. The anti-IAV activity of SLIGRL-amide was mimicked by the purinoceptor agonist ATP, a direct activator of mucus secretion from airway epithelial cells. Additionally, both SLIGRL-amide and ATP stimulated mucus secretion and inhibited IAV infectivity in mouse isolated tracheal segments. CONCLUSIONS: SLIGRL-amide inhibits IAV infection independently of MRGPRC11 and independently of capsaicin-sensitive, neuropeptide-releasing sensory nerves, and its secretory action on epithelial cells warrants further investigation.

Airborne acrolein induces keratin-8 (Ser-73) hyperphosphorylation and intermediate filament ubiquitination in bronchiolar lung cell monolayers.

The combustion product acrolein is a key mediator of pulmonary edema in victims of smoke inhalation injury. Since studying acrolein toxicity in conventional in vitro systems is complicated by reactivity with nucleophilic culture media constituents, we explored an exposure system which delivers airborne acrolein directly to lung cell monolayers at the air-liquid interface. Calu-3 lung adenocarcinoma cells were maintained on membrane inserts such that the basal surface was bathed in nucleophile-free media while the upper surface remained in contact with acrolein-containing air. Cells were exposed to airborne acrolein for 30 min before they were allowed to recover in fresh media, with cell sampling at defined time points to allow evaluation of toxicity and protein damage. After prior exposure to acrolein, cell ATP levels remained close to controls for 4h but decreased in an exposure-dependent manner by 24h. A loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-labeled dextran preceded ATP loss. Use of antibody arrays to monitor protein expression in exposed monolayers identified strong upregulation of phospho-keratin-8 (Ser(73)) as an early consequence of acrolein exposure. These changes were accompanied by chemical damage to keratin-8 and other intermediate filament family members, while acrolein exposure also resulted in controlled ubiquitination of high mass proteins within the intermediate filament extracts. These findings confirm the usefulness of systems allowing delivery of airborne smoke constituents to lung cell monolayers during studies of the molecular basis for acute smoke intoxication injury.

Early induction of uncoupling protein-2 in pulmonary macrophages in hyperoxia-associated lung injury.

CONTEXT: High concentrations of inspired oxygen contribute to the pathogenesis of neonatal bronchopulmonary dysplasia and adult acute respiratory distress syndrome. Animal models of hyperoxia-associated lung injury (HALI) are characterized by enhanced generation of reactive oxygen species (ROS) and an adaptive antioxidant response. ROS contribute to pathogenesis, partly through enhancing pro-inflammatory activity in macrophages. Uncoupling protein-2 (UCP2) is an inner mitochondrial membrane protein whose expression lowers mitochondrial superoxide (O2ⁱ⁻) production. UCP2, therefore, has potential to contribute to antioxidant response. It is inducible in macrophages. OBJECTIVES AND METHODS: We hypothesized that induction of UCP2 occurred in response to pulmonary hyperoxia in vivo and that expression localized to pulmonary macrophages. We then investigated mechanisms of UCP2 regulation in hyperoxia-exposed macrophages in vitro and correlated changing UCP2 expression with mitochondrial membrane potential (Δψm) and O2ⁱ⁻ production. RESULTS: UCP2 is induced in lungs of mice within 1 h of hyperoxia exposure. Induction occurs in pulmonary alveolar macrophages in vivo, and can be replicated in vitro in isolated macrophages. UCP2 mRNA does not change. UCP2 increases quickly after the first hyperoxia-induced burst of mitochondrial O2ⁱ⁻ generation. Suppression of Δψm and mitochondrial O2ⁱ⁻ production follow and persist while UCP2 is elevated. DISCUSSION AND CONCLUSIONS: Induction of UCP2 is an early response to hyperoxia in pulmonary macrophages. The mechanism is post-transcriptional. UCP2 induction follows a transient rise in mitochondrial ROS generation. The subsequent falls in Δψm and mitochondrial O2ⁱ⁻ support the notion that regulable UCP2 expression in macrophages acts to contain mitochondrial ROS generation. That, in turn, may limit inappropriate pro-inflammatory activation in HALI.

Prior exposure to acrolein accelerates pulmonary inflammation in influenza A-infected mice.

The combustion product acrolein contributes to several smoke-related health disorders, but whether this immunomodulatory toxicant alters pulmonary susceptibility to viruses has received little attention. To study the effects of prior acrolein dosing on the severity of influenza A viral infection, male BALB/c mice received acrolein (1mg/kg) or saline (control) via oropharyngeal aspiration either 4- or 7-days prior to intranasal inoculation with either influenza A/PR/8/34 virus or vehicle. At 0, 2, 4 and 7 days post-inoculation, lung samples were assessed for histological changes while pulmonary inflammation was monitored by estimating immune cell numbers and cytokine levels in bronchoalveolar lavage fluid (BALF). After viral challenge, animals that were exposed to acrolein 4 days previously experienced greater weight loss and exhibited an accelerated inflammatory response at 2 days after viral inoculation. Thus compared to saline-pretreated, virus-challenged controls, BALF recovered from these mice contained higher numbers of macrophages and neutrophils in addition to increased levels of several inflammatory cytokines, including IL-1α, IL-1ß, IL-6, TNF, IFN-γ, KC, and MCP-1. The acrolein-induced increase in viral susceptibility was suppressed by the carbonyl scavenger bisulphite. These findings suggest acute acrolein intoxication "primes" the lung to mount an accelerated immune response to inhaled viruses.

Inhibitors of prostaglandin transport and metabolism augment protease-activated receptor-2-mediated increases in prostaglandin E2 levels and smooth muscle relaxation in mouse isolated trachea.

Stimulants of protease-activated receptor-2 (PAR(2)), such as Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), cause airway smooth muscle relaxation via the release of the bronchodilatory prostanoid prostaglandin E(2) (PGE(2)). The principal aim of the current study was to determine whether compounds that inhibit PGE(2) reuptake by the prostaglandin transporter [bromocresol green and U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF2alpha) and PGE(2) metabolism by 15-hydroxyprostaglandin dehydrogenase (thiazolidenedione compounds rosiglitazone and ciglitazone) significantly enhanced the capacity of SLIGRL to elevate PGE(2) levels and produce relaxation in isolated segments of upper and lower mouse trachea. SLIGRL produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, although both effects were significantly greater in lower tracheal segments than in upper tracheal segments. SLIGRL-induced increases in PGE(2) levels were significantly enhanced in the presence of ciglitazone and rosiglitazone, and these effects were not inhibited by GW9662 (2-chloro-5-nitrobenzanilide), a peroxisome proliferator-activated receptor-gamma antagonist. SLI-GRL-induced relaxation responses were also significantly enhanced by ciglitazone and rosiglitazone, whereas responses to isoprenaline, a PGE(2)-independent smooth muscle relaxant, were unaltered. Ciglitazone and rosiglitazone alone produced concentration-dependent increases in PGE(2) levels and smooth muscle relaxation, and these responses were inhibited by indomethacin, a cyclooxygenase inhibitor. Bromocresol green, an inhibitor of prostaglandin transport, significantly enhanced SLIGRL-induced increases in PGE(2) levels and relaxation. Immunohistochemical staining for 15-hydroxyprostaglandin dehydrogenase was relatively intense over airway smooth muscle, as was staining for the prostaglandin transporter over both airway smooth muscle and epithelium. In summary, inhibitors of PGE(2) reuptake and metabolism significantly potentiate PAR(2)-mediated increases in PGE(2) levels and smooth muscle relaxation in murine-isolated airways.

Altered expression and in vivo lung function of protease-activated receptors during influenza A virus infection in mice.

Protease-activated receptors (PARs) are widely distributed in human airways, and recent evidence indicates a role for PARs in the pathophysiology of inflammatory airway disease. To further investigate the role of PARs in airway disease, we determined the expression and function of PARs in a murine model of respiratory tract viral infection. PAR-1, PAR-2, PAR-3, and PAR-4 mRNA and protein were expressed in murine airways, and confocal microscopy revealed colocalization of PAR-2 and cyclooxygenase (COX)-2 immunostaining in basal tracheal epithelial cells. Elevated levels of PAR immunostaining, which was particularly striking for PAR-1 and PAR-2, were observed in the airways of influenza A/PR-8/34 virus-infected mice compared with sham-infected mice. Furthermore, increased PAR-1 and PAR-2 expression was associated with significant changes in in vivo lung function responses. PAR-1 agonist peptide potentiated methacholine-induced increases in airway resistance in anesthetized sham-infected mice (and in indomethacin-treated, virus-infected mice), but no such potentiation was observed in virus-infected mice. PAR-2 agonist peptide transiently inhibited methacholine-induced bronchoconstriction in sham-infected mice, and this effect was prolonged in virus-infected mice. These findings suggest that during viral infection, the upregulation of PARs in the airways is coupled to increased activation of COX and enhanced generation of bronchodilatory prostanoids.

Role of endothelin-converting enzyme, chymase and neutral endopeptidase in the processing of big ET-1, ET-1(1-21) and ET-1(1-31) in the trachea of allergic mice.

The present study examined the roles of endothelin-converting enzyme (ECE), neutral endopeptidase (NEP) and mast cell chymase as processors of the endothelin (ET) analogues ET-1(1-21), ET-1(1-31) and big ET-1 in the trachea of allergic mice. Male CBA/CaH mice were sensitized with ovalbumin (10 microg) delivered intraperitoneal on days 1 and 14, and exposed to aerosolized ovalbumin on days 14, 25, 26 and 27 (OVA mice). Mice were killed and the trachea excised for histological analysis and contraction studies on day 28. Tracheae from OVA mice had 40% more mast cells than vehicle-sensitized mice (sham mice). Ovalbumin (10 microg/ml) induced transient contractions (15+/-3% of the C(max)) in tracheae from OVA mice. The ECE inhibitor CGS35066 (10 microM) inhibited contractions induced by big ET-1 (4.8-fold rightward shift of dose-response curve; P<0.05), but not those induced by either ET-1(1-21) or ET-1(1-31). The chymase inhibitors chymostatin (10 microM) and Bowman-Birk inhibitor (10 microM) had no effect on contractions induced by any of the ET analogues used. The NEP inhibitor CGS24592 (10 microM) inhibited contractions induced by ET-1(1-31) (6.2-fold rightward shift; P<0.05) but not ET-1(1-21) or big ET-1. These data suggest that big ET-1 is processed predominantly by a CGS35066-sensitive ECE within allergic airways rather than by mast cell-derived proteases such as chymase. If endogenous ET-1(1-31) is formed within allergic airways, it is likely to undergo further conversion by NEP to more active products.

Identification of PLA(2) and alpha-neurotoxin proteins in the venom of Pseudonaja affinis (dugite).

The Western brown snake Pseudonaja affinis (dugite), common to the Perth area of Western Australia, possesses one of the most lethal venoms in the world. Little is known, however, about the toxic protein constituents of the venom, other than those causing coagulopathic and procoagulant effects. The current study was therefore undertaken in order to identify other protein constituents and activities present. Crude venom induced a contraction in rat tracheal preparations through phospholipase A(2) (PLA(2)) activity, as shown by the complete and partial inhibition of contraction by PLA(2) inhibitors 4-bromophenacyl bromide and quinacrine. Further, a reduced degree of smooth muscle contraction in the presence of the leukotriene receptor antagonist SKF104353 suggested that this effect was mediated by leukotriene metabolites. The venom-induced contraction did not reoccur upon a second administration of the venom, despite the muscle retaining its contractile function and appearing histologically undamaged. Chromatographic separation of the protein constituents of the venom showed that PLA(2) activity was associated with all protein fractions. A low-molecular-weight component of the venom was further investigated through N-terminal sequencing and found to possess high identity to the short-chain alpha-neurotoxin family of toxins. Venom activity on cultured rat cardiac myocytes and cultured cortical neurons was also examined. The crude venom was found to temporarily inhibit the beating of the cardiac myocytes, after which the beating resumed erratically. Cortical neurons, however, were irreversibly affected, showing concentration-dependent cell death.