Figure 1.

GPR55 and CB₂R agonists induce the directional migration and polarization of neutrophils. (A) Human blood neutrophils were placed to the upper wells of a microBoyden chamber and (i) were allowed to migrate towards increasing concentrations of LPI (▪), AM251 (•) or 2-AG (♦) in the bottom wells for 1 h. Migrated cells in the bottom wells were counted by a flow cytometer. (ii) Neutrophils were pre-incubated with DMSO (0.05%), CBD (5 μM) or AM630 (5 μM) for 10 min at 37 °C and their migration towards LPI (3 μM) or 2-AG (1 μM) was assessed as in panel Ai. (iii) Chemotaxis of neutrophils towards DMSO (0.01%, white bar), LPI (3 μM, light gray bar), 2-AG (1 μM, dark gray bar) or LPI and 2-AG combined (3 μM and 1 μM, respectively, black bar) was assessed as in panel Ai. (iv) Chemotaxis of neutrophils was assessed as in panel iii, except that AM251 (3 μM) was used instead of LPI. (v) Neutrophils were pre-incubated with DMSO (0.05%), CBD (5 μM) and/or AM630 (5 μM) for 10 min at 37 °C and their migration towards DMSO (0.01%) and LPI (3 μM) + 2-AG (1 μM) was assessed as in panel Ai. Representatives of 3-6 independent experiments, performed in quadruplicates, are shown for all subpanels. Data are mean±SEM (^*P< 0.05; ^**P< 0.01; ^***P< 0.001). (B) Neutrophils were seeded on fibronectin-coated glass coverslips and treated with a gradient of 0.01% DMSO (control; i) or ligands for 5 min at 37 °C and stained with methanolic Texas-Red Phalloidin (red) and DAPI (blue). (ii) LPI treatment (3 μM) induced fuzzy protrusions (arrows), whereas (iii) 2-AG (1 μM) induced an elongation of the neutrophils (arrows). (iv) Extending head (arrow) and tail formation (dashed arrow) indicate a polarization of neutrophils in response to a mixture of LPI (3 μM) and 2-AG (1 μM). Cells were analyzed using a Zeiss LSM510 META Axioplan confocal microscope (original magnification: 100×). Scale bars: 10 μm. Representative images of three independent experiments are shown. (C) Neutrophils were seeded on fibronectin-coated glass coverslips and treated with LPI (3 μM) and 2-AG (1 μM) for 5 min at 37 °C and stained with Texas-Red Phalloidin (red) and DAPI (blue). (i) Cells displayed a clear directional and polarized structure when migrating towards a local source of LPI/2-AG (white dot). (ii) In contrast, no cytoskeleton remodeling occurred after a uniform addition of agonists to the medium. Cells were analyzed as in panel B (original magnification: 63×). Representative images of three independent experiments are shown. Scale bars: 10 μm.