Losartan does not inhibit cigarette smoke-induced lung inflammation in mice.

Chronic Obstructive Pulmonary Disease (COPD) is a progressive lung disease largely caused by cigarette smoking (CS) and is characterized by lung inflammation and airflow limitation that is not fully reversible. Approximately 50% of people with COPD die of a cardiovascular comorbidity and current pharmacological strategies provide little benefit. Therefore, drugs that target the lung and the cardiovascular system concurrently may be an advantageous therapeutic strategy. The aim of this study was to see whether losartan, an angiotensin-II AT1a receptor antagonist widely used to treat hypertension associated with cardiovascular disease, protects against CS-induced lung inflammation in mice. Male BALB/c mice were exposed to CS for 8 weeks and treated with either losartan (30 mg/kg) or vehicle daily. Mice were euthanized and bronchoalveolar lavage fluid (BALF) inflammation, and whole lung cytokine, chemokine and protease mRNA expression assessed. CS caused significant increases in BALF total cells, macrophages, neutrophils and whole lung IL-6, TNF-α, CXCL-1, IL-17A and MMP12 mRNA expression compared to sham-exposed mice. However, losartan only reduced CS-induced increases in IL-6 mRNA expression. Angiotensin-II receptor expression was reduced in lung tissue from CS-exposed mice. In conclusion, losartan did not inhibit CS-induced BALF cellularity despite reducing whole lung IL-6 mRNA and Ang-II receptor expression.

Apocynin and ebselen reduce influenza A virus-induced lung inflammation in cigarette smoke-exposed mice.

Influenza A virus (IAV) infections are a common cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Oxidative stress is increased in COPD, IAV-induced lung inflammation and AECOPD. Therefore, we investigated whether targeting oxidative stress with the Nox2 oxidase inhibitors and ROS scavengers, apocynin and ebselen could ameliorate lung inflammation in a mouse model of AECOPD. Male BALB/c mice were exposed to cigarette smoke (CS) generated from 9 cigarettes per day for 4 days. On day 5, mice were infected with 1 × 10(4.5) PFUs of the IAV Mem71 (H3N1). BALF inflammation, viral titers, superoxide production and whole lung cytokine, chemokine and protease mRNA expression were assessed 3 and 7 days post infection. IAV infection resulted in a greater increase in BALF inflammation in mice that had been exposed to CS compared to non-smoking mice. This increase in BALF inflammation in CS-exposed mice caused by IAV infection was associated with elevated gene expression of pro-inflammatory cytokines, chemokines and proteases, compared to CS alone mice. Apocynin and ebselen significantly reduced the exacerbated BALF inflammation and pro-inflammatory cytokine, chemokine and protease expression caused by IAV infection in CS mice. Targeting oxidative stress using apocynin and ebselen reduces IAV-induced lung inflammation in CS-exposed mice and may be therapeutically exploited to alleviate AECOPD.

Therapeutic potential of Panax ginseng and ginsenosides in the treatment of chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a major global health burden and will become the third largest cause of death in the world by 2030. It is currently believed that an exaggerated inflammatory response to inhaled irritants, in particular cigarette smoke, cause progressive airflow limitation. This inflammation, where macrophages, neutrophils and lymphocytes are prominent, leads to oxidative stress, emphysema, airways fibrosis and mucus hypersecretion. COPD responds poorly to current anti-inflammatory treatments including corticosteroids, which produce little or no benefit. Panax ginseng has a long history of use in Chinese medicine for respiratory conditions, including asthma and COPD. OBJECTIVES: In this perspective we consider the therapeutic potential of Panax ginseng for the treatment of COPD. RESULTS: Panax ginseng and its compounds, ginsenosides, have reported effects through multiple mechanisms but primarily have anti-inflammatory and anti-oxidative effects. Ginsenosides are functional ligands of glucocorticoid receptors and appear to inhibit kinase phosphorylation including MAPK and ERK1/2, NF-κB transcription factor induction/translocation, and DNA binding. They also inhibit pro-inflammatory mediators, TNF-α, IL-6, IL-8, ROS, and proteases such as MMP-9. Panax ginseng protects against oxidative stress by increasing anti-oxidative enzymes and reducing the production of oxidants. CONCLUSION: Given that Panax ginseng and ginsenosides appear to inhibit processes related to COPD pathogenesis, they represent an attractive therapeutic target for the treatment of COPD.

Influenza A virus and TLR7 activation potentiate NOX2 oxidase-dependent ROS production in macrophages.

Influenza A virus infects resident alveolar macrophages in the respiratory tract resulting in Toll like receptor 7 (TLR7) activation that triggers an inflammatory response to resolve the infection. Macrophages are also major sources of reactive oxygen species (ROS) via the NOX2-containing NADPH oxidase. Although ROS are crucial for pathogen clearance, in response to influenza A virus, ROS are touted as being culprit mediators of the lung tissue injury. The aim of the present study was to determine whether influenza A virus infection and TLR7 activation of macrophages, results in alterations in their ROS production. Here we demonstrate using immunofluorescence that influenza A virus (Hong Kong X-31 strain; H3N2) internalizes in RAW264.7 cells and mouse alveolar macrophages within 1 h, resulting in a significant enhancement in the stimulated NOX2 oxidase-dependent oxidative burst, although virus had no effect on basal ROS. The specific TLR7 agonist imiquimod (10 µg/ml) elevated basal superoxide production and, in a similar fashion to influenza A virus, enhanced NOX2 oxidase-dependent oxidative burst. By contrast, the TLR3 agonist, poly I:C (1-100 µg/ml) failed to influence the oxidative burst to NOX2 oxidase. A peptide corresponding to the region 337-348 on p47phox conjugated to a HIV-tat, designed to inhibit the phosphorylation of Ser346 on p47phox suppressed the influenza A virus- and imiquimod-induced enhancement in the oxidative burst. In conclusion, this study demonstrates for the first time that influenza A virus and TLR7 activation enhance the NOX2 oxidase-dependent oxidative burst in macrophages, which might underpin the acute lung injury to influenza A virus infection.

Therapeutic potential of treating chronic obstructive pulmonary disease (COPD) by neutralising granulocyte macrophage-colony stimulating factor (GM-CSF).

Chronic obstructive pulmonary disease (COPD) is a major incurable global health burden and will become the third largest cause of death in the world by 2020. It is currently believed that an exaggerated inflammatory response to inhaled irritants, in particular cigarette smoke, causes progressive airflow limitation. This inflammation, where macrophages and neutrophils are prominent, leads to oxidative stress, emphysema (loss of lung structure), small airways fibrosis and mucus hypersecretion. However, COPD responds poorly to current anti-inflammatory treatments including potent glucocorticosteroids, which produce little or no benefit. In this review we consider the therapeutic potential of targeting granulocyte macrophage-colony stimulating factor (GM-CSF) for the treatment of COPD. GM-CSF is a major regulator of both macrophage and neutrophil activation and survival in the lung-these cells are intimately linked to COPD. Animal data indicates that neutralisation of GM-CSF ameliorates experimental COPD and predicts therapeutic utility in treating stable COPD and treating exacerbations. As such, GM-CSF represents an attractive therapeutic target for the treatment of COPD.

Differential protease, innate immunity, and NF-kappaB induction profiles during lung inflammation induced by subchronic cigarette smoke exposure in mice.

Cigarette smoke exposure is a major determinant of adverse lung health, but the molecular processes underlying its effects on inflammation and immunity remain poorly understood. Therefore, we sought to understand whether inflammatory and host defense determinants are affected during subchronic cigarette smoke exposure. Dose-response and time course studies of lungs from Balb/c mice exposed to smoke generated from 3, 6, and 9 cigarettes/day for 4 days showed macrophage- and S100A8-positive neutrophil-rich inflammation in lung tissue and bronchoalveolar lavage (BAL) fluid, matrix metalloproteinase (MMP) and serine protease induction, sustained NF-kappaB translocation and binding, and mucus cell induction but very small numbers of CD3+CD4+ and CD3+CD8+ lymphocytes. Cigarette smoke had no effect on phospho-Akt but caused a small upregulation of phospho-Erk1/2. Activator protein-1 and phospho-p38 MAPK could not be detected. Quantitative real-time PCR showed upregulation of chemokines (macrophage inflammatory protein-2, monocyte chemoattractant protein-1), inflammatory mediators (TNF-alpha, IL-1beta), leukocyte growth and survival factors [granulocyte-macrophage colony-stimulating factor, colony-stimulating factor (CSF)-1, CSF-1 receptor], transforming growth factor-beta, matrix-degrading MMP-9 and MMP-12, and Toll-like receptor (TLR)2, broadly mirroring NF-kappaB activation. No upregulation was observed for MMP-2, urokinase-type plasminogen activator, tissue-type plasminogen activator, and TLRs 3, 4, and 9. In mouse strain comparisons the rank order of susceptibility was Balb/c > C3H/HeJ > 129SvJ > C57BL6. Partition of responses into BAL macrophages vs. lavaged lung strongly implicated macrophages in the inflammatory responses. Strikingly, except for IL-10 and MMP-12, macrophage and lung gene profiles in Balb/c and C57BL/6 mice were very similar. The response pattern we observed suggests that subchronic cigarette smoke exposure may be useful to understand pathogenic mechanisms triggered by cigarette smoke in the lungs including inflammation and alteration of host defense.

Modelling COPD in mice.

Chronic obstructive pulmonary disease (COPD) is characterised by persistent airflow limitation, neutrophilic inflammation, macrophage accumulation, and the production of cytokines, chemokines and proteases. Cigarette smoking is the major cause of COPD and there is currently no satisfactory therapy to help treat individuals with this disease. A better understanding of the cellular and molecular responses triggered by cigarette smoke may provide new molecular targets for the development of therapeutic agents. This brief review highlights some of the mouse models used to define the cellular, molecular and pathological consequences of cigarette smoke exposure.

Antigen-induced airway inflammation in the Brown Norway rat results in airway smooth muscle hyperplasia.

Asthma is characterized by chronic airways inflammation, airway wall remodeling, and airway hyperresponsiveness (AHR). An increase in airway smooth muscle has been proposed to explain a major part of AHR in asthma. We have used unbiased stereological methods to determine whether airway smooth muscle hyperplasia and AHR occurred in sensitized, antigen-challenged Brown Norway (BN) rats. Ovalbumin (OA)-sensitized BN rats chronically exposed to OA aerosol displayed airway inflammation and a modest level of AHR to intravenously administered ACh 24 h after the last antigen challenge. However, these animals did not show an increase in smooth muscle cell (SMC) number in the left main bronchus, suggesting that short-lived inflammatory mechanisms caused the acute AHR. In contrast, 7 days after the last aerosol challenge, there was a modest increase in SMC number, but no AHR to ACh. Addition of FCS to the chronic OA challenge protocol had no effect on the degree of inflammation but resulted in a marked increase in both SMC number and a persistent (7-day) AHR. These results raise the possibility that increases in airway SMC number rather than, or in addition to, chronic inflammation contribute to the persistent AHR detected in this model.

Thrombin-stimulated DNA synthesis in human cultured airway smooth muscle occurs independently of products of cyclo-oxygenase or 5-lipoxygenase.

Arachidonic acid (AA) liberation and metabolism via cyclo-oxygenase or lipoxygenases may be an important regulatory pathway for mitogenic signalling in human cultured airway smooth muscle (ASM) cells. In cytokine-treated cells, thrombin markedly enhances production of the anti-mitogenic arachidonic acid metabolite, PGE(2). In this study, in the absence of cytokines, we examined the role of endogenous AA metabolism in thrombin-stimulated ASM DNA synthesis. Selective inhibitors of cyclo-oxygenase of 5-lipoxygenase metabolism had no significant effect on 0.3 U/ml thrombin-stimulated DNA synthesis. However, the non-selective, redox-active lipoxygenase inhibitors NDGA and BWA4C inhibited thrombin-stimulated DNA synthesis. Under basal conditions, and following stimulation by thrombin, the levels of the AA metabolites PGE(2), TxA(2), and LTC(4), remained below assay detection limits. Exogenous addition of AA, LTD(4), or 5-, 12-, and 15-HETE and HpETE metabolites had no consistent or substantial stimulatory effect on either basal or thrombin-stimulated DNA synthesis. These data suggest that the non-selective lipoxygenase inhibitors influence DNA synthesis via effects unrelated to lipoxygenase inhibition. The lack of detection of AA metabolites, the lack of influence of selective antagonists/inhibitors of the AA pathway, and the failure of selected AA metabolites to either enhance or directly stimulate DNA synthesis suggest that in the absence of cytokines, cyclo-oxygenase and lipoxygenase metabolism has little role in signalling of human ASM DNA synthesis by thrombin.

Influence of the epithelium on acetylcholine release from parasympathetic nerves of the rat trachea.

1. The present study was undertaken to investigate the influence of the airway epithelium on the release of acetylcholine (ACh) from parasympathetic nerves of the rat trachea. Epithelium-intact and epithelium-denuded preparations of rat trachea were incubated with [3H]-choline to incorporate [3H]-ACh into the cholinergic transmitter stores. Release of radiolabelled transmitter ACh was evoked by electrical field stimulation (60 s trains of 1 ms pulses, 5 Hz, 15 V). 2. Field stimulation both of epithelium-intact and epithelium-denuded radiolabelled tracheal preparations evoked an increase in the efflux of radioactivity; however, the mean stimulation-induced (S-I) efflux from epithelium-denuded preparations (2932 +/- 190 d.p.m., n = 9) was approximately 60% of that from epithelium-intact preparations (4802 +/- 820 d.p.m., n = 11). We have shown previously that, in epithelium-intact (but not epithelium-denuded) tracheal preparations, a substantial proportion of the S-I efflux is resistant to tetrodotoxin (1 microM) and to the removal of extracellular Ca2+, indicating that much of the S-I efflux is not caused by exocytotic release of neuronal [3H]-ACh. In epithelium-denuded tracheal preparations, superfused individually, phosphorylcholine (1 and 100 microM) did not alter S-I efflux. In epithelium-intact tracheal preparations, both in the absence and in the presence of atropine (1 microM), neither N(G)-nitro-L-arginine (100 microM), superoxide dismutase (100 units ml(-1)), indomethacin (10 microM), capsaicin (30 microM) nor alpha-chymotrypsin (1 unit ml(-1)) altered S-I efflux. 3. Experiments were also performed using two tracheal preparations superfused in series. When unlabelled epithelium-intact preparations were present in the upper chamber (superfused first), the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber (superfused second) did not differ significantly from radiolabelled epithelium-denuded preparations superfused individually. Moreover, there was no significant difference in the S-I efflux from radiolabelled epithelium-denuded preparations in the lower chamber between experiments in which the upper chamber contained epithelium-intact or epithelium-denuded preparations. 4. Field stimulation of epithelium-intact tracheal preparations in the upper chamber with 90, 120 and 300-s periods (trains of 1 ms pulses, 5 Hz, 15 V) did not significantly alter the S-I efflux from radiolabelled epithelium-denuded tracheal preparations in the lower chamber. 5. When introduced into the upper (unlabelled epithelium-intact) and subsequently allowed to superfuse the lower (radiolabelled epithelium-denuded) tracheal preparations, the stable cholinomimetic carbachol (3 microM) markedly reduced the S-I efflux whereas ACh (0.1 and 1 microM) had no significant effect. However, in the presence of the anti-cholinesterase neostigmine (1 microM), ACh (1 microM) significantly reduced S-I efflux, indicating that ACh is subject to rapid hydrolysis by cholinesterase enzymes. When atropine (10 microM) was only exposed to radiolabelled epithelium-denuded preparations in the lower chamber, the inhibitory effects of ACh (1 microM) and carbachol (3 microM) on S-I efflux were prevented. 6. In conclusion, the findings of the present study do not support the notion that the airway epithelium exerts an inhibitory influence on ACh release from parasympathetic nerves of the rat trachea. Alternatively, if epithelium-dependent modulation of cholinergic transmission does occur in the rat trachea, then the mechanism does not appear to involve phosphorylcholine, nitric oxide, superoxide radicals, cyclo-oxygenase products of arachadonic acid, capsaicin-sensitive neuropeptides or vasoactive intestinal peptide. Moreover, the inhibitory effect of carbachol and ACh on transmitter ACh release in the rat trachea appears to be due solely to activation of prejunctional inhibitory muscarinic cholinoceptors on parasympathetic nerves and does not involve the liberation of a putative epithelium-derived inhibitory factor.

Interleukin-1alpha and tumour necrosis factor-alpha modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate.

1. Previous studies have established that glucocorticoids inhibit airway smooth muscle DNA synthesis. The effects of a combination of the pro-inflammatory cytokines, interleukin-1alpha (IL-1alpha) and tumour necrosis factor-alpha (TNF-alpha) on the inhibition of DNA synthesis by glucocorticoids in human cultured airway smooth muscle have now been investigated, since these cytokines are chronically expressed in asthmatic airways. 2. Thrombin (0.3 u ml(-1)) and basic fibroblast growth factor (bFGF, 300 pM) stimulated increases in DNA synthesis which were concentration-dependently inhibited by dexamethasone (1-1000 nM). 3. The cytokine mixture, comprising IL-1alpha (0.01 and 0.1 pM) and TNF-alpha (3 and 30 pM), directly evoked increases in DNA synthesis which were attenuated by dexamethasone. However, the cytokine mixture prevented responses to bFGF or thrombin. 4. Paradoxically, in the presence of the cytokine mixture and bFGF, dexamethasone (1-1000 nM) concentration-dependently increased DNA synthesis. Furthermore, neither dexamethasone (100 nM) nor fluticasone propionate (1 nM) inhibited DNA synthesized in response to bFGF/cytokine mixture combination and dexamethasone was similarly inactive against the thrombin/cytokine mixture. 5. The levels of prostaglandin E2 (PGE2), an established inhibitor of airway smooth muscle DNA synthesis, remained below the limits of assay detection (0.05 nM) under basal conditions or following stimulation with either thrombin or bFGF. In contrast, the cytokine mixture alone, and in the presence of thrombin or bFGF, induced biologically active levels of PGE2. Dexamethasone (100 nM), the non-selective cyclo-oxygenase (COX) inhibitor indomethacin (3 microM) or the selective COX-2 inhibitor L-745,337 (0.3 microM) completely inhibited synthesis of PGE2. 6. Neither indomethacin (3 microM) nor L-745,337 (0.3 microM) influenced thrombin- or bFGF-induced DNA synthesis. However, each COX inhibitor enhanced DNA synthesis in cytokine-treated cells. 7. In unstimulated airway smooth muscle cells, COX-1, but not COX-2 protein was detectable by Western blotting. The induction of COX-2 protein by the cytokine mixture was attenuated by dexamethasone (100 nM), whereas the level of COX-1 protein was unaffected by either the cytokines or by dexamethasone. 8. Cytokine-induced, COX-2-dependent eicosanoid production inhibits DNA synthesis. The paradoxical increase in DNA synthesis observed in glucocorticoid treated airway smooth muscle stimulated by cytokine/bFGF combinations may be explained by the ability of glucocorticoids to repress COX-2 induction and prevent cytokine-induction of the DNA synthesis inhibitor, PGE2.

Epithelium-dependent inhibition of cholinergic transmission in rat isolated trachea by potassium channel openers.

We have investigated the effects of the potassium channel openers, cromakalim and pinacidil, on cholinergic transmission in rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. In radiolabelled, epithelium-intact preparations, electrical field stimulation (60 s trains of 1 ms pulses, 5 Hz, 15 V) evoked an efflux of radioactivity that was unaffected by the removal of extracellular Ca2+ and, a large proportion of which was resistant to tetrodoxin (1 microM). In contrast, in epithelium-denuded preparations, both tetrodotoxin and Ca2+ withdrawal virtually abolished the stimulation-induced (S-I) efflux. Thus, with epithelium-denuded but not with epithelium-intact tracheal preparations, the S-1 efflux reflects the release of [3H]-acetylcholine from cholinergic nerves. Atropine (1 microM) markedly enhanced the S-I efflux in both epithelium-intact and epithelium-denuded preparations. In epithelium-intact preparations, the combination of atropine (1 microM) and tetrodotoxin (1 microM) reduced the S-I efflux to about the same level as did tetrodotoxin alone. Thus, in epithelium-intact tracheal preparations, when prejunctional muscarinic cholinoceptors subserving autoinhibition of transmitter release are blocked, S-I efflux may be taken as an index of transmitter acetylcholine release. Cromakalim (1 microM) had no effect on the S-I efflux from either epithelium-intact or epithelium-denuded tracheal preparations. However, in epithelium-intact preparations, when atropine (1 microM) was present, cromakalim (1 and 10 microM) and pinacidil (100 microM) significantly inhibited the S-I efflux. In epithelium-denuded preparations, in the presence of atropine (1 microM), cromakalim (1 microM) and pinacidil (100 microM) were without effect on S-I efflux. The inhibition of S-I efflux produced by cromakalim (1 microM) and pinacidil (100 microM) in epithelium-intact tracheal preparations (in the presence of atropine) was prevented by the ATP-sensitive potassium channel blocking drug glibenclamide (1 microM). Glibenclamide (1 microM) alone enhanced S-I efflux from epithelium-intact preparations in the absence but not in the presence of atropine (1 microM). Glibenclamide (1 microM) was without effect on S-I efflux in epithelium-denuded preparations both in the absence or presence of atropine (1 microM). In conclusion, the present study has provided additional evidence of an inhibitory action of the potassium channel openers, cromakalim and pinacidil, on the release of acetylcholine from parasympathetic nerves of the rat trachea which is dependent upon the functional integrity of the airway epithelium. The findings suggest that cromakalim and pinacidil may inhibit transmitter acetylcholine release by opening ATP-sensitive potassium channels, presumably on epithelial cells. In addition, the enhancement of S-I efflux from epithelium-intact tracheal preparations by glibenclamide may indicate that ATP-sensitive potassium channels on epithelial cells play a functional role in the modulation of transmitter acetylcholine release from parasympathetic cholinergic nerves of the airways.