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. 2016 Jul;71(7):944-56.
doi: 10.1111/all.12858. Epub 2016 Mar 16.

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

R B Frei  1 , P Luschnig  1 , G P Parzmair  1 , M Peinhaupt  1 , S Schranz  1 , A Fauland  2 , C E Wheelock  2 , A Heinemann  1 , E M Sturm  1
Affiliations

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

R B Frei et al. Allergy. 2016 Jul.

Abstract

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.

Keywords: Cannabinoid receptor 2; Eosinophils; Ovalbumin-induced asthma; Priming; airway hyperresponsiveness.

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Figures

Figure 1
Figure 1
CB 2 expression is enhanced on eosinophils from symptomatic allergic donors. Purified human eosinophils were stained with a polyclonal rabbit anti‐human CB 2 primary Ab or isotype control (1 : 50), followed by a goat anti‐rabbit secondary Ab (AF‐647; 1 : 500).
Figure 2
Figure 2
JWH‐133 differentially modulates shape change in human eosinophils, basophils and neutrophils. (A) Eosinophils were pretreated with JWH‐133 (100 nM) or vehicle and stimulated with eotaxin‐2/CCL24 (Eot‐2). (B) Prior to JWH‐133 treatment, eosinophils were incubated with SR144528 (1 μM) and then treated as described in (A). (C and D) Eosinophils were pretreated with 2‐AG or JWH‐133 and stimulated with eotaxin‐2/CCL24 (0.6 nM) or vehicle. (E) Basophils (HLA‐DR −/CD123+ PBMC) were treated as described in (A). (F) Neutrophils (CD16+ PMNL) were pretreated with JWH‐133 (100 nM) or vehicle and stimulated with IL‐8. Data are shown as mean ± SEM. *P < 0.05 vs vehicle n = 4–7.
Figure 3
Figure 3
JWH‐133 primes human and mouse eosinophils for an enhanced CD11b expression and adhesion. (A) Human whole blood samples were stained with anti‐CD11b‐PE and anti‐CD16‐PE‐Cy5 Ab and pretreated with JWH‐133 (1 μM) or vehicle and then stimulated with eotaxin‐2/CCL24. (B) bmEos were stained with anti‐mouse CD11b‐PE Ab, pretreated with JWH‐133 (250 nM) or vehicle and stimulated with eotaxin‐2/CCL24. (C, D and E) Eosinophils were pretreated with SR144528 (1 μM) or vehicle, treated with JWH‐133 (250 nM) or vehicle, and stimulated with eotaxin‐2/CCL24 (0.6 nM). ICAM‐1‐coated channels were superfused with human eosinophils, and tightly adherent eosinophils (black arrows) were counted. Data are shown as mean ± SEM, *P < 0.05 n = 4–8.
Figure 4
Figure 4
JWH‐133 enhances the migratory responsiveness of human and mouse eosinophils. (A) Chemotaxis: eosinophils were allowed to migrate toward serial dilutions of JWH‐133, vehicle, or eotaxin‐2/CCL24. Chemokinesis: Eosinophils were pretreated with serial dilutions of JWH‐133 or vehicle and were allowed to migrate toward assay buffer only. (B) eosinophils were pretreated with JWH‐133 (5 nM) and were allowed to migrate toward eotaxin‐2 (1 nM). (C) bmEos were pretreated with vehicle or SR144528 (1 μM), mixed with JWH‐133 (250 nM) or vehicle, and cells were allowed to migrate toward eotaxin‐2/CCL24 (100 nM). (D) IL‐5Tg BALB/c mice were treated i.p. with JWH‐133 (5mg/kg/d) or vehicle for 3 days. Five hours after intranasal application of 4 μg eotaxin‐2, BALF was collected and eosinophils were counted as Siglec F+/CD11c− cells. (E) bmEos were isolated from WT or CB 2‐KO mice, treated with JWH‐133 (250 nM) or vehicle, and were allowed to migrate toward serial dilutions of eotaxin‐2/CCL24. Data are shown as mean ± SEM, *P < 0.05, n = 4–7.
Figure 5
Figure 5
JWH‐133 enhances eosinophil respiratory burst but not degranulation. (A) Eosinophils were pretreated with JWH‐133 (250 nM) or vehicle, DCFDA (50 μM) was added, and cells were stimulated with eotaxin‐2/CCL24 for 30 min at 37°C. Data are shown as mean ± SEM, n = 6. (B and C) Eosinophils were pretreated with JWH‐133 (250 nM) or vehicle, and CD63 expression and EPO release were induced with serial dilutions of C5a. Data are shown as mean ± SEM, n = 5.
Figure 6
Figure 6
JWH‐133 modulates eosinophil function in a pertussis toxin (PTX)‐insensitive manner. Isolated human eosinophils were pretreated with vehicle (A and C) or PTX (5 μg/ml) (B and D) for 20 min at 37°C and exposed to JWH‐133 (100 nM) or vehicle for 5 min at room temperature. Shape change was stimulated for 4 min at 37°C with serial dilutions of PGD 2 (A and B) or eotaxin‐2/CCL24 (C and D). Isolated human (E) or mouse (F) eosinophils were loaded with Fluo3‐AM and incubated with 1 μM SR144528 or vehicle, mixed with 200 nM JWH‐133, and Ca2+ flux was measured by FACS. G: Ca2+ mobilization was elicited by 100 nM of JWH‐133 and 2‐AG. Data are shown as representative of 3–5 independent experiments. (H) Eosinophil were pretreated with 5 μg/ml PTX for 20 min, with 6 μM U‐73122 or its inactive form U73343, 50 μM 2‐APB, or vehicle, and Ca2+ flux was induced by 200 nM JWH, 200 nM 2‐AG, or 1 nM eotaxin‐2/CCL24. In some experiments, EGTA (3 mM) was added prior to stimulation. Data are shown as mean ± SEM, *P < 0.05, n = 5–10.
Figure 7
Figure 7
JWH‐133 deteriorates lung function and increases eosinophil counts in BALF. Eight‐week‐old female C57BL6 mice were sensitized and expose to ovalbumin and their lung function was assessed while applying increasing doses of methacholine (MCH) by a FlexiVent system. Mice were given either JWH‐133 (10 mg/kg) or vehicle from day 10 to 17 and airway resistance (A) and compliance (B) were measured. For (C and D), SR144528 (10 mg/kg) was applied alone or in combination with JWH‐133. (E–G) CysLT analysis in the BALF by mass spectrometry revealed slightly increased LTC4 but significantly enhanced LTD4 as well as LTE4 levels in the JWH‐133‐treated animals which could be reversed by SR144528. (H) Flow cytometric analysis of cells in the BALF of mice treated as in (A). Data are shown as mean ± SEM, * P < 0.05, n = 6–9.
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