Cannabinoid receptor 2 augments eosinophil responsiveness and aggravates allergen‐induced pulmonary inflammation in mice
Institute of Experimental and Clinical PharmacologyMedical University of GrazGrazAustria
Division of Physiological Chemistry IIDepartment of Medical Biochemistry and BiophysicsKarolinska InstitutetStockholmSweden
*CorrespondenceEva M. Sturm, PhD, Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Universitaetsplatz 4, 8010 Graz, Austria
Tel.: +43 316 380 4319
Fax : +43 316 380 9645
E‐mail: eva.sturm@medunigraz.at
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 CB 2 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 CB 2 receptor activation to human and mouse eosinophil effector function in vitro and in vivo.
Methods
In vitro studies including CB 2 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
CB 2 expression was significantly higher in eosinophils from symptomatic allergic donors. The selective CB 2 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 CB 2 knockout mice and by using the selective CB 2 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 CB 2‐specific manner. This effect was completely absent in eosinophil‐deficient ∆dblGATA mice.
Conclusion
Our data indicate that CB 2 may directly contribute to the pathogenesis of eosinophil‐driven diseases. Moreover, we provide new insights into the molecular mechanisms underlying the CB 2‐mediated priming of eosinophils. Hence, antagonism of CB 2 receptors may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophilic disorders.
Article notes
Frei RB , Luschnig P , Parzmair GP , Peinhaupt M , Schranz S , Fauland A , Wheelock CE , Heinemann A , Sturm EM . Cannabinoid receptor 2 augments eosinophil responsiveness and aggravates allergen‐induced pulmonary inflammation in mice. Allergy 2016; 71: 944–956.2685009410.1111/all.12858PMC5225803
Article notes
Footnote Group
- 2‐AG
- 2‐arachidonoylglycerol
- Ab
- antibody
- AF
- Alexa Fluor
- AHR
- airway hyperresponsiveness
- bmEos
- mouse bone marrow‐derived eosinophils
- CI
- chemotactic or chemokinetic index
- Cy5
- cyanine 5
- CysLTs
- cysteinyl leukotrienes
- DCF
- 2′7′‐dichlorofluorescein
- DCFDA
- 2′,7′‐dichlorofluorescin diacetate
- ERK
- extracellular signal‐regulated kinase
- FSC
- forward scatter
- IL
- interleukin
- LC‐MS
- liquid chromatography–mass spectrometry
- LT(x)
- leukotriene
- MAPK
- mitogen‐activated protein kinase
- MEK
- mitogen/extracellular signal‐regulated kinase
- OVA
- ovalbumin
- PAF
- platelet‐activating factor
- PBMC
- peripheral blood mononuclear cells
- PE
- phycoerythrin
- PG
- prostaglandin
- PI3K
- phosphoinositide 3‐kinase
- PMNL
- polymorphonuclear leukocytes
- PTX
- pertussis toxin
- ROCK
- rho‐associated protein kinase
- SSC
- side scatter
Eosinophils are potent effector cells in the pathogenesis of many disorders, ranging from allergy and bronchial asthma 1, to eosinophilic esophagitis 2, colitis ulcerosa 3, hypereosinophilic syndrome 4, and renal disease 5. Activated eosinophils are one of the primary sources of cytotoxic proteins, proinflammatory cytokines, and growth factors, such as IL‐4, IL‐5, IL‐10, and IL‐13 6, and promote numerous immunoregulatory functions that lead to the progression of inflammation, mucus secretion, tissue remodeling, and angiogenesis 7.
Regarding asthma, levels of eosinophil granule proteins such as major basic protein (MBP) or eosinophil peroxidase (EPO) broadly correlate with disease severity 8. Moreover, it has been shown that patients who receive treatment based on eosinophil counts in sputum have significantly fewer severe asthma exacerbations than patients treated according to standard management therapy 9.
Corticosteroids are currently the most effective treatment to reduce eosinophil numbers in the blood and tissue. However, the pleiotropic effects, especially of orally applied corticosteroids to control severe asthma, can result in potentially harmful side‐effects and thus limit their therapeutic use 10. Therefore, considerable effort has been invested in the development of drugs that can effectively control the trafficking and activation of eosinophils to ameliorate the inflammatory response. Current clinical trials with the eosinophil‐targeted mepolizumab revealed an oral glucocorticoid‐sparing effect and significantly reduced exacerbation rates in well‐selected patients with persistent eosinophilic asthma; however, FEV1 seemed to remain largely unaffected 11, 12. Of note, asthma is a heterogeneous condition with diverse characteristics and disease variants. Thus, stratification of patients by clinical characteristics (phenotypes) and pathogenetic mechanisms (endotypes) should lead to more targeted and personalized approaches to asthma therapy. Endocannabinoids are bioactive lipids released from the cell membrane upon cell activation. The two main endocannabinoids are the arachidonic acid (AA) derivatives 2‐arachidonoylglycerol (2‐AG) 13 and N‐arachidonoylethanolamine (anandamide (AEA)) 14. Most of their actions are mediated by two G‐protein‐coupled receptors, the cannabinoid receptors 1 and 2 (CB1 and CB2). In contrast to CB1, which is abundantly expressed in the nervous system 15, CB2 is mainly found in lymphoid organs and cells of the immune system 16. Among others, CB2 expression has been reported for B cells, monocytes/macrophages, and eosinophils, indicating a crucial immunoregulatory role for CB2 and its ligands 17.
Up to now, only three studies have examined the direct contribution of the endocannabinoid 2‐AG on eosinophil migration in vitro. Oka et al. demonstrated that 2‐AG induces the chemotaxis of EoL‐1 cells and human peripheral blood eosinophils in a CB2‐dependent manner, although the order of the pharmacologically effective concentration of 2‐AG was significantly higher than that of other chemoattractants 18. Interestingly, another group confirmed this minimal CB2‐mediated effect of 2‐AG and suggested that 2‐AG‐induced migration is a complex process that may involve other mechanisms, such as cytokine priming, rapid 2‐AG metabolism into eicosanoids, and further generation of 15‐lipoxygenase metabolites 19.
Moreover, due to the use of different rodent models and varying pharmacological approaches, the in vivo role of CB2 in eosinophilic disorders is still uncertain. Indeed, a CB2 inverse agonist was shown to block ovalbumin (OVA)‐induced lung eosinophilia in mice 20. Accordingly, the CB2 antagonist SR144528 mediated beneficial effects in oxazolone‐induced contact dermatitis 21. In contrast, Giannini et al. evaluated the effects of the CB1/CB2 receptor agonist CP55,940 on OVA‐induced asthma in guinea pigs and concluded that both CB1 and CB2 receptors are involved in lung protection 22. Similarly, THC reduced cytokine and IgE level as well as mucus production in OVA‐challenged mice 23; however, the use of CB1/CB2 knockout mice revealed that the effects of THC were cannabinoid receptor‐independent 24.
Because of the conflicting reports on the pathophysiological role of CB2 receptors, we set out to explore the direct contribution of specific CB2 activation on eosinophil effector function. To this end, the effects of the endocannabinoid 2‐AG and the potent and selective CB2 receptor agonist JWH‐133 were studied in vitro and in mouse models of acute OVA‐induced asthma and directed eosinophil migration. Our results provide clear evidence for a novel CB2‐induced eosinophil‐specific ‘priming mechanism’ that potentiates eosinophil effector function in vitro and enhances bronchial inflammation in vivo, reflected by both increased airway resistance and eosinophil influx into the airways.
Materials and methods
Detailed materials and methods are provided in the supplement.
Preparation of mouse eosinophils
Bone marrow‐derived eosinophils (bmEos) were differentiated ex vivo from unselected bone marrow progenitors using a well‐defined cytokine regimen 25.
Flow cytometric staining of CB2 receptors on eosinophils
Purified human eosinophils were stained with a polyclonal rabbit anti‐human CB2 primary Ab or isotype control, followed by a goat anti‐rabbit secondary Ab (AF‐647). CB2 expression was quantified by flow cytometry.
Shape change assay
Migration
Chemotaxis and Chemokinesis: Purified human eosinophils or bmEos were pretreated as indicated, placed into the top of a 48‐well micro‐Boyden chamber (human) or a 96‐well chemotaxis plate (bmEos), and were allowed to migrate toward the indicated chemoattractant or vehicle.
Calcium flux
Isolated human eosinophils or bmEos were loaded with Fluo‐3‐AM in the presence of 0.02% pluronic F‐127 28. Changes in intracellular Ca2+ were detected as fluorescence increase in the FL1‐(530/30 nm) channel.
Adhesion assay under flow
Vena8™ biochips (Cellix Ltd., Dublin, Ireland) were coated with ICAM‐1, and coated channels were superfused with purified eosinophils. Adhesion was monitored using a Hamamatsu ORCA‐03G digital camera and CellixVenaFlux software.
CD11b‐upregulation
Whole blood samples or PMNL were pretreated as indicated and were incubated with agonists for 30 min at 37°C 29. Samples were stained with anti‐CD16‐PE‐Cy5 and anti‐CD11b‐PE (ICRF44) Ab. CD11b upregulation was analyzed by flow cytometry.
In vivo chemotaxis
Eight‐week‐old IL‐5Tg mice were treated i.p. with JWH‐133 (5 mg/kg/day) or vehicle for three consecutive days. In vivo chemotaxis of eosinophils was induced by intranasal instillation of 4 μg eotaxin‐2/CCL24. Bronchoalveolar lavage fluid (BALF) was collected 5 h postinstillation, and migration was evaluated by flow cytometric counting of highly granular (high side scatter) CD11c−/Siglec F+ cells.
Mouse model of allergic lung inflammation
Eight‐week‐old female C57Bl6/N mice were immunized by i.p. injections of 10 μg of OVA adsorbed to Al(OH)3 on days 0 and 7. Mice were challenged by an aerosol of OVA in saline on days 14 and 16. Additionally, mice received a daily i.p. injection of CB2 agonist/antagonist (10 mg/kg) or vehicle on day 9 to day 16. On day 17, either airway hyperresponsiveness to methacholine was recorded with the FlexiVent system (Scireq, Montreal, QC, Canada) or BALF was taken and analyzed by flow cytometry.
Results
CB2 receptor expression is enhanced on eosinophils from allergic donors
CB2 expression has been reported for B cells, monocytes/macrophages, NK cells, basophils, and eosinophils, and at lower levels, for neutrophils and T cells 16, 30, 31. In this study, CB2 surface expression was confirmed by flow cytometric staining in‐ and off‐season in human peripheral blood eosinophils from allergic volunteers with seasonal respiratory symptoms and healthy subjects. Of note, quantification revealed ~3.4‐fold higher expression of CB2 on eosinophils from symptomatic allergic donors compared to healthy controls. Interestingly no CB2 upregulation was observed off‐season in eosinophils from asymptomatic allergic donors (Fig. 1).
The CB2 receptor agonist JWH‐133 specifically enhances human and mouse eosinophil function
Conflicting data exist regarding the effect of CB2 activation in eosinophils and neutrophils, and little is known about its impact on basophil function 32, 33. Thus, here we explored the effect of the selective CB2 agonist JWH‐133 on human eosinophils, neutrophils, and basophils by means of shape change. Having encountered a chemotactic factor in vivo, leukocytes immediately begin to rearrange their cytoskeleton and change their shape to facilitate their attachment to microvascular endothelial cells. Such morphological changes can be detected by flow cytometry as changes in the forward scatter properties of the cells 34. Representative plots are provided in Fig. S5. First, purified eosinophils were pretreated with JWH‐133 (100 nM) or vehicle, cells were stimulated with serial dilutions of eotaxin‐2/CCL24, and shape change was assessed by flow cytometry. As shown in Fig. 2A, JWH‐133 led to a significant increase of eosinophil responses; particularly, the sensitivity to eotaxin‐2/CCL24 was increased up to threefold. To verify that this effect was mediated through selective CB2 activation, eosinophils were exposed to the CB2 antagonist SR144528 (1 μM, 10 min at RT) prior to the assay. As illustrated in Fig. 2B, SR144528 totally prevented the enhancing effect of JWH‐133. Similarly, the endocannabinoid 2‐AG concentration‐dependently increased eotaxin‐2/CCL24‐induced shape change and elicited a weak, but dose‐dependent shape change by itself with a maximum response observed at 500 nM (Fig. 2C), similar to JWH‐133 (Fig. 2D). Emphasizing the cell type‐specific impact of selective CB2 activation, pretreatment with JWH‐133 only slightly enhanced basophil shape change as induced by eotaxin‐2/CCL24 (Fig. 2E) and did not affect neutrophil responses to IL‐8 (Fig. 2F).
Besides shape change, the chemoattractant‐induced upregulation of adhesion molecules such as αmβ2 integrins (CD11b/CD18; Mac‐1) is another prerequisite for eosinophil effector functions 35. Thus, human eosinophils in whole blood and mouse bmEos were pretreated with JWH‐133 (1 μM and 250 nM, respectively) or vehicle and stimulated with eotaxin‐2/CCL24. Interestingly, JWH‐133 clearly potentiated the ability of eotaxin‐2/CCL24 to upregulate CD11b in human eosinophils, whereas in mouse eosinophils, JWH‐133 directly induced CD11b surface expression. At lower concentrations of eotaxin‐2/CCL24, JWH‐133 led to a ~50% increase of CD11b in human eosinophils (Fig. 3A) and to a ~25% increase in bmEos (Fig. 3B). Similar modulatory effects were observed with 2‐AG (250 nM) (Fig. S1).
The same pattern of CB2‐mediated priming was observed in adhesion assays under flow conditions. Pretreatment of purified human eosinophils with 100 nM JWH‐133 led to a significant twofold increase of firm adherent cells on ICAM‐1 coated channels compared to eotaxin‐2/CCL24 alone. Again, this effect was completely prevented with the CB2 antagonist SR144528 (1 μM) (Fig. 3C). Representative pictures are provided in Fig. 3D and E.
To explore the direct contribution of CB2 activation to eosinophil recruitment, in vitro and in vivo migration experiments were performed. As shown in Fig. 4A, JWH‐133 itself elicited a moderate chemotactic response in human eosinophils but not in mouse eosinophils (Fig. S2), similar to the previously noted effect of the endogenous CB2 agonist 2‐AG 19. The maximal chemotactic response (CI ~3.9) was observed at ~100 nM of JWH‐133. At micromolar levels JWH‐133 evoked chemokinesis, with a maximal response (CI ~1.8) observed at 3 μM. Moreover, at low nanomolar concentrations JWH‐133 (5 nM) significantly increased the migratory capacity of human eosinophils (Fig. 4B) and bmEos (Fig. 4C) toward eotaxin‐2/CCL24 by ~80% and 28%, respectively. Emphasizing the physiological importance of these observations, systemic administration of JWH‐133 (5 mg/kg/day) for three consecutive days significantly enhanced the eotaxin‐2/CCL24‐directed accumulation of eosinophils in the airways of IL‐5 transgenic mice by ~29% (Fig. 4D). To further confirm the direct involvement of CB2 in the recruitment of mouse eosinophils, bmEos were isolated from WT and CB2‐KO mice, treated with vehicle or JWH‐133 (250 nM), and then allowed to migrate toward serial dilutions of eotaxin‐2. Figure 4E shows that the priming effect of the cannabinoid agonist is not present in CB2‐KO mice, whereas JWH‐133 treatment resulted in a significantly increased migratory response of bmEos from WT mice.
CB2 receptor activation enhances respiratory burst but not degranulation in human eosinophils
At sites of allergic inflammation, activated eosinophils cause tissue damage by the production of reactive oxygen species (ROS) and the release of toxic granule proteins 36. To explore the impact of selective CB2 activation on eosinophil respiratory burst, purified human eosinophils were applied to 250 nM JWH‐133 for 5 min at 37°C and respiratory burst was induced with eotaxin‐2/CCL24. As shown in Fig. 5A, JWH‐133 itself failed to induce respiratory burst, but significantly increased the eotaxin‐2/CCL24 induced ROS production up to ~25%.
Interestingly, although having significant effects on eosinophil recruitment and ROS production, JWH‐133 neither induced nor primed human eosinophil degranulation as assessed by means of CD63 upregulation and EPO release (Fig. 5B and C).
JWH‐133 modulates eosinophil responses in a pertussis toxin (PTX)‐insensitive manner
CB2 receptors are known to activate heterotrimeric Gi/o type G proteins leading to the inhibition of adenylyl cyclase (AC). Furthermore, CB2 signaling was shown to involve MAPK (p38 and p42/44) and PI3K activity 37. To explore whether the observed effects depend on Gαi activation, purified human eosinophils were incubated in the presence of pertussis toxin (PTX; 5 μg/ml for 20 min at 37°C) and shape change was induced with PGD2 (PTX‐insensitive) or eotaxin‐2/CCL24 (PTX‐sensitive). Notably, pretreatment with PTX did not affect the modulatory effects on PGD2‐induced shape change (Fig. 6A and B). As expected, PTX pretreatment almost completely blocked the Gαi‐dependent CCR3/CCL24 response but not the priming properties of JWH‐133 (Fig. 6C and D).
JWH‐133 induces calcium release in human and mouse eosinophils
Having confirmed that CB2 signaling in human eosinophils is transmitted in a Gαi‐independent manner, we next aimed to explore the role of Ca2+ in the CB2 pathway. To this end, responses of the endogenous ligand 2‐AG were compared with those of the synthetic agonist JWH‐133. At 100 nM, JWH‐133 only induced a weak Ca2+ signal in human eosinophils, whereas 2‐AG exhibited a robust fivefold stronger response (Fig. 6G). Consistent with JWH‐133 and 2‐AG sharing the same receptor, JWH‐133 (100 nM) desensitized the calcium response induced by 2‐AG (100 nM) (Fig. S3A). However, 100 nM of JWH‐133 or 2‐AG, concentrations that were effective in enhancing shape change responses in human eosinophils, did not increase eotaxin‐2/CCL24‐induced Ca2+ flux (Fig. S3B), neither did 200 nM of JWH‐133 (Fig. S3C), although at this concentration a robust and CB2‐dependent Ca2+ flux was found in human (Fig. 6E) and mouse eosinophils (Fig. 6F). To confirm the observed PTX insensibility of CB2 signaling, human eosinophils were treated with PTX (5 μg/ml, 20 min 37°C) and Ca2+ flux was induced with JWH‐133 (200 nM), 2‐AG (200 nM) or eotaxin‐2/CCL24 (1 nM), respectively. As expected, Ca2+ responses to eotaxin‐2/CCL24 were almost completely blocked, whereas JWH‐133‐ and 2‐AG‐induced Ca2+ responses remained unaffected (Fig. 6H). In contrast, pretreatment with the PLC inhibitor U‐73122 (but not its inactive form U73343), as well as the IP3 receptor antagonist 2‐APB abolished Ca2+ responses induced by JWH‐133 and 2‐AG indicating that CB2 interacts with Gαq rather than Gαi. 2‐APB is also capable of inhibiting TRP channels. Thus, to exclude the involvement of TRP, control experiments in the presence of 3 mM EGTA were conducted, which showed that Ca2+ influx from the extracellular space and hence TRP channels are not involved in the Ca2+ response to CB2 receptor activation (Fig. 6H).
Effects of JWH‐133 on human eosinophils are mediated via MEK1/2 and p160 ROCK
Human eosinophil migration is regulated by multiple signaling pathways involving PI3K, ROCK, ERK, and p38 MAPK 38. To assess the downstream components of the CB2 pathway in human eosinophils, cells were pretreated with LY‐294002 (PI3K, 10 μM), U‐0126 (MEK1/2, 10 μM), PD‐184161 (MEK1/2, 18 μM), SB202190 (p38 MAPK, 50 μM, Y‐27632 (ROCK, 200 nM) or vehicle, respectively. Concentrations were chosen according to the literature, and pretreatment was performed for 20 min at 37°C 38. Thereafter, eosinophil shape change was induced with eotaxin‐2/CCL24. As illustrated in Fig. S4, we found that the MEK1/2 inhibitors U‐0126 (B) and PD‐184161 (C) as well as the p160 ROCK inhibitor Y‐27632 (D) significantly reduced the priming effect of JWH‐133 for eotaxin‐2/CCL24 responses, whereas blockade of PI3K (E) and p38 MAP kinase (F) had no effect.
Having confirmed that JWH‐133 augments eosinophil responsiveness to other chemoattractants via MEK1/2 and ROCK signaling in shape change, we investigated the selective contribution of MEK1/2 and ROCK to JWH‐133‐induced chemotaxis. Similarly, pretreatment with U‐0126 and Y‐27632 prior to the chemotaxis assay effectively inhibited eosinophil migration toward serial dilutions of JWH‐133 (Fig. S4G and H).
Systemic application of JWH‐133 worsens airway hyperreactivity (AHR) in mice
Our in vitro data clearly show that CB2 ligands significantly contribute to eosinophil activation and responsiveness. To prove the in vivo relevance of these observations we used an acute model of OVA‐induced asthma in mice. In brief, mice were immunized to OVA on days 0 and 7, were treated i.p. with JWH‐133 (10 mg/kg), SR144528 (10 mg/kg), a combination of both, or vehicle from day 9 to day 16, and were challenged with inhaled OVA aerosol on days 14 and 16. On day 17, either airway hyperresponsiveness to methacholine was recorded, or BALF was taken. As illustrated in Fig. 7, JWH‐133 significantly impaired airway resistance and compliance compared to the vehicle group (Fig. 7A and B), whereas SR144528 pretreatment fully prevented these effects of JWH‐133 (Fig. 7C and D). Of the potent inflammatory lipid mediators comprising the cysteinyl leukotrienes (CysLTs: LTC4, LTD4, and LTE4), only LTE4 is stable and abundant in vivo. Besides mast cells, eosinophils are the main source of CysLTs which contribute not only to bronchoconstriction and airway hyperreactivity 39, but as in the case of LTE4 also to eosinophil recruitment 40. Increased levels of LTE4 can be detected in urine 41, BALF 42, and exhaled breath condensate 43 of patients after allergen challenge. Accordingly, mass spectrometric analysis of BALF from OVA‐challenged mice revealed slightly increased LTC4 levels (Fig. 7E) and significantly elevated concentrations of LTD4 and LTE4 in JWH‐133‐treated mice compared to vehicle‐treated controls. Again, the effect of JWH‐133 could be abolished with SR144528 pretreatment (Fig. 7F and G). We furthermore determined eosinophil counts in the BALF of JWH‐133‐ and vehicle‐treated mice. Consistent with our results from the in vitro and in vivo migration assays, flow cytometric analysis revealed threefold higher eosinophil counts in the JWH‐133‐treated group compared to controls (Fig. 7H). Representative histology images of lung sections are provided in Fig. S6. To confirm the eosinophil‐specific effect of the CB2 agonist to the pathogenesis of allergic asthma, further experiments in eosinophil‐deficient ∆dblGATA mice were conducted. Treatment with JWH‐133 worsened airway resistance in WT mice, but mediated beneficial effects on lung parameters in the ∆dblGATA group (Fig. S7A/B). Thus, our results show unequivocally that systemic CB2 activation directly contributes to the pathophysiology of asthma in mice by enhancing eosinophil migration and effector function.
Discussion
In this study, we propose a novel mechanism of CB2‐induced priming of eosinophils that may directly contribute to the pathogenesis of eosinophilic diseases. This notion is supported by the observation that pretreatment with the selective and stable CB2 receptor agonist JWH‐133 profoundly increased eosinophil responsiveness toward chemoattractants such as eotaxin‐2/CCL24 and PGD2, with respect to shape change, integrin expression, adhesion, chemotaxis and ROS production. The magnitude of primed responses was dependent on the concentration of JWH‐133 and of the chemoattractant. In line with these in vitro data, systemic application of JWH‐133 amplified the eotaxin‐2/CCL24‐directed recruitment of eosinophils into the airways of IL‐5Tg mice and exacerbated OVA‐induced asthmalike inflammation by increasing eosinophil influx into lungs and worsening of AHR. Oka et al. were the first who demonstrated that EoL‐1 cells and human peripheral blood eosinophils express CB2 but not CB1 receptors 30. Consistent with these data, we found that purified human eosinophils from healthy subjects express an appreciable amount of CB2 on mRNA (data not shown) and protein levels. Enhanced gene expression of CB2 has been shown for lung eosinophils in allergic patients after allergen challenge 44. Accordingly, we could demonstrate that also CB2 protein expression is significantly increased on the cell surface of peripheral blood eosinophils from symptomatic allergic donors, supporting a role for CB2 and its ligands in the regulation of allergen‐induced eosinophilic inflammation.
Activation with the selective agonist JWH‐133 at nanomolar levels only induced a slight chemotactic response in human eosinophils, but amplified eotaxin‐2/CCL24 induced eosinophil migration, especially at low concentrations. In contrast, IL‐8‐induced neutrophil activation remained unaffected by JWH‐133, which is in line with previous reports demonstrating that the effects of 2‐AG on neutrophils are mainly caused by the degradation of 2‐AG to AA and subsequent de novo synthesis of LTB4 32.
Preactivation or priming of eosinophils by proinflammatory cytokines in the peripheral blood is a crucial step in the pathogenesis of allergic diseases. Several priming‐dependent eosinophil responses, such as migration 45, adhesion 46, and degranulation 47, have been shown to be amplified in allergic patients. Moreover, a ‘hyperadhesive’ eosinophil phenotype, characterized by increased levels of the adhesion molecule integrin αMβ2 (CD11b/CD18) has been described in allergic patients after allergen challenge 48. Here we could show that exposure to the selective CB2 receptor agonist JWH‐133 prior to stimulation with chemoattractants yields significantly upregulated amounts of CD11b on the surface of both human and mouse eosinophils. Accordingly, JWH‐133 also primes eosinophils for an enhanced capacity to adhere to ICAM‐1 under physiological flow conditions, an important prerequisite of endothelial transmigration.
To further prove the in vivo relevance of the observed modulating activities of CB2, well‐established mouse models of directed eosinophil migration and OVA‐induced asthma were performed. Of note, we could show that daily treatment with JWH‐133 not only aggravated lung parameters, but also led to increased eosinophil counts in the airways of OVA‐challenged mice. Interestingly, these effects were completely absent in eosinophil‐deficient ∆dblGATA mice, indicating that eosinophils are the major target of JWH‐133 in allergic inflammation. Moreover, BALF of JWH‐133‐treated animals contained significantly higher CysLT levels compared to control animals; again, pointing to a more severe disease state due to systemic CB2 activation. Noteworthy, Larose et al. demonstrated that 2‐AG alone or in combination with platelet‐activating factor (PAF) induced CysLT biosynthesis in human eosinophils in vitro. However, 2‐AG/PAF‐induced synthesis was blocked by MAG lipase inhibitors, indicating that this effect is more related to 2‐AG degradation and metabolic transformation into eicosanoids than to selective CB2 activation 37. In contrast to 2‐AG, JWH‐133 is not degradable to AA, and therefore cannot be further used for CysLT synthesis indicating a direct correlation exists between the observed enhanced CysLT levels and activation of CB2. In accordance with our findings, recent work provided further evidence that CB2 receptor activation is capable of enhancing inflammatory processes. Treatment with the CB2 agonist JWH‐133 potentiated adipose tissue inflammation in mice on high fat diet 49. Notably, 2‐AG plasma levels were also found to positively correlate with the body mass index (BMI) in humans 50. Interestingly, the prevalence of asthma, its severity, and reduced responsiveness to standard medication seem to be associated with obesity and high BMI 51. However, the molecular mechanisms leading to these derangements are still poorly understood, but it is tempting to speculate that CB2 activation by systemically elevated 2‐AG levels and facilitation of eosinophil recruitment into the airways might be involved.
In both, human eosinophils and monocytes, 2‐AG‐induced CB2 signaling was reported to be Gαi/o‐dependent as pretreatment with PTX abrogated the chemotactic responses of these cells 30. Conversely, our data strongly suggest a Gαi/o/adenylyl cyclase‐independent pathway, substantiated by the fact that PTX was unable to prevent eosinophil priming and Ca2+ flux following CB2 activation. Furthermore, we observed that the PLC inhibitor U‐73122 and the IP3 receptor antagonist 2‐APB were capable of reducing JWH‐133‐ and 2‐AG‐induced Ca2+ release. Thus, eosinophil CB2 receptors seem to interact with Gαq rather than, or in addition to, Gαi proteins. Of note, Shoemaker et al. showed previously that cannabinoid agonists display different rank orders of potencies and receptor occupancies for regulation of intracellular effectors. Endogenous ligands such as 2‐AG are more ‘efficient’ agonists requiring only half the receptor occupancy to elicit same effects as synthetic agonists. Accordingly, we found that 2‐AG induced a much stronger Ca2+ response compared to JWH‐133 52. CB2 receptors have previously been shown to induce a Gβγ‐dependent MAPK/ERK signaling cascade 53. Here we found that the induction of MEK 1/2 and ROCK activity is likely to be a part of the signaling mechanism accounting for the priming effect of JWH‐133. Our observations are in line with previous findings indicating the involvement of MEK1/2‐ERK activation in IL‐5 and GM‐CSF induced priming of human eosinophils 53. Moreover, MEK1/2‐ERK and ROCK signaling regulates a variety of proinflammatory cellular processes such as eosinophil migration, degranulation, and respiratory burst 38, 55 which afford the progression of eosinophilic inflammation.
In summary, the results of the present study demonstrate for the first time that specific CB2 activation represents a novel priming process leading to enhanced migratory responsiveness of human and mouse eosinophils in vitro and in vivo. This CB2‐mediated amplification of eosinophil migration seems to occur Gi/o/adenylyl cyclase‐independent, but involves Gαq/MEK/ROCK signaling. Previous studies in patients with asthma showed a beneficial effect of inhaled or orally taken cannabinoids by dilating bronchial smooth muscles 56, 57. In contrast, our data provide evidence for the involvement of the endocannabinoid/CB2 axis in the progression of allergic inflammatory processes and indicate possible undesirable proinflammatory effects of long‐term cannabinoid use. Thus, specific CB2 receptor antagonism may open a new therapeutic approach for allergic disorders and other eosinophil‐driven diseases.
Declaration of funding
E.S. received funding from the Franz Lanyar Stiftung of the Medical University of Graz (Grant No. 344) and was supported by the Jubilaeumsfonds of the Austrian National Bank (Grant No. 14446). A.F. was funded by the Karen and Sten Mörtstedt Initiative on Anaphylaxis. R.F. and M.P. were supported by the Austrian Science Fund FWF (DK‐MOLIN W1241), and A.H. was supported by the Austrian Science Fund FWF (Grant No. P22521) and Jubilaeumsfonds of the Austrian National Bank (Grant No. 14263).
Conflicts of interest
The authors declare no conflict of interest.
Supporting information
Acknowledgments
We thank Birgit Brodacz, Iris Red, and Kathrin Rohrer for their skilled technical assistance.