Antoon J.M. Van Oosterhout

Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice

To investigate the mechanisms underlying airway hyperresponsiveness a murine model was developed with several important characteristics of human allergic asthma. Mice were intraperitoneally sensitized with ovalbumin and after 4 weeks challenge via an ovalbumin aerosol. After aerosol, lung function was evaluated with a non-invasive forced oscillation technique. The amount of mucosal exudation into the airway lumen and the presence of mast cell degranulation was determined. Tracheal responsiveness was measured at several time points after challenge. At these time points also bronchoalveolar lavage and histology were performed. Sensitization induced high antigen-specific IgE levels in serum. Inhalation of ovalbumin in sensitized mice induced an immediate but no late bronchoconstrictive response. During this immediate phase, respiratory resistance was increased (54%). Within the first hour after ovalbumin inhalation increased mucosal exudation and mast cell degranulation were observed. At 12 and 24 h after ovalbumin challenge, mice showed tracheal hyperresponsiveness (29% and 34%, respectively). However, no apparent inflammation was found in the lungs or bronchoalveolar lavage. From these results it can be concluded that hyperresponsiveness can develop via mechanisms independent of an inflammatory infiltrate. Since mast cell degranulation occurred after ovalbumin exposure, we hypothesize that mast cells are involved in the induction of airway hyperresponsiveness in this model.

Apocynin and 1400 W prevents airway hyperresponsiveness during allergic reactions in mice

The contribution of reactive nitrogen species to the development of airway hyperresponsiveness in a mouse model of allergic inflammation was investigated by the use of selective inhibitors of nitric oxide and superoxide formation. Sensitized mice, repeatedly challenged with ovalbumin showed a significant (P<0.001, n=9) increase in airway responsiveness measured using whole body plethysmography. This hyperresponsiveness was accompanied by an influx of eosinophils into the airway lumen and increased levels of ovalbumin‐specific serum IgE. Treatment of mice with the iNOS inhibitor 1400 W or the NADPH‐oxidase inhibitor apocynin did not significantly alter cellular influx into the airway lumen nor serum ovalbumin specific IgE. In contrast, apocynin as well as 1400 W inhibited ovalbumin‐induced airway hyperresponsiveness (P<0.001 and P<0.05 respectively, n=9). Furthermore, the airways of allergen challenged animals showed clear 3‐nitrotyrosine staining, which was mainly located in eosinophils. Remarkably, treatment with apocynin or 1400 W did not alter 3‐nitrotyrosine staining. These data suggest that the development of airway hyperresponsiveness during the airway inflammation upon ovalbumin challenge is dependent on the release of both superoxide and nitric oxide and is therefore likely to be dependent on reactive nitrogen species. This mechanism, however, is not reflected by 3‐nitrotyrosine formation in the airways.

Airway hyperresponsiveness: first eosinophils and then neuropeptides.

Airway hyperreactivity to bronchoconstrictor mediators is a main characteristic in the majority of asthmatic patients and correlates well with the severity of the disease. The airways of asthmatic patients are characterized by an inflammatory state resulting in activation of lung tissue cells and attraction and infiltration of leukocytes from the blood. The accumulation of eosinophilic leukocytes is a prominent feature of inflammatory reactions that occurs in allergic asthma. The increase in number of eosinophils is important since it correlates in time with an increase in bronchial hyperresponsiveness. Viral respiratory infections can also induce eosinophilia and airway hyperresponsiveness in humans and animals and can worsen asthmatic reactions. This report reviews current opinions on the relationship between inflammation-induced eosinophil accumulation/activation and the development of airway hyperresponsiveness and the possible role for sensory neuropeptides in this process. Firstly, CC chemokines play an important role in allergic airway inflammation and respiratory viral infections leading to eosinophil recruitment. Secondly, it can be concluded that IL5 is involved in the development in airway hyperresponsiveness. IL5 has profound effects on eosinophils as promoter of growth, differentiation and proliferation, chemoattractant, activator and primer. However, it is conceivable that in animal models for allergic asthma besides IL5 other regulatory mediators may be involved in eosinophil migration and activation in the lung, which in turn will lead to airway hyperresponsiveness. Recent data support the possible role of eotaxin and its eosinophil-specific receptor CCR-3 in eosinophil chemotaxis and activation in allergic asthma. Moreover, it is suggested that the development of airway eosinophilia in vivo involves a two-step mechanism, elicited by eotaxin and IL5. The precise mechanism by which eosinophils induce bronchial hyperresponsiveness is at present unknown. Sensory neuropeptides could be important mediators in this process, since it has been demonstrated that airway nerves are surrounded by and infiltrated with eosinophils after antigen challenge. Sensory neuropeptides could be the final, more downstream, common pathway after eosinophil infiltration and activation in inducing airway hyperresponsiveness due to allergen inhalation or respiratory viral infections. In conclusion, in the process of the development of airway hyperresponsiveness observed during viral infections or in allergic asthma, the IL5/eotaxin-induced infiltration and activation of eosinophils in the airways is evident. Following this step, eosinophil-derived inflammatory mediators will induce the release of sensory neuropeptides (possibly NK2-receptor activating tachykinins) which in turn will lead to airway hyperresponsiveness.

The Efficacy of Immunotherapy in an Experimental Murine Model of Allergic Asthma Is Related to the Strength and Site of T Cell Activation During Immunotherapy

In the present study, the relation between the efficacy of immunotherapy, and the strength and site of T cell activation during immunotherapy was evaluated. We used a model of allergic asthma in which OVA-sensitized and OVA-challenged mice display increased airway hyperresponsiveness, airway inflammation, and Th2 cytokine production by OVA-specific T cells. In this model, different immunotherapy strategies, including different routes of administration, or treatment with entire OVA or the immunodominant T cell epitope OVA323–339, or treatment with a peptide analogue of OVA323–339 with altered T cell activation capacity were studied. To gain more insight in how immunotherapy affects allergen-specific T cells, the site of Ag-specific T cell activation and the magnitude of the T cell response induced during different immunotherapy strategies were determined using an adoptive transfer model. Our data suggest that amelioration of airway hyperresponsiveness and inflammation is associated with the induction of a strong, synchronized, and systemic T cell response, resulting in a decreased OVA-specific Th2 response. In contrast, deterioration of the disease after immunotherapy is associated with the induction of a weak nonsynchronized T cell response, resulting in the enhancement of the OVA-specific Th2 response after challenge.

Involvement of the spleen in the endotoxin-induced deterioration of the respiratory airway and lymphocyte β-adrenergic systems of the guinea pig

The beta-adrenergic binding sites on splenic lymphocyte membranes of the guinea pig were characterized with the radio-ligand [125I]cyanopindolol and showed a maximal number of binding sites (Bmax) of 125 fmol/mg protein and an affinity (Kd) of 170 pM. The potency of various beta-adrenoceptor antagonists to compete for [125I]cyanopindolol binding suggested that the receptor is of the beta 2 subtype. Endotoxin (1 mg/kg) induced a 35% decrease in the number of beta-adrenergic binding sites on lymphocytes, 4 days after i.p. administration. The reduction in the number of beta-adrenoceptors on the lymphocytes was accompanied by a 30% decrease in the relaxation of isolated guinea pig tracheal spirals to isoprenaline and a 20% reduction in the number of beta-adrenergic binding sites in peripheral lung tissue. The endotoxin-induced deterioration of the beta-adrenergic system in the respiratory airways was completely prevented by splenectomy. It is concluded that the spleen, and or cells or products derived from the spleen, are involved in the changes of the beta-adrenergic system in the respiratory airways and lymphocytes.

Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice

BackgroundPreviously, we demonstrated that OVA-loaded macrophages (OVA-Mφ) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mφ start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mφ in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mφ could enhance these effects.MethodsPeritoneal Mφ were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mφ were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mφ-derived IL-10 mediates the immunosuppressive effects, Mφ isolated from IL-10-/- mice were used for treatment.ResultsWe found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mφ significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mφ suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mφ), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10-/-Mφ that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.ConclusionsThese results demonstrate that Mφ-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.

Specific and non-specific effects of β-adrenoceptor agonists on guinea pig alveolar macrophage function

The existence of beta-adrenoceptors on guinea pig alveolar macrophage membranes was determined by means of radioligand binding studies. Saturable binding with [125I]cyanopindolol demonstrated 38 +/- 6 fmol binding sites per 10(6) alveolar macrophages with a Kd of 0.85 +/- 0.15 nM. With timolol, atenolol and ICI 118.551 for competition of [125I]cyanopindolol binding it became clear that guinea pig alveolar macrophages possessed adrenergic binding sites of the beta 2-subtype. The cyclic AMP levels of alveolar macrophages could be increased by selective beta 2-adrenoceptor agonists but not by selective beta 1-adrenoceptor agonists. The influence of non-selective beta- and selective beta 1- and beta 2-adrenoceptor agonists on the phagocytic and metabolic responsiveness of alveolar macrophages was also studied. Addition of beta-adrenoceptor agonists had no effect on the uptake of bacteria by alveolar macrophages. Incubation of alveolar macrophages with increasing amounts of non-selective and selective beta 1-agonists resulted in a dose-dependent decrease in the detection of hydrogen peroxide released by alveolar macrophages. This effect was due to the scavenging properties of these drugs. The selective beta 2-receptor agonists, salbutamol and terbutaline, had no effect on the oxidative metabolism of alveolar macrophages. We conclude that guinea pig alveolar macrophages possess beta 2-adrenoceptors on their cell surface and that these receptors are not involved in the phagocytic activity of alveolar macrophages.

Effects of cytokines on β-adrenoceptor function of human peripheral blood mononuclear cells and guinea pig trachea

In asthma, a β-adrenoceptor dysfunction may be the consequence of an active disease state rather than a fundamental abnormality. In the present study the possible involvement of T lymphocytes in β-adrenergic impairment was investigated by studying the effects of lymphocyte-derived mediators on β-adrenoceptor function of human peripheral blood mononuclear cells (PBMCs) and guinea pig trachea. Supernatants of phytohemagglutinin-or concanavalin A-activated PBMCs from either persons with asthma or healthy persons inhibited isoprenaline stimulated cyclic adenosine 3′, 5′-monophosphate (cAMP) production of PBMCs after 20 hours of preincubation. These supernatants also inhibited β-adrenoceptor function of PBMCs from patients with asthma to the same extent. The isoprenaline stimulated cAMP production of PBMCs was not altered after a 2-hour preincubation period with human interleukin-1 (IL-1), IL-2, IL-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN-γ). In contrast, after 20 hours of preincubation, stimulated cAMP production of PBMCs was significantly diminished, with 63% by IL-1 (40 U/ml, p p p p 50 values or the maximal relaxation of isoprenaline dose response curves.

5-HT1-like receptors mediate potentiation of cholinergic nerve-mediated contraction of isolated mouse trachea

While it had no effect on the resting tension of mouse tracheal segments, 5-HT (10(-8)-10(-4) M) potentiated concentration dependently the contractions induced by electrical field stimulation (EFS). The maximal potentiation was 105 +/- 38% and the EC50 value was 1.4 +/- 0.6 x 10(-6) M (n = 6). The responsiveness of mouse trachea to acetylcholine was not altered by 5-HT (10(-5) M). The 5-HT1A,B antagonist pindolol (10(-6) M), the combined 5-HT2 and 5-HT1C receptor antagonist, ketanserin (10(-6) M), or the combined 5-HT1 and 5-HT2 receptor antagonist, methysergide (10(-6) M), all partially inhibited the effect of 5-HT on the twitch responses. Blockade of 5-HT3 receptors by GR 38032F (10(-6) M) did not affect the potentiation by 5-HT. Antagonism of 5-HT3 and 5-HT4 receptors by ICS 205,930 (3 x 10(-6) M) increased the potentiation of the twitch responses by 5-HT, this was probably due to a decrease of the baseline EFS-induced twitch response by ICS 205,930. Alkylation of the 5-HT2 receptor by phenoxybenzamine (3 x 10(-7) M) treatment did not significantly affect the potentiation of the twitch responses by 5-HT. The beta-adrenoceptor antagonist, timolol (10(-6) M), and the alpha-adrenoceptor antagonist, phentolamine (10(-6) M), did not influence the potentiation of the twitch responses by 5-HT, excluding the involvement of the adrenergic system.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of interleukin-16-blocking peptide on parameters of allergic asthma in a murine model

In this study, we examined whether peptides based on the hydrophilic Cluster of Differentiation (CD) 4-binding part of the amino acid sequence of human interleukin-16 can block interleukin-16-induced chemotaxis of murine lymphocytes in vitro. Peptide 3 was capable of inhibiting interleukin-16-induced chemotaxis of murine splenocytes in vitro. Next, we compared the effects of intra-airway administration of peptide 3 with those of antibodies to interleukin-16 on antigen-induced features in a murine model of allergic asthma. Intra-airway administration of peptide 3 largely inhibited the development of antigen-induced airway hyperresponsiveness while airway eosinophilia was not affected. Similar effects were observed after intranasal application of antibodies to interleukin-16. These results indicate that treatment with peptide 3 causes the same effects as do antibodies to interleukin-16, possibly via the inhibition of interaction between interleukin-16 and its receptor CD4. Therefore, peptide 3 could be useful as a lead compound in attempting to limit airway hyperresponsiveness via binding to CD4.