I. Van Ark

Effect of dexamethasone and endogenous corticosterone on airway hyperresponsiveness and eosinophilia in the mouse

1 Mice were sensitized by 7 intraperitoneal injections of ovalbumin without adjuvant (10 μg in 0.5 ml of sterile saline) on alternate days and after 3 weeks exposed to either ovalbumin (2 mg ml−1 in sterile saline) or saline aerosol for 5 min on 8 consecutive days. One day before the first challenge, animals were injected intraperitoneally on a daily basis with vehicle (0.25 ml sterile saline), dexamethasone (0.5 mg kg−1) or metyrapone (30 mg kg−1). 2 In vehicle‐treated ovalbumin‐sensitized animals ovalbumin challenge induced a significant increase of airway responsiveness to metacholine both in vitro (27%, P < 0.05) and in vivo (40%, P < 0.05) compared to saline‐challenged mice. Virtually no eosinophils could be detected after saline challenge, whereas the numbers of eosinophils were significantly increased (P < 0.01) at both 3 and 24 h after the last ovalbumin challenge (5.48 ± 3.8 × 103 and 9.13 ± 1.7 × 103 cells, respectively). Furthermore, a significant increase in ovalbumin‐specific immunoglobulin E level (583 ± 103 units ml−1, P < 0.05) was observed after ovalbumin challenge compared to saline challenge (201 ± 38 units ml−1). 3 Plasma corticosterone level was significantly reduced (−92%, P < 0.001) after treatment with metyrapone. Treatment with metyrapone significantly increased eosinophil infiltration (17.4 ± 9.93 × 103 and 18.7 ± 2.57 × 103 cells, P < 0.05 at 3 h and 24 h, respectively) and potentiated airway hyperresponsiveness to methacholine compared to vehicle‐treated ovalbumin‐challenged animals. Dexamethasone inhibited both in vitro and in vivo hyperresponsiveness as well as antigen‐induced infiltration of eosinophils (0, P < 0.05 and 0.7 ± 0.33 × 103 cells, P < 0.05 at 3 h and 24 h, respectively). Metyrapone as well as dexamethasone did not affect the increase in ovalbumin‐specific immunoglobulin E levels after ovalbumin challenge (565 ± 70 units/ml−1; P < 0.05; 552 ± 48 units ml−1, P < 0.05 respectively). 4 From these data it can be concluded that exogenously applied corticosteroids can inhibit eosinophil infiltration as well as airway hyperresponsiveness. Vise versa, endogenously produced corticosteroids play a down‐regulating role on the induction of both eosinophil infiltration and airway hyperresponsiveness.

Role of interleukin-5 and substance P in development of airway hyperreactivity to histamine in guinea-pigs

In this study, we examined the mechanism by which bronchoalveolar lavage (BAL) cells induced hyperreactivity of the trachea in vitro. As both interleukin-5 (IL-5) and substance P (SP) appeared to be involved, the effect of these mediators was examined in vivo. Tracheae were incubated with BAL cells from ovalbumin or saline challenged animals, and from naive animals, in the absence or presence of either IL-5, SP, or both. In addition, the effect of intra-airway application of IL-5, SP, both, or vehicle on tracheal hyperreactivity was examined. Incubation of tracheae with BAL cells from ovalbumin challenged animals induced an increase (30 +/- 10%) in the maximal response to histamine. The hyperreactivity could be completely inhibited by co-incubation with 5-lipoxygenase inhibitor, AA861. The hyperreactivity could be mimicked by incubation of tracheae with BAL cells from naive animals in the presence of IL-5 and SP. After in vivo administration of either IL-5 or SP, maximal responses to histamine were increased and amounted to 105 +/- 35 and 101 +/- 37%, respectively. Administration of IL-5 but not SP induced a significant increase in the number of eosinophils (67 +/- 22%) and eosinophil peroxidase (EPO) activity (94 +/- 33%) in BAL cells. The simultaneous administration of IL-5 and SP did not potentiate the hyperreactivity and eosinophilia observed with IL-5 alone. These data suggest that IL-5 is important in the recruitment of eosinophils, whereas both IL-5 and substance P are involved in the induction of airway hyperreactivity.

Salmeterol inhibits interferon-γ and interleukin-4 production by human peripheral blood mononuclear cells

T-lymphocytes play an important role in allergic asthma. In the present study, the effect of beta(2)-adrenoceptor agonists was examined on proliferation, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) production by human peripheral blood mononuclear cells (PBMC). The proliferation after 24 h phytohaemagglutinin (PHA) activation was significantly inhibited at high concentrations of salmeterol, isoprenaline and salbutamol (> or = 10(-6) M). A U-shaped concentration response curve was observed for the effect of all agonists on IL-4 production 24 h after PHA activation. Maximal inhibition occurred at 10(-9) M and amounted to 71% (P < 0.02), 38% (P < 0.01) and 49% (P < 0.01) for salmeterol, isoprenaline and salbutamol, respectively. In contrast, no significant effect of salmeterol (10(-11)-10(-5) M) on IL-4 production could be detected after 96 h. A biphasic concentration response curve was observed for the inhibitory activity of all beta-adrenoceptor agonists on IFN-gamma production by PBMC 24 h after PHA activation. The first phase reached a plateau at 10(-9) M and the inhibition amounted to 50% (P < 0.05), 33% (P < 0.01) and 44% (P < 0.05) for salmeterol, isoprenaline and salbutamol, respectively. At higher concentrations of the three beta-adrenoceptor agonists the inhibition was increased up to 80% (P < 0.05), 60% (P < 0.05) and 58% (P < 0.01), respectively. Similar to the results obtained after 24 h, IFN-gamma production after 96 h was biphasically inhibited by salmeterol, and this inhibition (60%) was significantly at 10(-5) M. Together, the present data provide clear evidence for concentration-dependent effects of beta-adrenoceptor agonists on the IL-4 and IFN-gamma production by human PBMC. These results suggest that beta-agonists, at low concentrations, predominantly inhibit IL-4 production and may therefore act as anti-inflammatory drugs in allergic asthma.

l-Arginine is not the limiting factor for nitric oxide synthesis by human alveolar macrophages in vitro

Unlike murine mononuclear phagocytes, human macrophages do not release high amounts of nitric oxide (NO) in vitro despite the presence of nitric oxide synthase (NOS). To determine whether this limited NO synthesis in vitro is due to limited availability of the NOS substrate L-arginine, and putative NOS inhibiting factors present in foetal serum preparations, both alveolar macrophages (AM) and monocyte derived macrophages (MDM) were incubated in various circumstances. Nitrite production measured using stimulated AM was typically <5 pmol x min(-1) x 10(-6) cells. A range of stimuli were tested, but without result. Furthermore, incubation of MDMs with normal human serum or purified bovine serum albumin instead of foetal calf serum failed to enhance NO production. Moreover, neither the use of arginase inhibitors nor the addition of surplus L-arginine resulted in an increased NO synthesis. Interestingly, addition of the NOS intermediate Nomega-hydroxy-L-arginine (100 microM) to AM led to nitrite release, which was unaffected by the NOS inhibitor amino guanidine showing that this effect is NOS independent. It is concluded that the limited nitric oxide production of human macrophages in vitro can neither be explained by limited availability of L-arginine, nor by nitric oxide synthase inhibiting substances in foetal serum. Furthermore, it is shown that nitrite release from Nomega-hydroxy-L-arginine by alveolar macrophages is nitric oxide synthase independent.

Antigen-stimulated lung CD4+ cells produce IL-5, while lymph node CD4+ cells produce Th2 cytokines concomitant with airway eosinophilia and hyperresponsiveness

Abstract.Objective and Design: We investigated whether airway inflammation in a mouse model of allergic asthma is related to antigen-specific T cell responses in the effector organ, the lung, and in the lung draining lymph nodes (LN). ¶Materials and Subjects: In BALB/c mice pathophysiological parameters were measured in vivo, and lung draining LN and lung cells were restimulated in vitro. ¶Treatment: Mice were sensitized with ovalbumin and repeatedly challenged with ovalbumin or saline inhalation. ¶Methods: Airway reactivity, inflammation in the airways, serum levels of IgE were measured, and cytokine levels and proliferative responses were determined in antigen-stimulated lymphocyte cultures. ¶Results and Conclusions: Sensitization results in antigen-specific Th0-like LN cells, despite the presence of antigen-specific IgE. Repeated antigen inhalation induced airway hyperresponsiveness and eosinophil infiltration concomitant with a shift towards Th2 cytokine production exclusively by lung draining LN T cells. Furthermore, these airway symptoms are associated with antigen-specific CD4+ effector T cells in the airway tissue producing only IL-5, but not IL-4, which are unable to proliferate.

Modulation of airway hyperresponsiveness by thiols in a murine in vivo model of allergic asthma.

Abstract:Objective and design: Since oxidative stress contributes to the pathogenesis of asthma, this study addressed the question whether supplementing the endogenous antioxidant, glutathione (GSH), would alleviate features of allergic asthma in the mouse.¶Material and methods: Ovalbumin-sensitized mice received aerosols of the GSH-donors, glutathione-ethyl ester (GSEt) or N-acetylcysteine, before or during respiratory allergen challenges, or during methacholine challenges given one day after the last allergen challenge. Lung GSH levels were measured shortly after allergen or methacholine challenge. In addition, the effect of GSH supplements on airway hyperresponsiveness and inflammatory cell numbers in the airway lumen was assessed.¶Results: GSEt decreased allergen-induced airway hyperresponsiveness when given in combination with methacholine. However, when given before or during allergen challenge, both GSH-donors failed to decrease the methacholine-induced airway contractility, change cell numbers in the airway lumen, or increase lung GSH levels. In addition, allergen challenges of sensitized mice did not decrease lung GSH levels.¶Conclusion: In contrast to guinea pigs and humans, allergen challenges in mice does not lead to acute oxidative stress.

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.