Effects of Peripheral Cannabinoid Receptor Ligands on Motility and Polarization in Neutrophil-like HL60 Cells and Human Neutrophils

Abstract

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.The peripheral cannabinoid receptor (CB2) 2 was cloned in 1993 (1) after cloning of the central cannabinoid receptor (CB1) in 1990 (2). It has been suggested that the gene encoding CB2 is a protooncogene and that aberrant expression of CB2 in myeloid precursor cells results in the development of leukemia by blocking neutrophil differentiation (3, 4). CB2 is expressed predominantly in immune cells (5), and because of the diversity of immune cells, it is assumed that CB2 is involved in various activities in addition to inhibition of neutrophil differentiation (6 -9). Steffens et al. (10) recently reported that doses of ⌬ 9 -tetrahydrocannabinol (the most psychoactive component of marijuana) too low to have psychotropic effects inhibit the progression of atherosclerosis via immunomodulatory effects on lymphoid and myeloid cells. This report indicates that CB2 may be involved in a wide range of physiologic phenomena related to immunity and that some CB2 ligands may have application in the treatment of inflammatory disease. However, research into CB2 is still in its early stages. In particular, the involvement of only a few molecules, G␣ i /G␣ o protein, phosphatidylinositol 3-kinase (PI3K), and members of the mitogen-activated protein kinase and nuclear factor-B families, in the CB2 signaling pathways has been reported (6 -8, 11).Among the possible roles of CB2 in immunity is the induction of leukocyte migration to sites of infection and inflammation, an important step in the host defense against pathogenic microorganisms. CB2 is a seven-transmembrane, G␣ i /G␣ o protein-coupled receptor, as ar...

The Endocannabinoid 2-Arachidonoyl-Glycerol Activates Human Neutrophils: Critical Role of Its Hydrolysis and De Novo Leukotriene B4 Biosynthesis

“…Interestingly, endocannabinoids were reported to inhibit the fMLP-induced migration and polarization (54,56) of neutrophils, in contrast to our results on MPO release (Fig. 2).…”

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

“…Conflicting data exist regarding the effect of CB 2 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 CB 2 agonist JWH‐133 on human eosinophils, neutrophils, and basophils by means of shape change.…”

Inhibition of Human Neutrophil Chemotaxis by Endogenous Cannabinoids and Phytocannabinoids: Evidence for a Site Distinct from CB1and CB2

“…2-AG is reported to exert CB 2 -mediated stimulation of hematopoietic cell migration (Jordà et al, 2002;Kishimoto et al, 2003;Walter et al, 2003;Oka et al, 2004;Kishimoto et al, 2005;Sacerdote et al, 2005). Conversely, inhibition of migration by 2-AG, AEA, synthetic cannabinoids, and the phytocannabinoid ⌬ 9 -THC has also been reported previously (Sacredote et al, 2000;Steffens et al, 2005;Ghosh et al, 2006;Kurihara et al, 2006).…”