Allergen-induced Early Asthmatic Reactions Research Articles

A whey-based glutathione-enhancing diet decreases allergen-induced airway contraction in a guinea-pig model of asthma

Since an allergen-induced early asthmatic reaction is likely to be accompanied by oxidative stress and since levels of the endogenous antioxidant glutathione can be enhanced by a whey-based diet (undenatured whey protein concentrate, UWPC), it was investigated whether UWPC could alleviate allergen-induced lung contractions. Guinea pigs were fed water or UWPC twice a day starting at day - 3 up to day 20. The animals were sensitised to ovalbumin or received saline on day 0. Serum samples were taken at several days after sensitisation to measure allergen-specific IgG. On day 20, lungs were isolated and perfused with buffer containing the allergen ovalbumin. Airway contractions were assessed, and mediators and indicators for oxidative stress were measured in the lung effluent. Moreover, glutathione levels were determined in the liver. The indicator of oxidative stress and airway contractile mediator, 8-iso-PGF(2α), was increased upon ovalbumin challenge in ovalbumin-sensitised groups. Furthermore, thiobarbituric acid-reactive substances (TBARS) were increased as well. Sensitisation with ovalbumin increased IgG levels from day 12 up to day 20, which were not influenced by the UWPC diet. In contrast, the UWPC diet significantly enhanced glutathione levels in the liver. Moreover, the UWPC diet significantly reduced the ovalbumin-induced anaphylactic response by 45 % and decreased PGE2 levels by 55 % in the effluent fluid. We show for the first time that during anaphylaxis, there is acute oxidative stress in the respiratory tract. The UWPC diet did not influence the sensitisation response to the allergen but did increase endogenous glutathione levels. The UWPC diet profoundly reduces allergen-induced airway constrictions, which opens new avenues for dietary management of allergic diseases.

Role of the l-citrulline/ l-arginine cycle in iNANC nerve-mediated nitric oxide production and airway smooth muscle relaxation in allergic asthma

Nitric oxide synthase (NOS) converts l-arginine into nitric oxide (NO) and l-citrulline. In NO-producing cells, l-citrulline can be recycled to l-arginine in a two-step reaction involving argininosuccinate synthase (ASS) and -lyase (ASL). In guinea pig trachea, l-arginine is a limiting factor in neuronal nNOS-mediated airway smooth muscle relaxation upon inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation. Moreover, in a guinea pig model of asthma iNANC nerve-induced NO production and airway smooth muscle relaxation are impaired after the allergen-induced early asthmatic reaction, due to limitation of l-arginine. Using guinea pig tracheal preparations, we now investigated whether (i) the l-citrulline/ l-arginine cycle is active in airway iNANC nerves and (ii) the NO deficiency after the early asthmatic reaction involves impaired l-citrulline recycling. Electrical field stimulation-induced relaxation was measured in tracheal open-rings precontracted with histamine. l-citrulline as well as the ASL inhibitor succinate did not affect electrical field stimulation-induced relaxation under basal conditions. However, reduced relaxation induced by a submaximal concentration of the NOS inhibitor N ω-nitro- l-arginine was restored by l-citrulline, which was prevented by the additional presence of succinate or the ASS inhibitor α-methyl- d, l-aspartate. Remarkably, the impaired iNANC relaxation after the early asthmatic reaction was restored by l-citrulline. In conclusion, the l-citrulline/ l-arginine cycle is operative in guinea pig iNANC nerves in the airways and may be effective under conditions of low l-arginine utilization by nNOS (caused by NOS inhibitors), and during reduced l-arginine availability after allergen challenge. Enzymatic dysfunction in the l-citrulline/ l-arginine cycle appears not to be involved in the l-arginine limitation and reduced iNANC activity after the early asthmatic reaction.

Arginase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma

BackgroundUsing guinea pig tracheal preparations, we have recently shown that endogenous arginase activity attenuates inhibitory nonadrenergic noncholinergic (iNANC) nerve-mediated airway smooth muscle relaxation by reducing nitric oxide (NO) production – due to competition with neuronal NO-synthase (nNOS) for the common substrate, L-arginine. Furthermore, in a guinea pig model of allergic asthma, airway arginase activity is markedly increased after the early asthmatic reaction (EAR), leading to deficiency of agonist-induced, epithelium-derived NO and subsequent airway hyperreactivity.In this study, we investigated whether increased arginase activity after the EAR affects iNANC nerve-derived NO production and airway smooth muscle relaxation.MethodsElectrical field stimulation (EFS; 150 mA, 4 ms, 4 s, 0.5 – 16 Hz)-induced relaxation was measured in tracheal open-ring preparations precontracted to 30% with histamine in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to EFS-induced relaxation was assessed by the nonselective NOS inhibitor Nω-nitro-L-arginine (L-NNA, 100 μM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA, 10 μM). Furthermore, the role of substrate availability to nNOS was measured in the presence of exogenous L-arginine (5.0 mM).ResultsAt 6 h after ovalbumin-challenge (after the EAR), EFS-induced relaxation (ranging from 3.2 ± 1.1% at 0.5 Hz to 58.5 ± 2.2% at 16 Hz) was significantly decreased compared to unchallenged controls (7.1 ± 0.8% to 75.8 ± 0.7%; P < 0.05 all). In contrast to unchallenged controls, the NOS inhibitor L-NNA did not affect EFS-induced relaxation after allergen challenge, indicating that NO deficiency underlies the impaired relaxation. Remarkably, the specific arginase inhibitor nor-NOHA normalized the impaired relaxation to unchallenged control (P < 0.05 all), which effect was inhibited by L-NNA (P < 0.01 all). Moreover, the effect of nor-NOHA was mimicked by exogenous L-arginine.ConclusionThe results clearly demonstrate that increased arginase activity after the allergen-induced EAR contributes to a deficiency of iNANC nerve-derived NO and decreased airway smooth muscle relaxation, presumably via increased substrate competition with nNOS.

Role of nitric oxide in the development and partial reversal of allergen-induced airway hyperreactivity in conscious, unrestrained guinea-pigs.

1. Using a conscious, unrestrained guinea-pig model of allergic asthma, we investigated the role of endogenous nitric oxide (NO) in the regulation of airway (hyper)reactivity to histamine before and after the allergen-induced early and late asthmatic reactions, by examining the effect of inhalation of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 12 mM, 15 min) on the histamine-induced airway obstruction of ovalbumin-sensitized guinea-pigs before, and at 5.5 h and 23.5 h after allergen challenge. 2. Before allergen challenge, inhaled L-NAME caused a significant 2.02+/-0.25 fold increase (P<0.01) in airway reactivity to histamine; this effect was reversed within 2.5 to 6 h after administration. 3. After the allergen-induced early asthmatic reaction at 5 h after ovalbumin provocation, a significant 3.73+/-0.67 fold increase (P<0.01) of the airway reactivity to histamine was observed; subsequent inhalation of L-NAME at 5.5 h had no effect on the airway hyperreactivity, reassessed at 6 h. 4. After the late asthmatic reaction, at 23 h after ovalbumin provocation, a reduced, but still significant airway hyperreactivity to histamine (2.18+/-0.40 fold; P<0.05) was observed. Subsequent inhalation of L-NAME now significantly potentiated the partially reduced airway hyperreactivity 1.57+/-0.19 fold (P<0.05) to the level observed after the early asthmatic reaction. 5. When administered 30 min before allergen exposure, L-NAME significantly enhanced the allergen-induced early asthmatic reaction. However, when administered at 5.5 h after allergen provocation, L-NAME did not affect the subsequent late asthmatic reaction. 6. These results indicate that endogenous NO is involved the regulation of histamine- and allergen-induced bronchoconstriction and that a deficiency of cNOS-derived NO contributes to the allergen-induced airway hyperreactivity to histamine after the early asthmatic reaction, while a recovery of NO deficiency may account for the partial reversal of the allergen-induced airway hyperreactivity after the late asthmatic reaction.

Tolerance to the Protective Effect of Salmeterol on Allergen Challenge

Long-term treatment with inhaled beta 2-agonists may be associated with a deterioration in asthma control, potentially due to tolerance. Regular use of short-acting beta 2-agonists has been shown to induce tolerance to allergen or adenosine 5'-monophosphate challenge. The aim of the study was to detect the efficacy of a single dose and a short-term treatment with salmeterol, a long-acting beta 2-agonist, to protect against early asthmatic reaction (EAR) to allergen. Eight subjects with mild allergic asthma underwent two treatment periods in which subjects performed an allergen challenge (specific bronchial provocation test) protected by a single dose (50 micrograms) of salmeterol (Salm-1) followed by a second specific bronchial provocation test after regular treatment with salmeterol for 1 week (Salm-2), or a single dose of placebo (Plac-1) and regular treatment (1 week) with placebo (Plac-2). Each subject performed both treatments in a randomized order. Each time allergen challenge was performed 1 h after last drug inhalation and it was stopped when the same provocative dose of allergen of a previous screening allergen challenge was achieved. The maximum decrease in FEV1 and area under curve in the first hour after allergen inhalation were significantly lower in Salm-1 (max delta FEV1 %, median [range]: 4%[0 to 9]) with respect to Salm-2, Plac-1, Plac-2 (24%[13 to 38], 31%[19 to 50], 30%[6 to 44], respectively, p < 0.001); there was no difference among Salm-2, Plac-1 and Plac-2. In Salm-1, all subjects were protected against EAR, whereas in Salm-2 only 2 subjects showed a partial protection. In conclusion the protective effect of a single dose of salmeterol against allergen-induced EAR was lost after regular treatment with salmeterol for 1 week. The clinical relevance of this mechanism remains to be elucidated.

The effect of MK-0591, a novel 5-lipoxygenase activating protein inhibitor, on leukotriene biosynthesis and allergen-induced airway responses in asthmatic subjects in vivo

Background: The 5-lipoxygenase metabolites of arachidonic acid are likely to be involved in the pathophysiology of atopic asthma. We investigated the effect of pretreatment with MK-0591, a novel 5-lipoxygenase activating protein inhibitor, on allergen-induced early asthmatic reactions (EARs) and late asthmatic reactions (LARs), and subsequent airway hyperresponsiveness to histamine. Methods: Eight atopic men with mild to moderate asthma aged 19 to 31 years, (forced expiratory volume in 1 second [FEV 1] ≥67% of predicted value, histamine provocative concentration causing a 20% fall in FEV 1 [PC 20] <4 mg/ml) and documented EAR and LAR to house dust mite extract participated in a two-period, double-blind, placebo-controlled, crossover study. During each study period histamine PC 20 was measured 2 days before and 1 day after a standardized allergen inhalation challenge test. MK-0591 was administered in 3 oral doses of 250 mg each at 24, 12, and 1.5 hours before inhalation of allergen. Biochemical activity of MK-0591 was determined by calcium ionophore A-23187–stimulated leukotriene (LT)B 4 biosynthesis in whole blood ex vivo and by urinary LTE 4 excretion. Airway response to allergen was measured by FEV 1 (percent fall from baseline). The EAR (0 to 3 hours) and the LAR (3 to 8 hours) were expressed as corresponding areas under the time-response curves. Results: MK-0591 and placebo did not differ in their effects on prechallenge FEV 1 ( p = 0.10). As compared with the value before pretreatment, MK-0591 blocked LTB 4 biosynthesis and LTE 4 excretion by a mean of 98% (range, 96% to 99%; p < 0.002) and 87% (range, 84% to 96%; p < 0.046), respectively, from 0 to 24 hours after allergen challenge. Both the EAR and the LAR were significantly reduced after administration of MK-0591 as compared with placebo, with a mean inhibition of 79% ( p = 0.011) and 39% ( p = 0.040), respectively. Allergen-induced airway hyperresponsiveness was not significantly different between the two pretreatment periods ( p = 0.37). Conclusions: In this study oral MK-0591 prevented leukotriene biosynthesis after allergen challenge in patients with mild to moderate asthma. The results of our study indicate that 5-lipoxygenase products play an important role during the EAR, whereas their contribution to the pathophysiology of the LAR seems to be of less importance. (J A LLERGY C LIN I MMUNOL 1995;95:42-51.)

Relationships among allergen-induced early and late phase airway obstructions, bronchial hyperreactivity, and inflammation in conscious, unrestrained guinea pigs

The relationships among allergen-induced early asthmatic reactions (EARs) and late asthmatic reactions (LARs), early (between EAR and LAR) and late (after LAR) changes in bronchial reactivity to histamine and infiltration of inflammatory cells into the airways were investigated with a new model of chronically instrumented, unrestrained, and ovalbumin-sensitized guinea pigs. Two different provocation strategies were examined. With the use of stepwise increasing allergen concentrations, all 21 animals responded with an EAR, which in 15 animals (71%) was followed by an LAR. By inhalation of a single allergen concentration for up to 15 minutes, 11 of 14 animals showed an EAR, which in 10 animals (71%) was followed by an LAR. One animal did not respond, whereas the remaining two showed only an LAR. At 6 hours (after the EAR) and 24 hours (after the LAR) after allergen provocation, a significant bronchial hyperreactivity (BHR) toward histamine aerosol was observed in the dual responding animals (both protocols), but no significant changes were observed in animals with a single EAR or a single LAR. Significant correlations were found between the initial increase in airway obstruction after allergen provocation and the severity of the EAR and LAR, as well as the early and late BHR; in addition, a significant correlation was found between the early and late BHR. In contrast, the severity of the LAR did not correlate with the BHR at 6 hours and 24 hours. At 6 hours, there was a marked tendency to an increase in the number of eosinophils and a significant increase in the number of neutrophils in the bronchoalveolar lavage. At 24 hours after provocation, the number of eosinophils and neutrophils was significantly enhanced. These data suggest that early activation of mast cells and/or inflammatory leukocytes may determine the development of the LAR, as well as the early and late BHR, although there appears to be no causal relationship between the BHR at both time points and the severity of the LAR. The relationships among allergen-induced EAR and LAR, early and late BHR, and airway inflammation observed in this new guinea pig model are strikingly similar to those observed in patients with asthma. (J A LLERGY C LIN I MMUNOL 1994;93:1021-30.)

Immunophenotyping of eosinophils recovered from blood and BAL of allergic asthmatics.

Studies of bronchoalveolar lavage (BAL) fluid from patients with allergic asthma have demonstrated active migration of eosinophils into the bronchial lumen after allergen challenge. The mechanisms mediating this eosinophil infiltration and cell activation are largely unexplained. The expression of several cell-surface molecules was measured on eosinophils derived from blood and BAL fluid 4 h after an allergen-induced early asthmatic reaction in order to find indications for a role of these molecules during extravasation to and activation in the bronchial compartment. Nine patients with allergic asthma participated in the study. An eosinophil-specific, high-depolarization signal enabled us to measure expression on eosinophils in a fluorescence activated cell sorter (FACS) analysis without isolation of these cells. Eosinophils recovered from BAL showed a different phenotype than blood eosinophils; upregulation of CR-3, p150/95, CD67, and CD63, and downregulation of L-selectin indicate that the cells are activated in terms of degranulation. Up-regulation of intercellular adhesion molecule-1 (ICAM-1), LFA-3, and human leukocyte antigen II (HLA-II) might enable cell-cell contact between T-lymphocytes and eosinophils, probably leading to immunomodulation and cell activation. The finding that eosinophils in BAL are activated and can interact with T cells is further evidence for the proinflammatory role of these cells in allergic asthma.

Effect of inhaled furosemide on urinary leukotriene E 4 in allergen-induced early asthmatic reactions

Effect of inhaled furosemide on urinary leukotriene E 4 in allergen-induced early asthmatic reactions

Venous blood platelets decrease during allergen‐induced asthmatic reactions

To determine whether circulating platelets alter during asthmatic reactions induced by allergens, we studied nine subjects previously shown to develop an early or dual asthmatic reaction after inhalation challenge with extracts of house dust mite or grass pollen. In each subject, FEV1, circulating platelets and leucocytes were measured before, 15, 30 and 60 min, and 2, 4, 6 and 8 hr after inhalation of allergen and diluent control administered in a single-blind, randomized fashion. The same procedure was repeated in six of the nine subjects after bronchoconstriction induced by methacholine. Each subject developed an early asthmatic reaction after allergen inhalation challenge, which was followed by a late asthmatic reaction in six subjects and by an equivocal late asthmatic reaction in two of them (fall in FEV1 of 15 and 17% respectively). Compared with the control day, circulating platelets significantly decreased during the allergen-induced early asthmatic reaction (P less than 0.025, at 30 min). Platelet counts returned to baseline values within 4 hr and remained steady thereafter both in subjects who did and did not develop a late asthmatic reaction. No changes in platelet counts occurred after bronchoconstriction induced by methacholine. Diurnal increase of leucocyte numbers occurred after challenge with both allergen and diluent control. These results suggest that platelets may be involved in the pathogenesis of allergen-induced asthmatic reactions.