Maiko Gokoh

2‐Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL‐1 human eosinophilic leukemia cells and human peripheral blood eosinophils

2‐Arachidonoylglycerol (2‐AG) is an endogenous cannabinoid receptor ligand. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, abundantly expressed in the brain, and the CB2 receptor, expressed in various lymphoid tissues such as the spleen. The CB1 receptor has been assumed to play an important role in the regulation of synaptic transmission, whereas the physiological roles of the CB2 receptor remain obscure. In this study, we examined whether the CB2 receptor is present in human eosinophils and found that the CB2 receptor is expressed in human peripheral blood eosinophils. In contrast, human neutrophils do not contain a significant amount of the CB2 receptor. We then examined the effect of 2‐AG on the motility of eosinophils. We found that 2‐AG induces the migration of human eosinophilic leukemia EoL‐1 cells. The migration evoked by 2‐AG was abolished in the presence of SR144528, a CB2 receptor antagonist, or by pretreatment of the cells with pertussis toxin, suggesting that the CB2 receptor and Gi/o are involved in the 2‐AG‐induced migration. The migration of EoL‐1 cells induced by 2‐AG was suggested to be a result of chemotaxis. In contrast to 2‐AG, neither anandamide nor free arachidonic acid elicited the migration. Finally, we examined the effect of 2‐AG on human peripheral blood eosinophils and neutrophils and found that 2‐AG induces migration of eosinophils but not neutrophils. These results suggest that the CB2 receptor and its endogenous ligand 2‐AG may be closely involved in allergic inflammation accompanied by the infiltration of eosinophils.

Involvement of the Cannabinoid CB2 Receptor and Its Endogenous Ligand 2-Arachidonoylglycerol in Oxazolone-Induced Contact Dermatitis in Mice

The possible involvement of 2-arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), in contact dermatitis in mouse ear was investigated. We found that the level of 2-AG was markedly elevated in the ear following a challenge with oxazolone in sensitized mice. Of note, the swelling following the challenge was suppressed by either the administration of SR144528, a CB2 receptor antagonist, immediately after sensitization, or the administration of SR144528 upon the challenge. The effect of AM251, a CB1 receptor antagonist, was marginal in either case. It seems apparent, therefore, that the CB2 receptor and its endogenous ligand 2-AG are closely involved in both the sensitization phase and the elicitation phase of oxazolone-induced contact dermatitis. In line with this, we found that Langerhans cells (MHC class II+) contain a substantial amount of CB2 receptor mRNA, whereas keratinocytes (MHC class II−) do not. We also obtained evidence that the expression of mRNAs for proinflammatory cytokines following a challenge with oxazolone was markedly suppressed by treatment with SR144528. We next examined whether the CB2 receptor and 2-AG participate in chronic contact dermatitis accompanied by the infiltration of tissues by eosinophils. The amount of 2-AG in mouse ear dramatically increased following repeated challenge with oxazolone. Importantly, treatment with SR144528 attenuated both the recruitment of eosinophils and ear swelling in chronic contact dermatitis induced by repeated challenge with oxazolone. These results strongly suggest that the CB2 receptor and 2-AG play important stimulative roles in the sensitization, elicitation, and exacerbation of allergic inflammation.

2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces rapid actin polymerization in HL-60 cells differentiated into macrophage-like cells.

Delta9-Tetrahydrocannabinol, a major psychoactive constituent of marijuana, interacts with specific receptors, i.e. the cannabinoid receptors, thereby eliciting a variety of pharmacological responses. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, which is abundantly expressed in the nervous system, and the CB2 receptor, which is predominantly expressed in the immune system. Previously, we investigated in detail the structure-activity relationship of various cannabinoid receptor ligands and found that 2-AG (2-arachidonoylglycerol) is the most efficacious agonist. We have proposed that 2-AG is the true natural ligand for both the CB1 and CB2 receptors. Despite the potential physiological importance of 2-AG, not much information is available concerning its biological activities towards mammalian tissues and cells. In the present study, we examined the effect of 2-AG on morphology as well as the actin filament system in differentiated HL-60 cells, which express the CB2 receptor. We found that 2-AG induces rapid morphological changes such as the extension of pseudopods. We also found that it provokes a rapid actin polymerization in these cells. Actin polymerization induced by 2-AG was abolished when cells were treated with SR144528, a CB2 receptor antagonist, and pertussis toxin, suggesting that the response was mediated by the CB2 receptor and G(i/o). A phosphoinositide 3-kinase, Rho family small G-proteins and a tyrosine kinase were also suggested to be involved. Reorganization of the actin filament system is known to be indispensable for a variety of cellular events; it is possible that 2-AG plays physiologically essential roles in various inflammatory cells and immune-competent cells by inducing a rapid actin rearrangement.

Chemotaxis of Human Peripheral Blood Eosinophils to 2-Arachidonoylglycerol: Comparison with Other Eosinophil Chemoattractants

Background: 2-Arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we found that human eosinophilic leukemia EoL-1 cells and human peripheral blood eosinophils express the CB2 receptor. We also found that 2-AG induces the migration of these cells in a CB2 receptor-dependent manner. In this study, we investigated whether the 2-AG-induced migration of human eosinophils is due to chemotaxis or chemokinesis. We also compared the ability of 2-AG to induce the migration of eosinophils with those of other eosinophil chemoattractants. Methods: Eosinophils were separated from the peripheral blood of healthy donors. The migration of eosinophils to various stimulants was examined using Transwell™ inserts. In view of the fact that 2-AG is rapidly metabolized by cells, we employed 2-AG ether, an ether-linked nonhydrolyzable analog of 2-AG, instead of 2-AG to determine whether the 2-AG-induced migration is due to chemotaxis or chemokinesis. Results: 2-AG ether induced the migration of human eosinophils, like 2-AG. The 2-AG ether-induced migration was reduced by the coincubation of eosinophils with 2-AG ether in the upper compartment of the Transwell inserts, indicating that the migration is attributable to chemotaxis. The concentration of 2-AG required to induce the eosinophil migration appears to be pathophysiologically relevant, although the order of the pharmacologically effective concentration of 2-AG was approximately ten times lower than those of platelet-activating factor, RANTES and eotaxin. Conclusion: These results strongly suggest that 2-AG is involved in the infiltration of eosinophils during allergic inflammation.

2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, enhances the adhesion of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes.

2‐Arachidonoylglycerol (2‐AG), an endogenous cannabionoid receptor (CB1 and CB2) ligand, enhanced the adhesion of HL‐60 cells differentiated into macrophage‐like cells to fibronectin and the vascular cell adhesion molecule‐1. The CB2 receptor, Gi/Go, intracellular free Ca2+ and phosphatidylinositol 3‐kinase were shown to be involved in 2‐AG‐induced augmented cell adhesion. 2‐AG also enhanced the adhesion of human monocytic leukemia U937 cells and peripheral blood monocytes. These results strongly suggest that 2‐AG plays some essential role in inflammatory reactions and immune responses by inducing robust adhesion to extracellular matrix proteins and adhesion molecules in several types of inflammatory cells and immune‐competent cells.

Metabolism and physiological significance of anandamide and 2-arachidonoylglycerol, endogenous cannabinoid receptor ligands

Arachidonic acid is known to be metabolized into a number of bioactive eicosanoids such as prostaglandins, thromboxanes, leukotrienes, lipoxins and mono-and dihydroxyeicosatetraenoic acids. It is well known that these bioactive eicosanoids are involved in diverse physiological and pathophysiological processes in mammalian tissues. In the last decade of the 20th century, two remarkable derivatives of arachidonic acid, i.e., N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) (Fig. 1) were reported to be new members of the bioactive lipids. Anandamide and 2-AG have unique structural characteristic in that they contain an intact arachidonoyl moiety in their molecules, thus differing from other eicosanoids. Both anandamide and 2-AG have been shown to act as endogenous cannabinoid receptor ligands. In this review, we focused on anandamide and 2-AG and described the metabolism and possible physiological significance of these molecules in mammalian tissues and cells including inflammatory cells and immune competent cells.