Cannabinoid receptor 2 augments eosinophil responsiveness and aggravates allergen-induced pulmonary inflammation in mice.

BACKGROUND: Accumulation of activated eosinophils in tissue is a hallmark of allergic inflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) has been proposed to elicit eosinophil migration in a CB2 receptor/Gi/o -dependent manner. However, it has been claimed recently that this process may also involve other mechanisms such as cytokine priming and the metabolism of 2-AG into eicosanoids. Here, we explored the direct contribution of specific CB2 receptor activation to human and mouse eosinophil effector function in vitro and in vivo. METHODS: In vitro studies including CB2 expression, adhesion and migratory responsiveness, respiratory burst, degranulation, and calcium mobilization were conducted in human peripheral blood eosinophils and mouse bone marrow-derived eosinophils. Allergic airway inflammation was assessed in mouse models of acute OVA-induced asthma and directed eosinophil migration. RESULTS: CB2 expression was significantly higher in eosinophils from symptomatic allergic donors. The selective CB2 receptor agonist JWH-133 induced a moderate migratory response in eosinophils. However, short-term exposure to JWH-133 potently enhanced chemoattractant-induced eosinophil shape change, chemotaxis, CD11b surface expression, and adhesion as well as production of reactive oxygen species. Receptor specificity of the observed effects was confirmed in eosinophils from CB2 knockout mice and by using the selective CB2 antagonist SR144528. Of note, systemic application of JWH-133 clearly primed eosinophil-directed migration in vivo and aggravated both AHR and eosinophil influx into the airways in a CB2 -specific manner. This effect was completely absent in eosinophil-deficient ∆dblGATA mice. CONCLUSION: Our data indicate that CB2 may directly contribute to the pathogenesis of eosinophil-driven diseases. Moreover, we provide new insights into the molecular mechanisms underlying the CB2 -mediated priming of eosinophils. Hence, antagonism of CB2 receptors may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophilic disorders.

The basophil activation test in the diagnosis of allergy: technical issues and critical factors.

BACKGROUND: The basophil activation test (BAT) is a widely validated and reliable tool especially for the diagnosis of hymenoptera venom allergy. Nevertheless, several pitfalls have to be considered and outcomes may differ because of diverse in-house protocols and commercially available kits. We aimed to identify the factors that may influence results of the CD63-based BAT. METHODS: Basophil responses to monoclonal anti-IgE (clone E124.2.8) and bee and wasp venom were determined by BAT based on CD63. The effect of stimulating factors such as, IL-3, cytochalasin B and prewarming of the samples was investigated. Furthermore, we compared two different flow cytometer systems and evaluated the influence of storage time, different staining protocols and anti-allergic drugs on the test results. RESULTS: Interleukin-3 enhanced the reactivity of basophils at 300 pM, but not at 75 and 150 pM. Prewarming of samples and reagents did not affect basophil reactivity. CD63 expression assayed after storage time of up to 48 h showed that basophil reactivity already started to decline after 4 h. Basophils stained with HLA-DR-PC5 and CD123-PE antibodies gated as HLA-DR(neg)/CD123(pos) cells showed the highest reactivity. No effect on test outcomes was observed at therapeutic doses of dimetindene and desloratadine. Finally, slight differences in the percentage of activated basophils, depending on the cytometer system used, were found. CONCLUSION: Basophil activation test should be performed as early as possible after taking the blood sample, preferably within 4 h. In contrast to the skin test, BAT can be performed in patients undergoing treatment with antihistamines. For reasons of multiple influencing factors, BAT should be performed only at validated laboratories.

Influence of total IgE levels on the severity of sting reactions in Hymenoptera venom allergy.

BACKGROUND: Detection of specific IgE for Hymenoptera venoms and skin tests are well established diagnostic tools for the diagnosis of insect venom hypersensitivity. The aim of our study was to analyze the effect of total IgE levels on the outcome of generalized anaphylactic reactions after a Hymenoptera sting. METHODS: Two hundred and twenty patients allergic to bee, wasp, or European hornet venom were included in the study. Their specific and total IgE levels, serum tryptase levels, skin tests, and sting history were analyzed. RESULTS: In patients with mild reactions (grade I, generalized skin symptoms) we observed higher total IgE levels (248.0 kU/l) compared to patients with moderate reactions (grade II, moderate pulmonary, cardiovascular, or gastrointestinal symptoms; 75.2 kU/l) and severe reactions (grade III, bronchoconstriction, emesis, anaphylactic shock, or loss of consciousness; 56.5 kU/l; P < 0.001). Accordingly, 25% of the patients with low levels of total IgE (<50 kU/l), but no individual with total IgE levels >250 kU/l, developed loss of consciousness (P = 0.001). Additionally, specific IgE levels were related to total IgE levels: Specific IgE levels increased from 1.6 to 7.1 kU/l in patients with low (<50 kU/l) and high (>250 kU/l) total IgE levels, respectively (P < 0.001). Specific IgE levels correlated inversely to the clinical reaction grades, however, this trend was not statistically significant (P = 0.083). CONCLUSION: Patients with Hymenoptera venom allergy and high levels (>250 kU/l) of total IgE, predominantly develop grade I and grade II reactions and appear to be protected from grade III reactions. However, this hypothesis should be confirmed by extended studies with sting challenges.