Bronchial hyperresponsiveness, epithelial damage, and airway eosinophilia after single and repeated allergen exposure in a rat model of anhydride-induced asthma

Bronchial hyperresponsiveness (BHR) and damage of the epithelium, as well as eosinophilia in the airway wall, induced by trimellitic anhydride (TMA) in sensitized brown Norway rats were studied. Rats were challenged once or seven times with aerosol of TMA conjugated to rat serum albumin (TMA-RSA) 3 weeks after intradermal TMA sensitization. Airway responsiveness (-log PC₃₀₀ of acetylcholine i.v.) was measured 24 h after allergen challenge. Epithelial lesion and eosinophil infiltration in the airway walls were quantified under light microscopy, and TMA-specific IgE and IgG in serum were evaluated with ELISA. High levels of TMA-specific IgE and IgG were found in all rats in the sensitized groups compared to nonsensitized groups ( P < 0.001). Repeated allergen challenges of 0.03% TMA-RSA for 7 consecutive days enhanced the level of TMA-specific IgG, compared to single challenge ( P < 0.05). Single allergen challenge of 0.3% TMA-RSA had a nonsignificant tendency to produce BHR in sensitized rats compared to nonsensitized rats ( P =0.06). However, repeated allergen challenges (0.003% and 0.03% TMA-RSA for 7 consecutive days) produced significant BHR in sensitized rats ( P < 0.05). Furthermore, repeated low-dose (0.003%) TMA-RSA challenge produced more BHR than a 10 times higher single dose (0.03%) ( P < 0.05). Slight damage of the airway epithelium was seen in sensitized and repeat-challenged groups. However, bronchial eosinophilia was found in the sensitized and single-challenged groups, but not in nonsensitized nonchallenged, and sensitized repeat-challenged groups ( P < 0.005). We conclude that the brown Norway rat can be sensitized with TMA, and that repeated low-dose allergen challenges produce slight epithelial damage and BHR which is independent of ongoing eosinophilia in the airway wall.     

Bradykinin-induced release of thromboxane B2 into bronchoalveolar lavage fluid of guinea pigs: relationship to airflow obstruction

The aim of this study was to evaluate the role of thromboxane A₂ in bradykinin-induced airflow obstruction in guinea pig in vivo. Airway insufflation pressure (P_i) was measured to assess airflow obstruction and the thromboxane B₂ (a stable metabolite of thromboxane A₂) concentration in bronchoalvelolar lavage fluid was determined by radioimmunoassay. The animals were pretreated with propranolol (1 mg/kg i.v.) and suxamethonium (5 mg i.v.) prior to bradykinin administration. Bradykinin instillation into the trachea (300 nmol) induced a P_i increase (47.5 ± 8.3 cm H₂O versus 23.8 ± 1.5 in sham) and significant thromboxane B₂ release into bronchoalveolar lavage fluid (79 ± 19 pg/ml versus 19 ± 6 in sham). A thromboxane synthase inhibitor (OKY-046, 30 mg/kg i.v.; ((E-E)-3-[p(1H-imidazole-1-yl-methyl) phenyl]-2-propenoic acid hydrochloride mono-hydrate)) or a thromboxane A₂ receptor antagonist (ICI192,605, 0.5 mg/kg i.v.; (4-(Z)-6-(2-o-chloro-phenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl)hexenoic acid)) reduced the P_i increase evoked by bradykinin (38.7 ± 3.8 and 40.6 ± 3.8 cm H₂O, respectively). OKY-046 abolished the thromboxane B₂ release. A platelet-activating factor receptor antagonist, WEB2086 (1 mg/kg i.v.; (3-[4-(chlorophenyl)-9-methyl-6H-thienol [3,2-f][1,2,4]trizolo-[4,3-a][1,4] diazepin-2-yl]1-4-(4-morpholinyl)-1-propanon) did not significantly affect any measured parameter. We conclude that, in guinea pigs, bradykinin-induced airway effects are associated with a local thromboxane A₂ release.     

Relaxation kinetics of formoterol and salmeterol in the guinea pig trachea in vitro

The mechanisms producing long duration of action for formoterol and salmeterol are not fully understood. The aim of the current study was to examine how the concentration of long and short acting ₂-adrenoceptor agonists affects their relaxation kinetics in airway smooth muscle. Onset (time to peak relaxation) and offset of action (reassertion of reversible relaxation following repeated -adrenoceptor blockade and washout) were measured in the guinea pig trachea precontracted postjunctionally by carbachol 0.3 M in vitro. At 10–1,000% (C _1O–C _1,000) of the maximally effective concentration (C ₁₀₀: 150 nM formoterol, 10 M salbutamol, 30 M salmeterol), salbutamol had a shorter time to peak relaxation than did salmeterol. Formoterol and salmeterol had a similar time to peak relaxation at C ₁₀, but, in contrast to salmeterol, formoterol's time to peak relaxation became markedly shorter and similar to that of salbutamol as the concentration was increased up to C _1,000. Significant reversible reasserted relaxation was demonstrated for salmeterol alone at C ₁₀. At C ₃₀–C _1,000, however, salmeterol produced irreversible relaxation only, in spite of repeated -adrenoceptor blockade by sotalol 10 M followed by washout. In contrast, formoterol produced an increasing reversible reasserted relaxation at C ₃₀–C _1,000. Salbutamol produced significant, reversible reasserted relaxation at C _1,000 only. In conclusion, the concentration determines the onset and offset of action for formoterol and to a lesser extent for salbutamol, but not for salmeterol. To cause sustained action, a submaximally effective concentration is sufficient for salmeterol, whereas formoterol requires a maximally effective concentration. The rank order of concentration dependence for the relaxation kinetics is not paralleled by the rank order of lipophilicity for formoterol, salbutamol, and salmeterol. Therefore, factors other than lipophilicity may also play a role in determining the relationship between concentration and relaxation kinetics for the investigated ₂-agonists. Offprint requests to: Anders Linden, MD, PhD     

The long and short of beta2-agonists

For patients whose asthma is not adequately controlled with inhaled corticosteroid (ICS) therapy alone, increasing the dose of ICS or the addition of a long-acting beta(2)-agonist is recommended. Greater improvements in lung function are achieved with the addition of a long-acting beta(2)-agonist to ICS therapy, rather than doubling the dose of ICS. Formoterol and salmeterol have a similarly long duration of effect (up to 12 h). However, as a result of their different chemical structures, there are marked pharmacological differences in the mechanism of action which affect their speeds of onset. These differences amount to a more rapid onset of effect for formoterol compared with salmeterol. Long-acting beta(2)-agonists appear to be well tolerated at elevated doses. These two features (tolerability at high doses and rapid onset of effect) support the use of formoterol as a reliever medication in addition to use in maintenance therapy. The long-acting beta(2)-agonists can be considered as beneficial additions to ICS therapy for the management of moderate-to-severe asthma.     

Regulation of allergen-induced bone marrow eosinophilopoiesis: role of CD4+ and CD8+ T cells

BACKGROUND: The mechanisms of the distant stimulation of the bone marrow (BM) after airway allergen exposure remain largely obscure. T cells have been implicated in allergic airway inflammation but their role in allergen-induced BM eosinophilopoiesis is poorly understood. The aim of this study was to determine the role of CD4(+) and CD8(+) T cells in allergen-induced BM eosinophilopoiesis. METHODS: Ovalbumin (OVA)-sensitized wild type (WT), CD4 knockout (CD4-/-) and CD8 knockout (CD8-/-) mice were exposed intranasally to OVA or saline. Bromo-deoxyuridine (BrdU) was used to label newly produced cells. Bone marrow, blood and bronchoalveolar lavage (BAL) were sampled 24 h after the final exposure. Immunostaining for newly produced eosinophils (i.e. BrdU(+)/MBP(+)) and BM eosinophil progenitor [CD34(+)/CD45(+)/interleukin-5 (IL-5)Ralpha(+)] cells was performed. RESULTS: The number of newly produced BM eosinophils (BrdU(+)/MBP(+) cells) was significantly reduced in allergen exposed CD4-/- or CD8-/- mice compared with allergen exposed WT mice, which was followed by a subsequent decrease in newly produced blood and airway eosinophils. Furthermore, BM eosinophil progenitors were significantly reduced in allergen exposed CD4-/- and CD8-/- mice compared with WT mice. Finally, serum IL-5 and Bronchoalveolar lavage fluid eotaxin-2 levels were abolished in allergen exposed CD4-/- mice to levels seen in saline exposed WT mice. CONCLUSIONS: These data suggests that both CD4(+) and CD8(+) T cells have a regulatory role in allergen-induced BM eosinophilopoiesis, whereas CD4(+) T cells are obligatory for allergen-induced airway eosinophilia. The subsequent traffic of eosinophils to the airways is likely to be at least partly regulated by a CD4(+) T-cell-dependent local airway eotaxin-2 production.     

Ultra long-acting beta 2-agonists in development for asthma and chronic obstructive pulmonary disease

After the discovery of formoterol and salmeterol, new candidates for long-acting beta2-adrenoceptor agonists (LABAs) have emerged from various companies. In particular, once-daily beta2-adrenoceptor agonists such as arformoterol, carmoterol, indacaterol, GSK-159797, GSK-597901, 159802, 642444 and 678007 are under development for the treatment of asthma and chronic obstructive pulmonary disease. The majority of these compounds are (R,R)-isomers in order to control desensitisation and accumulation. Several options for combination products are currently being evaluated in parallel with the development of these ultra LABAs. Once-daily dosing of an ultra LABA would be a significant convenience and probably a compliance-enhancing advantage leading to improved overall clinical outcomes in patients with asthma and chronic obstructive pulmonary disease. The only limits set for the development of a LABA with a new product profile are medical needs and marketing opportunities.     

Interleukin-17 as a recruitment and survival factor for airway macrophages in allergic airway inflammation

Recent data indicate that the proinflammatory cytokine, interleukin (IL)-17, stimulates certain effector functions of human macrophages. We evaluated whether IL-17 mediates allergen-induced accumulation of airway macrophages and, if so, whether such an effect relates to the control of macrophage recruitment and survival. BALB/c mice were sensitized and challenged with ovalbumin. Three hours before challenge an anti-mouse IL-17 mAb (a-IL-17) was administered. Sampling was conducted 24 h after the allergen challenge. In vitro chemotaxis assay for blood monocytes and culture of airway macrophages, immunocytochemistry for Fas-antigen, and matrix metalloproteinase-9 (MMP-9) were used to determine the effect of IL-17 on the recruitment, survival, and activity of airway macrophages. A-IL-17 reduced the number of airway neutrophils and macrophages after allergen challenge. In vitro, recombinant IL-17 induced migration of blood monocytes and prolonged survival of airway macrophages. A-IL-17 also increased the expression of Fas-antigen in airway macrophages in vivo. Finally, the expression of MMP-9 by airway neutrophils and macrophages in vivo was downregulated by a-IL-17. This study indicates that endogenous IL-17 mediates the accumulation of macrophages during allergen-induced airway inflammation. IL-17 exerts its effects by acting directly on airway macrophages by promoting their recruitment and survival. Furthermore, IL-17 is involved in controlling the proteolytic activity of macrophages and neutrophils in allergen-induced airway inflammation.