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.
Highlights
Diversity of immune cells, it is assumed that CB2 is involved in various activities in addition to inhibition of neutrophil differentiation (6 –9)
CB2 Was Detected on the Surface of Neutrophil-like HL60 Cells and Human Neutrophils—HL60 cells are known to differentiate morphologically and functionally into neutrophil-like cells when treated with all-trans-retinoic acid (ATRA) [27]
We examined CB2 expression in HL60 cells, in HL60 cells 4 days after exposure to 1 M ATRA (ATRA-treated HL60 cells), and in human polymorphonuclear granulocytes (PMNs) isolated from whole blood
Summary
Diversity of immune cells, it is assumed that CB2 is involved in various activities in addition to inhibition of neutrophil differentiation (6 –9). In cells that exhibited typical morphologic alterations in response to CB2 stimulation, GFP-PKB/Akt-PH domain accumulated in one or more regions, mostly in pseudopods (Fig. 3B, panels b and c). Neutrophils showed no motility or morphologic alterations in response to either ligand (Fig. 6A, panels b and c), whereas Rac1 and Rac2 activity showed an increase in response to CB2 stimulation.
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