Suppression of allergen-induced respiratory dysfunction and airway inflammation in sensitized guinea pigs by MnII(Me2DO2A), a novel superoxide scavenger compound

Reactive oxygen species produced during allergic inflammation are key players of the pathophysiology of asthma, leading to oxidative tissue injury and inactivation of endogenous manganese superoxide dismutase (MnSOD). On this ground, removal of excess superoxide anion by scavenger molecules would be beneficial and protective. Here we show that a novel manganese(II)-containing polyamine-polycarboxylic compound, termed Mn^II(Me₂DO2A), with potent superoxide dismuting properties decreases the respiratory and histopathological lung abnormalities due to allergen inhalation in a model of ovalbumin (OA)-induced allergic asthma-like reaction in sensitized guinea pigs.Severe respiratory dysfunction in response to OA aerosolic challenge arose rapidly in the sensitized animals and was accompanied by bronchoconstriction, alveolar hyperinflation, mast cell activation, increased leukocyte infiltration (evaluated by myeloperoxidase assay), oxidative lung tissue injury (evaluated by the thiobarbituric-acid-reactive substances and nitrotyrosine immunostaining), decay of endogenous MnSOD activity, production of pro-inflammatory prostaglandins, and lung cell apoptosis. Treatment with Mn^II(Me₂DO2A) (15 mg/kg, given 1 h before allergen challenge), but not the inactive congener Zn^II(Me₂DO2A) lacking redox-active metal site, significantly attenuated all the above functional, histopathological and biochemical parameters of allergic inflammation and restored the levels of MnSOD activity. In conclusion, our findings support the potential therapeutic use of Mn^II(Me₂DO2A) as novel superoxide scavenger drug in asthma and anaphylactic reactions.     

Prevention of antigen-induced bronchial hyperreactivity and airway inflammation in sensitized guinea-pigs by tacrolimus

We examined the effect of the immunosuppressive agent, tacrolimus (FK506), on antigen-induced bronchial hyperreactivity to acetylcholine and leukocyte infiltration into the airways of ovalbumin-challenged guinea-pigs. Subcutaneous injection of 0.5 mg/kg of FK506, 1 h before and 5 h after intra-nasal antigen challenge prevented bronchial hyperreactivity to aerosolized acetylcholine, eosinophilia in bronchoalveolar lavage (BAL) fluid and bronchial tissue and the invasion of the bronchial wall by CD4+ T-lymphocytes. FK506 also suppressed ovalbumin-induced increase in the number of leukocytes adhering to the pulmonary vascular endothelium and expressing alpha4-integrins. Inhibition by FK506 of antigen-induced bronchial hyperreactivity in sensitized guinea-pigs may thus relate to its ability to prevent the emergence of important inflammatory components of airway inflammation, such as eosinophil accumulation, as well as CD4+ T-lymphocyte infiltration into the bronchial tissue.     

Effect of a peptide leukotriene antagonist, ONO-1078 on antigen-induced airway microvascular leakage in actively sensitized guinea pigs.

We examined the effect of ONO-1078, a peptide leukotriene antagonist, on antigen-induced airway microvascular leakage in ovalbumin-sensitized guinea pigs. When guinea pigs were pretreated with mepyramine, ovalbumin challenge increased vascular permeability to Evans blue dye in trachea, main bronchi and intrapulmonary airways. Oral administration of ONO-1078 significantly reduced microvascular leakage in intrapulmonary airways at doses more than 3 mg/kg, but not in trachea. Moreover, oral administration of ONO-1078 significantly reduced SRS-A mediated microvascular leakage into all airway tissues and was more effective in intrapulmonary airways at 3 mg/kg. Simultaneously, ONO-1078 also inhibited SRS-A mediated bronchoconstriction. On the other hand, azelastine (10 mg/kg, p.o.), an anti-asthma agent, failed to inhibit microvascular leakage into the airways. These results suggest that peptide leukotrienes may be important mediators of airway microvascular leakage, and that the inhibitory effect of ONO-1078 on antigen-induced airway microvascular leakage in addition to the blockade of bronchoconstriction may have therapeutic implications for bronchial asthma.     

Ebselen suppresses late airway responses and airway inflammation in guinea pigs

Although ebselen, a seleno-organic compound, inhibits inflammation in various animal models, its efficacy as an anti-asthma drug remains to be clarified. In this study, we investigated the inhibitory effect of ebselen on a guinea pig asthma model. Ebselen was orally administered at dosages of 1-20 mg/kg 2 h before an ovalbumin (OA) challenge, and then airway responses, airway inflammation, the generation of superoxide, H(2)O(2), and nitrotyrosine, and the induction of inducible nitric oxide synthase (iNOS) were evaluated. Sensitized animals challenged with OA aerosol showed dual airflow limitations, i.e., immediate and late airway responses (IAR and LAR). Ebselen significantly inhibited LAR at dosages greater than 10 mg/kg, but did not inhibit IAR at any dosage. Bronchoalveolar lavage (BAL) examination showed that airway inflammation was significantly suppressed by ebselen at 10 mg/kg. The generation of superoxide and H(2)O(2) occurred on endothelial cells of LAR bronchi, and was inhibited by 10 mg/kg of ebselen. Superoxide generation was inhibited by diphenyleneiodonium chloride (DPI), a NAD(P)H oxidase inhibitor, but not by allopurinol, a xanthine oxidase inhibitor. Immunoreactivities for iNOS and nitrotyrosine were also observed on endothelial cells of LAR bronchi and were abolished in ebselen-treated animals. The present findings suggest that ebselen can be applied as a new therapeutic agent for asthma. The possible mechanisms by which ebselen inhibits LAR likely involve suppression of oxidant formation and iNOS induction in endothelial cells.     

Heme oxygenase attenuates allergen-induced airway inflammation and hyperreactivity in guinea pigs

Heme oxygenase (HO), the heme-degrading enzyme, has shown anti-inflammatory effects in several models of pulmonary diseases. HO is induced in airways during asthma; however, its functional role is unclear. Therefore, we evaluated the role of HO on airway inflammation [evaluated by bronchoalveolar lavage (BAL) cellularity and BAL levels of eotaxin, PGE(2), and proteins], mucus secretion (evaluated by analysis of MUC5AC gene expression and periodic acid-Schiff staining), oxidative stress (evaluated by quantification of 4-hydroxynonenal adducts and carbonylated protein levels in lung homogenates), and airway responsiveness to histamine in ovalbumin (OVA)-sensitized and multiple aerosol OVA or saline-challenged guinea pigs (6 challenges, once daily, OVA group and control group, respectively). Airway inflammation, mucus secretion, oxidative stress, and responsiveness were significantly increased in the OVA group compared with the control group. HO upregulation by repeated administrations of hemin (50 mg/kg i.p.) significantly decreased airway responsiveness in control animals and airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These effects were reversed by the concomitant administration of the HO inhibitor tin protoporphyrin-IX (50 micromol/kg i.p.). Repeated administrations of tin protoporphyrin-IX alone significantly increased airway responsiveness in control animals but did not modify airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These results suggest that upregulation of the HO pathway has a significant protective effect against airway inflammation, mucus hypersecretion, oxidative stress, and hyperresponsiveness in a model of allergic asthma in guinea pigs.     

Reduction of paraquat toxicity by superoxide dismutase

The effect of intravenously administered superoxide dismutase on paraquat-treated rats kept either in air or an atmosphere of 90%–95% oxygen was investigated. Of those rats maintained in the oxygen-enriched atmosphere, 50% died within 30 hours whereas, 50 hours elapsed before 50% mortality was observed for the superoxide dismutase-treated rats. Those animals allowed to remain in air were more responsive to superoxide dismutase treatment. Of those animals for which paraquat was fatal, untreated rats showed 50% cumulative mortality within 35 hours after paraquat administration, whereas those rats treated with superoxide dismutase showed 50% mortality after 80 hours. Sections of lung tissue examined at low magnification indicated that the extensive alveolar and vascular damage caused by paraquat was ameliorated with the administration of superoxide dismutase. These findings may have particular relevance in the treatment of paraquat intoxication in humans.     

Inhibition of airway inflammation and hyperreactivity by an antioxidant mimetic

A catalytic antioxidant, AEOL 10113, was used in a murine model of asthma to test the hypothesis that oxidants contribute to the pathogenesis of asthma. Balb/c mice were immunized and challenged with ovalbumin. AEOL 10113 was administered to the mice by intratracheal instillation during ovalbumin challenges. Enhanced pause as an indicator of airway reactivity and differential cell count of lavage cells as an indicator of airway inflammation were assessed. Lung expressions of the adhesion molecules VCAM-1 and ICAM-1 were measured. We found that treatment of ovalbumin-challenged mice with AEOL 10113 drastically reduced the severity of airway inflammation as evidenced by the reduced numbers of eosinophils, neutrophils, and lymphocytes found in bronchoalveolar lavage fluid. Inhibition of ovalbumin-induced airway inflammation is associated with inhibited expression of VCAM-1, which is a key adhesion molecule responsible for the recruitment of inflammatory cells into the lungs of ovalbumin-challenged mice. In addition, treatment with AEOL 10113 reduced the magnitude of ovalbumin-induced airway hyperreactivity to methacholine. These results suggest that oxidative stress is an important factor in the pathogenesis of asthma and that a synthetic catalytic antioxidant could be effective in the treatment of asthma.     

Effects of hyperoxia and allergic airway inflammation on cough reflex intensity in guinea pigs

Toxic influence of high oxygen concentration on pulmonary function and structures has been known for many years. However, the influence of high oxygen concentration breathing on defensive respiratory reflexes is still not clear. In our previous experiments, we found an inhibitory effect of 100 % oxygen breathing on cough reflex intensity in healthy guinea pigs. The present study was designed to detect the effects of hyperoxia on cough reflex in guinea pigs with allergic airway inflammation. In the first phase of our experiment, the animals were sensitized with ovalbumin. Thirty-two sensitized animals were used in two separate experiments according to oxygen concentration breathing: 100 % or 50 % oxygen for 60 h continuously. In each experiment, one group of animals was exposed to hyperoxia, another to ambient air. The cough reflex was induced both by aerosol of citric acid before sensitization, then in sensitized animals at 24 h and 60 h of exposition to oxygen/air in awake animals, and by mechanical stimulation of airway mucosa in anesthetized animals just after the end of the experiment. In contrast to 50 % oxygen, 100 % oxygen breathing leads to significant decrease in chemically induced cough in guinea pigs with allergic inflammation. No significant changes were present in cough induced by mechanical stimulation of airways.     

Neurokinins and inflammatory cell iNOS expression in guinea pigs with chronic allergic airway inflammation

In the present study we evaluated the role of neurokinins in the modulation of inducible nitric oxide synthase (iNOS) inflammatory cell expression in guinea pigs with chronic allergic airway inflammation. In addition, we studied the acute effects of nitric oxide inhibition on this response. Animals were anesthetized and pretreated with capsaicin (50 mg/kg sc) or vehicle 10 days before receiving aerosolized ovalbumin or normal saline twice weekly for 4 wk. Animals were then anesthetized, mechanically ventilated, given normal saline or N(G)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg ic), and challenged with ovalbumin. Prechallenge exhaled NO increased in ovalbumin-exposed guinea pigs (P < 0.05 compared with controls), and capsaicin reduced this response (P < 0.001). Compared with animals inhaled with normal saline, ovalbumin-exposed animals presented increases in respiratory system resistance and elastance and numbers of total mononuclear cells and eosinophils, including those expressing iNOS (P < 0.001). Capsaicin reduced all these responses (P < 0.05) except for iNOS expression in eosinophils. Treatment with l-NAME increased postantigen challenge elastance and restored both resistance and elastance previously attenuated by capsaicin treatment. Isolated l-NAME administration also reduced total eosinophils and mononuclear cells, as well as those cells expressing iNOS (P < 0.05 compared with ovalbumin alone). Because l-NAME treatment restored lung mechanical alterations previously attenuated by capsaicin, NO and neurokinins may interact in controlling airway tone. In this experimental model, NO and neurokinins modulate eosinophil and lymphocyte infiltration in the airways.     

Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide

The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H(2)O(2) and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H(2)O(2)- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the "untargeted" carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.     

Cardioprotection by chronic estrogen or superoxide dismutase mimetic treatment in the aged female rat

Aging and estrogen deficiency increase the risk for developing cardiovascular disease (CVD). Oxidative stress has also been implicated in the pathophysiology of CVD and in ischemia-reperfusion (I/R) injury. We tested the hypothesis that chronic in vivo estrogen treatment or superoxide inhibition with the SOD mimetic EUK-8 improves cardiac functional recovery after I/R in the aged female rat. Sprague-Dawley rats (12-14 mo) were used as follows: intact (n = 6), ovariectomized + placebo (OVX, n = 6), OVX + EUK-8 (EUK-8, 3 mg/kg, n = 6), and OVX + estrogen (1.5 mg/pellet, 60 days release, n = 6). Perfused isolated hearts were subjected to global ischemia (25 min) followed by reperfusion (40 min). Functional recovery after I/R and myocardial protein expression of NADPH oxidase (p22, p67, and gp91(phox)), inducible nitric oxide synthase (NOS), endothelial NOS, and SOD1, as well as nitrotyrosine levels (as a marker for peroxynitrite), were assessed. Compared with OVX, EUK-8 and estrogen markedly improved functional recovery after I/R, which was associated with a decrease in NADPH oxidase expression and nitrotyrosine staining. However, estrogen increased inducible NOS expression, whereas EUK-8 had little effect. There were no significant changes in endothelial NOS and SOD1 expression among the groups. These results indicate that EUK-8 and estrogen improved cardiac recovery after I/R. Given the controversy surrounding hormone replacement therapy, EUK-8 may be an alternative to estrogen in protecting those at risk for myocardial ischemia in the aging population.     

TNF-alpha-induced mitochondrial oxidative stress and cardiac dysfunction: restoration by superoxide dismutase mimetic Tempol

Mitochondria are indispensable for bioenergetics and for the regulation of physiological/signaling events in cellular life. Although TNF-alpha-induced oxidative stress and mitochondrial dysfunction are evident in several pathophysiological states, the molecular mechanisms coupled with impaired cardiac function and its potential reversal by drugs such as Tempol or apocyanin have not yet been explored. Here, we hypothesize that TNF-alpha-induced oxidative stress compromises cardiac function by altering the mitochondrial redox state and the membrane permeability transition pore (MPTP) opening, thereby causing mitochondrial dysfunction. We measured the redox states in the cytosol and mitochondria of the heart to understand the mechanisms related to the MPTP and the antioxidant defense system. Our studies demonstrate that TNF-alpha-induced oxidative stress alters redox homeostasis by impairing the MPTP proteins adenine nucleotide translocator and voltage-dependent anion channel, thereby resulting in the pore opening, causing uncontrolled transport of substances to alter mitochondrial pH, and subsequently leading to dysfunction of mitochondria and attenuated cardiac function. Interestingly, we show that the supplementation of Tempol along with TNF-alpha restores mitochondrial and cardiac function.     

Inhibition of airway hyperreactivity, edema, and lung cell infiltration by compound U-83836E in sensitized guinea pigs

Sensitized guinea pigs were used to assess the effect of treatment with the compound U-83836E ((-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]methyl]-3 ,4-dihydro-2,5,7,8-tetramethyl-2H--benzopyran-6-ol, dihydrochloride) on the antigen-induced late-phase (16 h) airway hyperreactivity, increase in inflammatory cell number, edema, and release of inflammatory mediators in the bronchoalveolar lavage (BAL) fluid. After antigen challenge, an increase of the in vitro reactivity of the trachea and upper bronchi to acetylcholine and histamine and an increase in the number of leukocytes in the BAL fluid, mainly eosinophils and mononuclear cells, were observed. The concentrations of proteins, histamine, and PGE2 in the BAL fluid were also significantly increased by 53, 57, and 216%, respectively, after antigen challenge. Treatment with U-83836E (10 mg/kg) given i.p. 17 and 3 h before and 6 h after antigen challenge inhibited by approximately 80% the total cell number in the airways and the BAL fluid protein content. Moreover, this treatment totally inhibited airway hyperreactivity. Histamine and PGE2 levels in the BAL fluid were not significantly affected by U-83836E treatment. These results indicate that U-83836E is effective against some of the characteristic features of asthma in ovalbumin-sensitized guinea pigs.     

Determination of plasma leukotrienes in antigen-induced bronchoconstrictive guinea pigs.

Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and the extract was washed with dichloromethane then adjusted to pH 3.5 or 6.0, respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantitated by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025-1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 +/- 0.036 ng/ml) and LTD4 (1.399 +/- 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)- 4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone ) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors.     

The primary structure of porcine Cu-Zn superoxide dismutase. Evidence for allotypes of superoxide dismutase in pigs

Cu-Zn superoxide dismutase was purified to homogeneity from mixed pig blood and from a single pig. The isolated product had an absorption ratio 280/260 nm of 0.91, a specific activity of 3 000 +/- 200 units (cytochrome c reduction test), and an isoelectric point of 7.5 (chromatofocusing) or 7.25 (isoelectric focusing), respectively. Sequence determination was performed by automated solid-phase Edman degradation of fragments of the reduced S-carboxymethylated proteins obtained by digestion with trypsin or Staphylococcus aureus proteinase V8 or treatment with cyanogen bromide. Acetylation of the N-terminus was confirmed by comparing high performance liquid chromatography retention times of N-terminal peptides with those of authentic samples. Sequencing of the superoxide dismutase of mixed porcine blood revealed heterogeneity (70% Leu; 30% Val at position 29), whereas the sample derived from a single French pig proved to be homogeneous (100% Leu at position 29). The complete sequence of pig superoxide dismutase comprised 152 amino-acid residues, which corresponds to a theoretical molecular mass of 15 800 Da per subunit, and exhibited the expected high homology with those of other mammals. The aspartate and all 7 histidine residues known to complex the metal ions in bovine superoxide dismutase are conserved in the porcine sequence at the homologous positions Asp82 and His45, His47, His62, His70, His79, His119, respectively.     

Staphylococcal infection in guinea pigs sensitized with a commercial staphylococcal allergen

Guinea pigs, previously injected with commercial staphylococcal allergen to induce delayed hypersensitivity, were infected by the intramuscular injection of S. aureus in a nonlethal dose. For control, the animals receiving only S. aureus were used. The dynamic study of the degree of septicemia and some lymphocytic characteristics in the animals was made. The study revealed that delayed hypersensitivity did not aggravate the course of the main disease; on the contrary, it rendered protection against the subsequent infection. Increased resistance to infection was manifested by a decrease in the degree of septicemia, determined from the decreased number of colony-forming units of S. aureus in the splenic tissue as assessed by inoculation into agar, as well as from a higher level of the activation of lymphocytes as assessed by rosette formation.     

Pulmonary responses to tracheal or esophageal acidification in guinea pigs with airway inflammation.

The association between asthma and gastroesophageal reflux has been attributed to microaspiration of gastric contents and/or vagally mediated reflex bronchoconstriction. In previous experimental studies concerning the pulmonary effects of tracheal or esophageal acid infusion, only animals without airway inflammation have been studied. We assessed the effects of esophageal and tracheal administration of hydrochloric acid (HCl) on normal guinea pigs (GP) and GP with airway inflammation induced by repeated ovalbumin exposures. These GP were anesthetized (pentobarbital sodium) and received 1) 20 microl of either 0.2 N HCl or saline into the trachea, or 2) 1 ml of either 1 N HCl or saline into the esophagus. Intratracheal HCl resulted in a significant increase in both respiratory system elastance and resistance (P < 0.001). There were no significant changes in respiratory mechanics when HCl was infused into the esophagus. In conclusion, we observed that infusion of large volumes of HCl into the esophagus did not change pulmonary mechanics significantly, even in guinea pigs with chronic allergen-induced airway inflammation. In contrast, intratracheal administration of small amounts of acid had substantial effects in normal GP and GP with airway inflammation.     

Lack of adrenomedullin on antigen-induced bronchoconstriction in guinea pigs in vivo

Antigen challenge can provoke acute bronchoconstriction, recognized as immediate asthmatic response (IAR), but the evolving events in this reaction are not well defined. Recently, a novel peptide, designated adrenomedullin, was isolated from human pheochromocytoma, and has been shown to have potent systemic and pulmonary vasodilator activity.The purpose of this study was to elucidate the influence of adrenomedullin in the development of IAR. Passively sensitized guinea pigs were anesthetized and treated with diphenhydramine hydrochloride, and then artificially ventilated. Ovalbumin was inhaled after an intravenous administration of adrenomedullin. Other studies were performed in naive guinea pigs to investigate the airway responses to inhaled methacholine or histamine after an intravenous administration of adrenomedullin. Antigen challenge caused bronchoconstriction in sensitized guinea pigs. Adrenomedullin did not inhibit the antigen-induced bronchoconstriction in sensitized guinea pigs or the dose-dependent responses to inhaled methacholine or histamine in naive animals in spite of its vasodilating effect. We conclude that an intravenous administration of adrenomedullin does not influence antigen-induced bronchoconstriction or bronchial responsiveness to inhaled methacholine or histamine in vivo.