Homoegonol attenuates the asthmatic responses induced by ovalbumin challenge.

Homoegonol is a lignan derived from styraxlignolide A, which was isolated from Styrax japonica, a medicinal plant widely used for treatment of inflammatory diseases in Korea. We investigated the efficacy of homoegonol for the treatment of allergic asthma using an ovalbumin (OVA)-induced murine asthma model. The mice were sensitized through intraperitoneal injections of OVA on days 0 and 14. On days 21, 22 and 23 after the initial OVA sensitization, the mice were received OVA airway challenge. Homoegonol was administered by oral gavage at a dose of 30 mg/kg 1 h prior to the OVA challenge. The homoegonol-treated mice exhibited reduced inflammatory cell counts and Th2 cytokines in BALF, AHR, and IgE in the serum compared with the OVA-sensitized/challenged mice. The histological analysis of the lung tissue revealed that the administration of homoegonol attenuated the airway inflammation and the mucus overproduction in airway epithelial lesions induced by OVA through a reduction in expression of inducible nitric oxide synthase and matrix metalloproteinase-9. These findings indicate that homoegonol effectively suppresses the asthmatic responses induced by OVA challenge and suggests that homoegonol exhibits potential as therapeutic drug for allergic asthma.

ß-carotene regulates expression of ß-carotene 15,15'-monoxygenase in human alveolar epithelial cells.

ß-Carotene 15,15'-monooxygenase (CMO1, BCMO1) converts ß-carotene to retinaldehyde (retinal) and is a key enzyme in vitamin A metabolism. CMO1 activity is robust in the intestine and liver, where cmo1 gene transcription may be subject to negative feedback by accumulation of its metabolic products. Evidence from CMO1 null animals also indicates that non-gastrointestinal CMO1 may be required for tissue-specific conversion of ß-carotene into vitamin A. The aim of this study was to investigate the effects of the enzymatic substrate, ß-carotene, on regulation of CMO1 in a cell model of human alveolar pneumocytes. We demonstrate that CMO1 is expressed in human alveolar epithelial (A549) cells and converts ß-carotene into retinal and biologically active retinoic acids (RA). Exposure to ß-carotene suppresses CMO1 expression at both mRNA and protein levels. ß-Carotene, but not all-trans RA, decreases CMO1 promoter activity in a time- and dosage-dependent manner. This ß-carotene-mediated inhibition of CMO1 expression results from decreased binding of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα) in the CMO1 promoter. ß-Carotene treatment also antagonizes PPARγ activity in HEK293 cells that stably express CMO1 wild-type, but not in cells that express the CMO1 mutant or vector alone. These findings have implications for local vitamin A synthesis in the lung, especially during systemic vitamin A insufficiency and may also help to explain, in part, the mechanism underlying the increased lung cancer risk upon ß-carotene supplementation in smokers.

Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation.

Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits.

Cyclooxygenase-2/prostaglandin E2 inducing effects of α-tocopheryl polyethylene glycol succinate in lung epithelial cells.

Tocopherol analogs are known to have pleiotropic effects due to its interaction with diverse intracellular targets. Previously we reported that low/subapoptotic dose of α-tocopheryl succinate (αTOS) inhibits cyclooxygenase (COX) and prostaglandin E2 (PGE2) production in lung epithelial cells, while high dose of αTOS induces the reactive oxygen species (ROS) generation and apoptosis. In our separate study, we demonstrated that α-tocopheryl polyethylene glycol succinate (αTPGS), a polyethylene glycol (PEG)-conjugated derivative of αTOS, is a more potent ROS/apoptosis inducer compared with αTOS. The present study was prompted to examine whether PEG conjugation to αTOS also enforced its COX inhibitory activity. Of interest, we found that αTPGS failed to inhibit COX activity regardless of doses, suggesting that PEG conjugation to αTOS resulted in the loss of its COX inhibitory activity. Unexpectedly, αTPGS rather induced the COX-2 protein expression at higher/apoptotic doses. αTPGS-induced COX-2 expression was inhibited by antioxidant pretreatment. These data indicate that the COX-2 induction by αTPGS is mediated through increased ROS generation. Since the use of αTPGS as a surfactant component of dispersive drug delivery systems is frequently considered, caution should be taken when the drugs involved in COX signaling are loaded in αTPGS-included delivery systems.

Airway surface liquid contains endogenous DNase activity which can be activated by exogenous magnesium.

INTRODUCTION: The removal of highly viscous mucus from the airways is an important task in the treatment of chronic lung disease like in cystic fibrosis. The inhalation of recombinant human DNase- I (rhDNase-I) is used to facilitate the removal of tenacious airway secretions in different lung diseases and especially in CF. Little is known about endogenous DNase activity in the airway surface liquid. Therefore, we analysed bronchoalveolar lavage fluid (BAL) and exhaled breath condensate (EBC) for the presence of endogenous DNase activity. METHODS: The degradation of plasmid DNA by BAL from patients who had diagnostic bronchoscopy and bronchoalveolar lavage was analyzed. In a group of CF patients and healthy control volunteers the exhaled breath condensate was obtained and also analyzed for the ability to degrade plasmid DNA. In addition, the ability of magnesium to activate endogenous DNase activity in BAL and exhaled breath condensate was investigated. RESULTS: The analyzed BAL samples degraded plasmid DNA only after preincubation with magnesium. When analyzing the exhaled breath condensate the samples obtained from the healthy volunteers showed no DNase activity even after preincubation with magnesium, whereas in one of the two samples obtained from CF patients we found a DNase activity after preincubation with magnesium. CONCLUSION: Increasing the magnesium concentration in the airway surface liquid by aerosolisation of magnesium solutions or oral magnesium supplements could improve the removal of highly viscous mucus in chronic lung disease by activating endogenous DNase activity.

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells.

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.

Dexamethasone upregulates 11beta-hydroxysteroid dehydrogenase type 2 in BEAS-2B cells.

The actions of natural and synthetic glucocorticoids are in part determined by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). We examined whether carbenoxolone, a potent inhibitor of 11beta-HSD, would potentiate the inhibitory action of dexamethasone on interleukin-8 release from BEAS-2B cells, and whether prolonged treatment with dexamethasone at therapeutic doses would upregulate 11beta-HSD2 in the cells. We found that carbenoxolone increased the potency of dexamethasone almost 10-fold. Reverse transcription-polymerase chain reaction and Western blot revealed that BEAS-2B cells expressed 11beta-HSD2, but not 11beta-HSD1. An enzyme activity assay of the cell homogenate demonstrated only NAD+-dependent dehydrogenase activity. The Km value for cortisol in intact BEAS-2B cells was estimated to be 42 nM. When the cells were incubated with dexamethasone for up to 72 hours at increasing concentrations (10(-9) to 10(-5) M), there were considerable increases in mRNA and protein levels of 11beta-HSD2. Prolonged treatment with dexamethasone also increased the enzyme activity of 11beta-HSD in the cells in a dose- and time-dependent manner, with complete inhibition by RU38486. These results suggest that bronchial epithelial cells possess an autoregulatory system for glucocorticoids in the control of their own bioactive levels by inducing the expression of 11beta-HSD2, and that 11beta-HSD2 in the bronchial epithelium may play a role in the local regulation of inhaled glucocorticoid actions.

Gene expression and immunolocalization of 15-lipoxygenase isozymes in the airway mucosa of smokers with chronic bronchitis.

15-lipoxygenase (15-LO) has been implicated in the inflammation of chronic bronchitis (CB), but it is unclear which of its isoforms, 15-LOa or 15-LOb, is primarily involved. To detect 15-LO gene (mRNA) and protein expression, we have applied in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, to bronchial biopsies obtained from 7 healthy nonsmokers (HNS), 5 healthy smokers (HS), and 8 smokers with CB, and additionally include the airways of lungs resected from 11 asymptomatic smokers (AS) and 11 smokers with CB. Compared with HNS, biopsies in CB demonstrated increased numbers of 15-LOa mRNA+ cells (median: HNS = 31.3/mm(2) versus CB = 84.9/mm(2), P < 0.01) and protein+ cells (HNS = 2.9/mm(2) versus CB = 32.1/mm(2), P < 0.01). The HS group also showed a significant increase in protein+ cells (HNS = 2.9/mm(2) versus HS = 14/mm(2), P < 0.05). In the resected airways, 15-LOa protein+ cells in the submucosal glands of the CB group were more numerous than in the AS group (AS = 33/mm(2) versus CB = 208/mm(2); P < 0.001). 15-LOa mRNA+ and protein+ cells consistently outnumbered 15-LOb by approximately 7- and 5-fold, respectively (P < 0.01). Quantitative reverse transcriptase polymerase chain reaction of complementary biopsies confirmed the increased levels of 15-LOa in CB compared with that in either HNS or HS (P < 0.05). There was no difference between the subject groups with respect to 15-LOb expression. The numbers of cells expressing mRNA for 15-LOa in CB showed a positive association with those expressing interleukin (IL)-4 mRNA (r = 0.80; P < 0.01). We conclude that the upregulation of 15-LO activity in the airways of HS and of smokers with CB primarily involves the 15-LOa isoform: the functional consequences of its association the upregulation of IL-4 in chronic bronchitis requires further study.

House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1.

In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR-2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca(2+) concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR-2 agonist peptide-induced Ca(2+) release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1.