Inhibition of anti-IgE induced skin response in normals by formoterol, a new β2 adrenoceptor agonist, and terbutaline

The intention of the present study was to compare formoterol and terbutaline regarding ability to inhibit immediate wheal and flare responses (WFR) to anti-human IgE with focus on the duration of anti-WFR action. Formoterol is a novel beta 2-adrenergic agonist with a prolonged duration of bronchodilation capacity after inhalation. The drugs injected intradermally 2 min prior to challenge with anti-IgE in volunteers produced a dose-dependent inhibition of the WFR in the range 1pg-100ng (formoterol) and 1ng-1 microgram (terbutaline). Formoterol was 70 times (flare) and 25 times (wheal) more potent (ID40) than terbutaline on a weight basis. The duration of the anti-WFR action for formoterol, injected in a 25 times lower dose than terbutaline, was significantly longer, namely greater than 24 h versus 8 h for terbutaline. The histamine-induced wheal reaction was attenuated by 15% and 25% by terbutaline and formoterol, respectively. The results indicate a higher beta 2-receptor activity for formoterol with respect to inhibition of IgE-dependent mast cell mediator release in addition to an anti-leak effect exerted by both drugs. The prolonged duration of antagonistic effect by formoterol on the WFR to anti-IgE might be due to the lipophilic property of the drug, with an expected higher retention of formoterol at the target tissue compared with the more hydrophilic compound terbutaline.     

Formoterol fumarate, a new β2-adrenoceptor agonist

Four double-blind, double-dummy, randomized, crossover studies were performed in nine asthmatic patients to evaluate beta 2-adrenoceptor selectivity and the duration of effect of formoterol. One study with cumulatively increasing doses of formoterol and salbutamol showed that with the inhaled route, formoterol was 5-15 times more potent than salbutamol with respect to bronchodilation. In a second study, 6 micrograms formoterol and 0.1 mg salbutamol were given for comparison of effect duration. Five hours after salbutamol inhalation the FEV1 values were back to basal. Eight hours after formoterol inhalation about 75% of the maximum bronchodilation remained and the FEV1 was significantly higher than after salbutamol inhalation. By oral route, a study with cumulatively increasing doses showed that as a bronchodilator formoterol was about 50 times more potent than salbutamol. The same potency difference was seen for increase of heart rate and decrease of diastolic blood pressure, indicating that the clinical selectivity for beta 2-adrenoceptors is equal for the two drugs. In a fourth study comparable doses by oral route did not show any difference in the duration of bronchodilation. We conclude that inhaled formoterol is 5-15 times more potent than salbutamol. Inhaled formoterol produces longer duration of bronchodilation, with clinically relevant bronchodilation for at least 8 h. The prolonged duration of formoterol was not seen with oral treatment.     

Sustained Inhibition of Antigen-Induced Histamine Release from Human Lung by the β2-Adrenoceptor Agonist Terbutaline

Antigen-induced histamine release from passively sensitized human lung tissue was inhibited in the presence of the β₂-adrenoceptor agonist, terbutaline. A sustained and statistically significant suppression was detected in the concentration interval 3 × 10⁻⁸-1 × 10⁻⁶ M. Fifty per cent inhibition IC₅₀, was obtained at an interpolated concentration of 5.3 × 10⁻⁸± 0.4 × 10⁻⁸ M ( n = 13), when the histamine secretion was elicited with optimum concentration of antigen. Histamine release induced with a suboptimum concentration of antigen was inhibited to a greater extent than release initiated with optimum concentration. The data in the present investigation support the concept that terbutaline-induced inhibition of mediator release from human lung tissue can contribute to the clinical effectiveness of the drug during treatment of allergic asthma.     

Effects of salmeterol and terbutaline on IgE-mediated dermal reactions and inflammatory events in skin chambers in atopic patients

The objective of the present study was to investigate the potential of the long-acting p-aonist salmeterol as an inhibitor of various components of IgE-mediated inflammation in man. For this purpose, we measured gross skin reactivity (diameters of wheal and flare reaction [WFR] and late cutaneous reaction [LCR]) as well as inflammatory cells, mediators, and protein in cutaneous suction blister chambers in eight subjects with allergic rhinitis. Blisters were induced, two on each forearm, by gentle suction and heating, and were unroofed 12 h later, after which plastic chambers were placed over the denuded area. The chambers were challenged for 2 h with antihuman IgE (titer 1:10) in the presence and absence of salmeterol or terbutaline. Normal goat IgG served as negative control. Chamber fluids were removed hourly for the first 4 h, and this was followed by a 4-h incubation before final collection. Salmeterol (10^-6M) and terbutaline (10^-5 M) injected intradermally 30 min before, as well as together with anti-IgE (titer 1:100), inhibited the WFRs by up to 30%. The effect of salmeterol on the ensuing LCR (75% inhibition at 24 h) tended to be more pronounced than the corresponding inhibition by terbutaline. Both salmeterol and terbutaline very effectively inhibited the anti-IgE-induced extravasation of α₂-macroglobulin into skin chambers, with a significantly more sustained effect by salmeterol. Interestingly, only terbutaline reduced the histamine release evoked by anti-IgE. With the present experimental design, where both drugs were washed out from the chambers after 2 h, neither drug inhibited recruitment of leukocytes (including eosinophils). Taken together, salmeterol had a more sustained inhibitory effect than terbutaline on indices of IgE-mediated edema formation (late induration and plasma protein extravasation). On the other hand, under the present experimental conditions, salmeterol failed to reduce the histamine release (in contrast to terbutaline), and neither salmeterol nor terbutaline affected the recruitment of leukocytes.     

Reversibility of the allergen-provoked late asthmatic response by an inhaled β2-adrenoceptor agonist

There is uncertainly about the relative contributions of bronchial smooth muscle contract ion. mucosal oedema and mucus plugging lo airflow obstruct ion in the allergen induced late asthmatic response (LAR). We systematically studied the ability of the inhaled β₂-agonist salbutamol to reverse the LAR in eight subjects after allergen bronchoprovocation. Salbutamol reversed the LAR by restoring FEV₁, to a level similar to the initial value measured at the same time of day (18.00 h)on the previous evening. For the eight subjects studied, this initial FEV₁ value, measured after abstaining from β-agonists for 8 h, was a mean ±SEM 90.7 ± 5.6% of predicted, which suggests further bronchodilation may have been possible at this time. We then studied six of the eight subjects in an identical protocol with saline challenge substituted for allergen bronchoprovocation to answer the question whether further bronchodilation was possible at that time after salbutamol in the absence of an LAR. After salbutamol on the allergen challenge day the FEV₁ for the six subjects was 84.1 ± 7.0% of predicted, compared with 94.0 ± 3.7% of predicted at the same point on the saline challenge day (p < 0.05). We conclude that, although the LAR may be effectively reversed by β₂-agonists, there is evidence for some residual airway narrowing, presumably related lo mucosal oedema, exudate and mucus plugging.     

Association between bronchodilating response to short-acting β-agonist and non-synonymous single-nucleotide polymorphisms of β2-adrenoceptor gene

BACKGROUND: With beta-agonists being the most widely used agents in the treatment of asthma, in vitro studies reported that beta(2)-adrenergic receptor (ADRB2) polymorphisms are associated with agonist-promoted down-regulation. OBJECTIVE: The present population-based study aimed to evaluate the association between bronchodilating response to inhaled short-acting beta-agonist and two non-synonymous single-nucleotide polymorphisms (SNPs) of ADRB2 (ADRB2-16 and ADRB2-27). METHODS: Two hundred and nine children with reduction in forced expiratory volume in 1 s of more than 20% on methacholine bronchial challenge underwent bronchodilating response testing 5 min after the inhalation of 200 mug of albuterol. Of these 209, 195 gave peripheral blood for genotyping of ADRB2 polymorphisms. RESULTS: The bronchodilating response was significantly higher in subjects with the homozygous Arg16 than in those with the homozygous Gly16. It was further demonstrated that haplotype pairs of the homozygous Arg16Gln27 and of the heterozygous Arg16Gln27/Gly16Glu27 showed the highest bronchodilating responses, and the haplotype pairs of the homozygous Gly16Gln27 the lowest response. As a whole, the bronchodilating response was more positively associated with the combined quantity of Arg16 and Glu27 polymorphisms than with that of Arg16 alone. CONCLUSION: Non-synonymous SNPs of ADRB2 at codons 16 and 27 is significantly associated with bronchodilating response to inhaled short acting beta-agonists.     

Histopathological Support of Antagonism of Allergen-Induced Mast Cell Mediator Release in Human Skin by a Beta2-Agonist

The site of antagonistic action on allergen-induced early skin reactions by the beta 2-agonist terbutaline was studied by light microscopy in 10 atopic subjects. Pretreatment with 1 microgram terbutaline intradermally 5 min prior to challenge with horse dander allergen produced an approximate inhibition of 85 and 55% of flare and wheal responses respectively (P less than 0.001). Biopsy specimens obtained from skin sites injected with allergen alone showed a reduced number of remaining stainable mast cells as compared to sites injected with terbutaline prior to allergen (P less than 0.05, Sign test). The data support the concept of in vivo inhibition of the mast cell mediator release reaction in atopic skin by terbutaline.     

Two New β2-Adrenoceptor Agonists, D-2343 and QH 25, Studied in Asthmatic Patients

Two new beta 2-adrenoceptor agonists, D-2343 and QH 25, have been studied in asthmatic patients. Animal experiments indicated less effect on skeletal muscle than on bronchial muscle. D-2343 (four i.v. cumulatively increasing doses, 0.21--6.3 micrograms kg-1 min-1) and i.v. terbutaline (four cumulative doses 0.07--2.1 micrograms kg-1 min-1) were single-blindly compared in a randomized crossover study in eight asthmatics. It was shown that D-2343 is an effective beta 2-adrenoceptor agonist with 5--6 times lower potency than terbutaline. The tremor-inducing effect of D-2343 was the same as that of terbutaline, when the same degree of bronchodilation was obtained. The effect of QH 25 (0.25, 0.75 and 2.25 mg cumulatively) administered orally, was single-blindly compared with the effect of oral salbutamol (2, 6 and 18 mg) in a randomized crossover study in eight asthmatics. QH 25 was shown to be an effective beta 2-adrenoceptor stimulant about 12 times more potent than salbutamol. The tremor-inducing capacity was the same as for salbutamol when equipotent bronchodilating doses were studied.     

Association studies on β2-adrenoceptor polymorphisms and enhanced IgE responsiveness in an atopic population

The beta2 adrenergic receptor 2 represents a cell surface receptor responsible for the binding of endogenous catecholamines and their exogenously administered agonists and antagonists, mediating their effects to the interior of the cell. On the basis of these functions, the observed association of two of its polymorphisms, Gly16 and Gln27, with nocturnal- and steroid-dependent asthma has been discussed. It has recently been suggested that Gln27 contributes to IgE variability in families with asthma. The aim of this study was to investigate possible influences of the polymorphisms Arg16Gly and Glu27Gln on IgE levels in families recruited through an atopic index case without regard to the presence of clinical symptoms. We employed linkage analysis in affected sibpairs characterized by elevated total IgE concentrations or sensitization to common inhalant allergens. Furthermore, we tested 258 children for association of any of the polymorphisms with enhanced IgE responsiveness. We could find neither linkage at the locus 5q31 nor significant association of the polymorphisms with elevated total IgE concentrations or specific sensitization. We conclude from our data that the polymorphisms Gln27Glu and Arg16Gly of the beta2-adrenergic receptor do not play a significant role in the pathogenesis of enhanced IgE responsiveness in an atopic population in general.     

Effect of Terbutaline, a β2-adrenergic Receptor Stimulating Compound, on Cutaneous Responses to Histamine, Allergen, Compound 48/80, and Trypsin

The beta-adrenoceptor stimulating agent terbutaline (2 ng-2 microgram) injected intradermally in eight atopic subjects produced a dose-dependent inhibition of the skin reactions induced by subsequently injected allergen. After injection of 0.5 microgram terbutaline inhibition of the flare and weal responses was demonstrable throughout the observation period of 90 min. The flare response induced by histamine, the histamine liberator compound 48/80 and the proteolytic enzyme trypsin was not inhibited by terbutaline in the doses used, suggesting a selective action of terbutaline on the allergen-induced response. The weal response elicited by histamine and compound 48/80 was slightly reduced by 2 microgram terbutaline. It is suggested that pretreatment of the skin with terbutaline interferes with the ability of the cutaneous mast cells to respond to challenge with allergen and that terbutaline produces this effect in doses lower than those needed to counteract the permeability increasing effect of released mediator substances.     

Increased expression of collagen receptors: α1β1 and α2β1 integrins on blood eosinophils in bronchial asthma

BACKGROUND: Eosinophils are one of the major effector cells in bronchial asthma. Their infiltration of airways correlates with the asthma severity. Recruitment and activation of eosinophils are partially mediated by integrins alpha4beta1 and alpha4beta7. Collagens type I and IV constitute important components of extracellular matrix and vascular basement membrane, respectively. Therefore, collagen-binding integrins (alpha1beta1 and alpha2beta1) may also play a role in eosinophil lung infiltration. OBJECTIVE: To evaluate the possible presence of alpha1beta1 and alpha2beta1 integrins on peripheral blood eosinophils from asthmatic subjects. METHODS: Collagen receptors were studied on eosinophils separated by immunomagnetic CD16-negative method from healthy donors (n=13) and patients with moderate persistent atopic bronchial asthma (n=15). Surface receptor identification was performed by flow cytometry and cell adhesion assay. RESULTS: Eosinophils isolated from the patients showed increased expression of both alpha1beta1 and alpha2beta1 integrins as compared with healthy controls. Moreover, adhesive function of eosinophils to collagen type IV was inhibited by snake venom disintegrins: viperistatin and obtustatin. These disintegrins contain KTS active motif and are specific inhibitors of alpha1beta1 integrin. CONCLUSION: We demonstrated for the first time that collagen receptors: alpha1beta1 and alpha2beta1 integrins are overexpressed on the surface of peripheral blood eosinophils of asthmatic subjects. Further studies may reveal potential application of KTS-disintegrins or their structural analogs for therapy of bronchial asthma.     

Adhesion of Activated T Lymphocytes to Vascular Smooth Muscle Cells and Dermal Fibroblasts is Mediated by β1- and β2-Integrins

Several studies during recent years have demonstrated the potential for vascular smooth muscle cells (SMC) and dermal fibroblasts to participate in immune interactions such as antigen presentation and alloreactivity. The molecular interactions mediating lymphocyte adhesion to these mesenchymal cells have, however, not previously been characterized in detail. In the present study we demonstrate ICAM-1 (CD54) expression by cultured human SMC and its up-regulation by IL-1, IFN-gamma, and bacterial lipopolysaccharide. Monoclonal antibodies were used to define the molecular interactions in the adhesion of 51Cr-labelled T lymphoblasts to adherent SMC and fibroblasts. ICAM-1 appeared to mediate adhesion of T lymphocytes by binding to the beta 2-integrin CD11a/CD18 (LFA-1) expressed by the lymphoblasts. We present evidence for the involvement of at least three different mechanisms in the adhesion of activated T lymphocytes to cultured fibroblasts. It was found that beta 2-integrin-mediated interaction could only account for less than half of the binding activity. The remaining adhesion was partly mediated by beta 1-integrins, presumably via VLA-5 since an anti-VLA-5 antibody and an RGD-containing peptide blocked adhesion to the same degree. However, antibodies to beta 1-, beta 2-, and beta 3-integrin subunits added together only inhibited adhesion by approximately 50%. The residual adhesion could be blocked by inhibition of cell metabolism and was increased by stimulation of the lymphocytes with phorbol ester, suggesting involvement of other, as yet undefined, adhesion molecules. The molecular interactions between lymphocytes and mesenchymal cells demonstrated in this study may have implications in several inflammatory conditions such as vasculitis, atherosclerosis, and connective tissue diseases.     

A new β2 microglobulin mutation found in a melanoma tumor cell line

Beta2 microglobulin mutations are an important mechanism for HLA class I total loss, (phenotype No. I) and have been described in colon carcinomas, melanomas and lymphomas. We describe a new beta2 microglobulin mutation detected in the melanoma cell line GR-34. The new mutation reported here was identified as a deletion of 4 bases (TTCT) in the highly repetitive sequence CTCTCTCTTTCT located in the leader sequence of the beta2 microglobulin gene at codon 15-16 of exon 1. The mutation produces a frameshift in the open reading frame sequence with the appearance of a stop codon at position 42. We also demonstrate that the second beta2 microglobulin gene is deleted. Comparisons with beta2 microglobulin mutations in other tumor cell lines suggest a mutation hot spot in exon 1.     

A method for canine nasal mucosa superfusion, in vivo; evidence that pseudomonas-induced neutrophil migration is inhibited by α2-adrenoceptor agonist

Methods and Results A method for isolating and superfusing a segment of the nasal cavity in the anaesthetized dog was developed. Introduction of Pseudomonas aeruginosa supernatant (PA), which is known to induce IL-8 release, resulted in a marked time-dependent exponential increase in neutrophil recruitment to the nasal chamber. When oxymetazoline (final concentration. 10^-5 M), was added to the superfusate, the PA induced-neutrophil migration was inhibited; the addition of benzalchoniul chloride to the superfusate had no effect on the PA induced-neutrophil migration. Conclusion We conclude that a α₂ adrenoceptor agonist profoundly inhibits neutrophil migration in response to bacterial products. Our novel method allows continuous introduction of stimuli and monitoring of responses in the nasal mucosa.     

Suppression of Immediate and Late Anti-IgE-Induced Skin Reactions by Topically Applied Alcohol/Onion Extract

In a double blind study, alcohol/onion extract (5% ethanol) was injected simultaneously with 20 IU and 200 IU rabbit anti-human-IgE intradermally in 12 adult volunteers (6 atopics, 6 non-atopics). Diameters of wheals and flares were measured 10 min after and compared with control sites challenged with 20 IU and 200 IU anti-IgE in a 5% ethanol solution. The skin sites were then treated epidermally with 45% alcohol/onion extract and 45% ethanol under occlusion. Diameters of late cutaneous reactions were measured hourly. Oedema formation was clinically estimated according to an arbitrary scale and skin thickness measured with a calliper. In the onion-treated skin sites the wheal areas were significantly reduced (20 IU: control: 108 +/- 53 mm2; onion 69 +/- 42 mm2, P less than 0.05; 200 IU anti-IgE: control: 152 +/- 25 mm2, onion: 138 +/- 26 mm2, P less than 0.02). The oedema formation during the late phase skin reaction was markedly depressed (P less than 0.005 at 2 h, P less than 0.01 at 4 and 6 h, P less than 0.02 at 8 h). The extent of late skin reactions was slightly, but not significantly reduced. Obviously, onions contain pharmacologically active substances with anti-inflammatory and/or allergic properties.     

Activation of Human T and B Cells by Rabbit Anti-Human β2-Microglobulin

Rabbit anti-human β₂-microglobulin (anti-β₂m) increased the highest DNA synthesis in un-separated lymphocytes or artificially composed mixtures enriched in T and B cells. In enriched T and B cells no or low stimulation was seen. The maximal response by IgG anti-β₂m was seen in proportions of enriched T/B cells, being 3:1 for blood lymphocytes and 1:1 for spleen cells, which are the same as the physiological proportions of T and B cells in these lymphoid organs. Whereas unseparated lymphocytes gave a peat response on day 3, enriched B cells had a peak response on day 6. Fab anti-β₂m did not activate enriched T cells but increased DNA synthesis in enriched B cells to about the same extent as unseparated lymphocytes and mixtures of enriched T and B cells. The proportion of sheep erythrocyte rosette-forming cells (E-RFC) decreased after stimulation by anti-β₂m and increased after stimulation by phytohaemagglutinin. However, as revealed by autoradiography, a proportion of lymphocytes activated by anti-β₂m were E-RFC and this proportion increased with increasing stimulation by anti-β₂m. DNA synthesis induced by anti-β₂m was unchanged for spleen cells and slightly decreased for blood lymphocytes when phagocytic cells were removed by iron treatment. Supernatants from lymphocytes activated by anti-β₂m only induced low DNA synthesis in enriched T or B cells. Experiments with mitomycin treated cells indicate that cooperation and close contact between T and B cells are needed for activation by IgG anti-β₂m. T cells are needed for B cell activation by anti-β₂m and B cells are required for T cell activation to occur.