signature=49a139e45aae48095ba948d9ce05c91d,GPR55 regulates cannabinoid 2 receptor-mediated responses...

Chemotaxis and polarization of neutrophils are dependent on the gradient of GPR55 and CB2R agonists

It has previously been suggested that there is a site distinct from CB1R and CB2R, which affects CB2R-mediated migration in human blood neutrophils and that this site could be GPR55 Figure 1Ai shows, neutrophils readily migrated towards a gradient of LPI (Figure 1Ai, ▪). Likewise, the more stable synthetic cannabinoid GPR55 agonist and CB1R antagonist AM251 Figure 1Ai, •). The chemotactic properties of these ligands were comparable to that of the CB2R agonist 2-AG (Figure 1Ai, ♦). Next, we tested whether the migration of neutrophils towards either LPI or 2-AG could be blocked by pretreating the cells with selective receptor antagonists. Pre-incubation of neutrophils with either the GPR55 antagonist cannabidiol (5 μM) Figure 1Aii, CBD) or the selective CB2R antagonist AM630 (Figure 1Aii, 5 μM) for 10 min significantly diminished the migratory properties of neutrophils towards LPI (3 μM) or 2-AG (1 μM), respectively. In contrast, neutrophil migration was not impaired when cells were pretreated with the 'wrong' antagonists, i.e., 5 μM of CBD followed by 1 μM 2-AG stimulation or 5 μM AM630 followed by 3 μM LPI stimulation, respectively (data not shown).

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.

In most of the previous studies, 2-AG has been used alone Figure 1Aiii). More prominently, the migration of neutrophils towards AM251 (3 μM), when combined with 2-AG (1 μM), was synergistically enhanced compared to neutrophils migrating towards AM251 or 2-AG alone (Figure 1Aiv). These effects could be significantly diminished by pretreating the cells with the GPR55 antagonist CBD (5 μM), the CB2R antagonist AM630 (5 μM) or a combination of both for 10 min (Figure 1Av).

In response to chemokines, neutrophils undergo cytoskeletal rearrangement and shape change, which culminates in cellular polarization and thereby enables the cells to migrate efficiently Figure 1Bi). LPI (3 μM) induced random non-directional protrusions in the neutrophils (Figure 1Bii, arrows). Consistent with previous reports Figure 1Biii, arrows). Only when neutrophils were concomitantly incubated with a gradient of LPI (3 μM) and 2-AG (1 μM), the typical polarity of migrating neutrophils – i.e. extending head (arrow) and retracting tail (dashed arrow) – could be detected (Figure 1Biv). This effect was not due to the higher concentration of the combined ligands per se, since neither LPI nor 2-AG alone, up to 5 μM, could evoke the same cytoskeletal rearrangement (data not shown), but it was dependent on the gradient of compounds (see Figure 1C).

When LPI (3 μM) and 2-AG (1 μM) were simultaneously applied to the media using the top of a narrow tip (Figure 1Ci, white dot), a directional movement towards the source of ligands was observed. The uniform addition of ligands into the culture medium, however, did not evoke any shape change in the neutrophils (Figure 1Cii). Likewise, when tested in a Boyden-migration assay, no migration of neutrophils could be observed in the absence of a ligand gradient (Supplementary information, Figure S1).

Taken together, these data show that LPI, AM251 or 2-AG alone could each evoke neutrophil migration – albeit to a lesser extent than when used in combination – and without inducing the typical morphology of migrating neutrophils. In fact, only the combination of LPI and 2-AG evoked a strong migratory response in human blood neutrophils via the establishment of a rear-front asymmetric morphology.

GPR55 is expressed in human blood neutrophils

Next, we investigated the expression of the cannabinoid receptors CB1 and CB2 as well as that of GPR55 in human blood neutrophils at both mRNA and protein levels. It has previously been reported that the pattern of expression levels of CB1 and CB2 receptors in neutrophils depends on the isolation procedure 2+ and Mg2+ ions to prevent a stimulation of the cells. GPR55 mRNA copy numbers (Figure 2Ai, black bar) were significantly higher than those of the CB2R (Figure 2Ai, white bar). Indeed, both CB2R and GPR55 mRNA could also be detected by RT-PCR (Figure 2Aii). The expression of CB1R was not detectable in either RT-PCR or real time PCR (data not shown). In addition, we tested the expression of GPR55 in differentiated neutrophil-like HL60 cells (dHL60), which expressed the differentiation marker CD11b at levels comparable to those of neutrophils (Supplementary information, Figure S2). Real time PCR analysis showed a 5.5-fold increase in GPR55 mRNA levels in dHL60 cells (Figure 2Aiii, black bar) compared to undifferentiated HL60 cells (uHL60) (Figure 2Aiii, white bar).

Figure 2

GPR55 is highly expressed in human blood neutrophils and neutrophil-like HL60 cells. (A) GPR55 and CB2R mRNA expression in freshly isolated human blood neutrophils was assessed by (i) quantitative real-time PCR and (ii) RT-PCR. PCR products were analyzed on a 3% agarose gel. (iii) Relative expression of GPR55 mRNA in undifferentiated (uHL60) and HL60 cells differentiated with 1.75% DMSO for 4 days (dHL60) was measured by real-time PCR. Representatives of three independent experiments are shown for all subpanels. Data are mean±SEM (**P< 0.01). (B) (i) GPR55 and CB2R protein expression in neutrophils, uHL60 and dHL60 cells was assessed by western blotting using rat anti-GPR55 and rabbit anti-CB2R antibodies. Lysates were probed for β-actin as a loading control. A representative blot of three independent experiments is shown. (ii) Western blotting of GPR55 and CB2R in lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells was performed as in panel Bi. A representative blot of three independent experiments is shown. (C) GPR55 expression in human blood neutrophils, uHL60 and dHL60 cells was confirmed with the rat anti-GPR55 antibody (1:250) and an Alexa Fluor-594 goat anti-rat secondary antibody (1:250). The dHL60 cells show multilobular nuclei (arrows). Cells were analyzed using a Zeiss LSM510 META Axioplan confocal microscope (original magnification: 100×). Scale bars: 10 μm. Representative images of 2-3 independent experiments are shown.

We next assessed the protein levels and cellular expression of both GPR55 and CB2 receptors in neutrophils, as well as in uHL60 and dHL60 cells. HEK293 cell lines stably expressing the CB2 receptor (HEK-CB2R), the GPR55 receptor (HEK-GPR55) 2 receptor (HEK-CB2R/GPR55) served as controls. Using antibodies specifically targeting the respective receptors, we found that GPR55 and CB2R proteins were expressed in neutrophils, uHL60 and dHL60 cells at the appropriate protein sizes (Figure 2Bi, ∼37 kDa for GPR55 and ∼45 kDa for CB2R). The specificity of the antibodies was confirmed by western blot using the lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells. As Figure 2Bii shows, the antibodies reacted with their respective targets only. Moreover, we confirmed the expression of GPR55 in freshly isolated neutrophils and HL60 cells by immunofluorescence using the rat anti-GPR55 antibody (Figure 2C). As observed in other primary cells Supplementary information, Figure S3).

Gα13/RhoA and Gαi mediate GPR55 and CB2R cytoskeletal remodeling/chemotactic effects, respectively

It has been demonstrated that GPR55 mediates its downstream signaling events via Gα13 and the RhoA small GTPase in HEK293 cells 13 in HEK293 cells 2R mediated signaling effects in neutrophils, we used the toxins C3 and pertussis toxin to inhibit the activity of Gα13/RhoA and Gαi, respectively. Pre-incubation of neutrophils with C3 toxin (3 μg/ml, 2 h) significantly inhibited the LPI-induced migration of neutrophils, but showed no effect on the 2-AG-stimulated migration (Figure 3Ai and 3Aii). On the other hand, pertussis toxin (3 μg/ml, 2 h) prevented the migration of neutrophils towards 2-AG, but had no significant effect on LPI-induced migration (Figure 3Ai and 3Aii). Furthermore, migration of neutrophils towards a combination of LPI and 2-AG was significantly inhibited when the cells were pre-incubated with either C3 or pertussis toxin (Figure 3Aiii).

Figure 3

GPR55 and CB2R mediate chemotaxis and cytoskeletal remodeling via coupling to Gα13/RhoA and Gαi proteins. Neutrophils were pre-incubated with cell-permeable C3 toxin (C3, 3 μg/ml) or pertussis toxin (PTX, 3 μg/ml) for 2 h at 37 °C in PBG buffer. (A) Cells were allowed to migrate towards (i) LPI (3 μM), (ii) 2-AG (1 μM) or (iii) a combination of LPI (3 μM) and 2-AG (1 μM) for 1 h. Migrated cells in the bottom wells were counted by a flow cytometer. The chemotactic index was calculated as number of cells migrated towards agonists divided by the number of cells migrated towards vehicle (DMSO 0.01%). Representatives of two independent experiments, performed in quadruplicates, are shown. Data are mean±SEM (*P< 0.05; **P< 0.01). n.s.: not significant. (B) Cells were seeded on fibronectin-coated glass coverslips and treated with 0.01% DMSO (Control), LPI (3 μM), 2-AG (1 μM) or a mixture of LPI (3 μM) and 2-AG (1 μM) for 5 min at 37 °C and stained with Texas-Red Phalloidin (red) and DAPI (blue). Cells were analyzed using an OLYMPUS fluorescence microscope equipped with a Hamamatsu ORCA CCD camera (original magnification: 60×). Scale bars: 10 μm. Representative images from 2 independent experiments are shown.

Next, we tested whether the impact of these inhibitors on neutrophil migration was due to an impairment of cytoskeletal remodeling. C3 toxin (3 μg/ml, 2 h) inhibited the formation of protrusions induced by LPI (compare Figure 3Bvi and 3Bii), but had no effect on the elongated morphology of 2-AG-treated neutrophils (compare Figure 3Bvii and 3Biii). Moreover, inhibition of RhoA prevented the polarization of neutrophils in response to a combination of LPI and 2-AG (compare Figure 3Bviii and 3Biv). Pertussis toxin (3 μg/ml, 2 h) inhibited the elongation of 2-AG-stimulated cells (compare Figure 3Bxi and 3Biii), but did not affect the non-directional protrusions stimulated by LPI (compare Figure 3Bx and 3Bii). Inhibition of Gαi signaling abrogated the polarized head/tail morphology of neutrophils upon treatment with a combination of LPI and 2-AG (compare Figure 3Bxii and 3Biv).

In summary, the exclusive inhibitory effects either (i) of C3 toxin on GPR55-mediated responses or (ii) of pertussis toxin on CB2R-mediated responses, provide evidence for the involvement of Gα13/RhoA in GPR55-mediated and Gαi in CB2R-mediated signaling in neutrophils.

Rac1 and Cdc42 are involved in the cytoskeletal rearrangement of neutrophils after concomitant activation of GPR55 and CB2R

The migration of leukocytes towards chemotactic agents occurs through a coordinated series of events, typically including a cytoskeletal rearrangement that relies on the function of the Rho family of small GTPases 2R agonists (i.e. JWH015 and 2-AG), thereby activating Rac1 and Cdc42, while repressing RhoA

We tested whether Rac1 and Cdc42 were differentially activated by either LPI, 2-AG or the combined application of both ligands in neutrophils. Consistent with previous reports of GPR55-mediated Rac1 activation in HEK293 cells Figure 4Ai, lane 2). The 2-AG (1 μM) induced a rapid and significant increase in activated Rac1 (Figure 4Aii, lane 3), whereby a maximum of Rac1 activity was reached within 60 s (Supplementary information, Figure S4). This time frame of activation is consistent with CB2R-mediated Rac1 activation in dHL60 cells Figure 4Ai, lane 4).

Figure 4

Cytoskeletal rearrangement of (A) neutrophils and (B) HEK293 cells requires the concomitant activation of GPR55 and CB2R. (A) Neutrophils were stimulated with LPI (1 μM), 2-AG (1 μM) and LPI (1 μM) + 2-AG (1 μM) for 1 min at 37 °C. Active GTP-bound Rac1 and Cdc42 GTPases were extracted from the lysates with PAK domain-gluthatione agarose beads. GTP-bound and total GTPase levels were visualized by western blotting using mouse anti-Rac1 (i) and rabbit anti-Cdc42 (ii) antibodies. The β-actin served as a loading control. The ratio of GTP-bound vs total GTPase levels was assessed with ImageJ software (graphs). Representative blots from 3-4 independent experiments are shown. Data are mean±SEM. (*P< 0.05; ***P< 0.001). (B) HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells were seeded on 1% PDL-coated glass coverslips. Serum-starved cells were incubated with agonists (1 μM) for 10 min in a serum-free medium. The fixed cells were stained for F-actin by methanolic Texas-Red Phalloidin (red) and with DAPI (blue). Cells were analyzed using an OLYMPUS fluorescence microscope equipped with a Hamamatsu ORCA CCD camera (original magnification: 60×). Scale bars: 20 μm. Representative images from 3-4 experiments are shown. (C) (i) HEK-CB2R/GPR55 cells were transfected with 200 ng of NFAT-luciferase reporter plasmid and 24 h later cells were stimulated with agonists (1 μM) for 3 h in a serum-free medium. The luciferase activity was visualized using a steadylite plus kit (PerkinElmer). Luminescence (relative light units (RLU)) was measured in a TopCounter (Top Count NXT; Packard) for 5 s. Data are mean±SEM from three independent experiments performed in quadruplicate (**P< 0.01) (ii) HEK-CB2R/GPR55 cells were challenged with ligands (1 μM) and the resulting picometer shifts of reflected light wavelength against the time (s) were monitored. Transformation of optical signatures were made by using the area under the curve (AUC) values between the 1 200 and 3 600 s time points. Data were normalized and expressed as percent of maximum activation induced by LPI + 2-AG. Data are mean ±SEM from three independent experiments performed in quadruplicate (***P< 0.001).

Activated Cdc42 was reported to be involved in the polarization and directional migration of neutrophils Figure 4Aii, lane 2), whereas 1 μM 2-AG was able to promote GTP-binding to Cdc42 in neutrophils within 1 min (Figure 4Aii, lane 3). Interestingly, incubation of neutrophils with both agonists led to a further increase in Cdc42 activity compared to 2-AG alone (Figure 4Aii, lane 4).

In summary, the combined application of both the GPR55 agonist LPI and the CB2R agonist 2-AG further potentiated Cdc42 activity. This process may thus underlie the polarized morphology (see Figure 1Biv and 4A, top of lane 4) and the trafficking of neutrophils towards a gradient of both ligands (see Figure 1Aiii).

RhoA-dependent F-actin formation and activation of the downstream transcription factor NFAT are mediated by GPR55 and enhanced in the presence of activated CB2R

Next, we wanted to test the extent to which each of the respective receptors – i.e. GPR55 or CB2R – is involved in the formation of RhoA-dependent F-actin. Since the short life-time of purified neutrophils does not allow for manipulations such as siRNA knockdown, we took advantage of HEK293 cells stably expressing these receptors alone, or a combination thereof. We and others have previously shown that in HEK293 cells, GPR55 stimulation leads to the activation of RhoA, Rac1 and Cdc42

The GPR55 agonist LPI (1 μM, 10 min) induced prominent F-actin fibers in HEK-GPR55 (Figure 4Biv) but not in HEK-CB2R cells (Figure 4Bv). This effect was dependent on the activity of the Gα13/RhoA axis, since the transient transfection of HEK-GPR55 cells with dominant negative mutants of Gα13 (Supplementary information, Figure S5i) and RhoA (Supplementary information, Figure S5ii) or a 10-min pretreatment with 10 μM ROCK inhibitor Y27632 (Supplementary information, Figure S5iii) prevented actin polymerization in response to 1 μM LPI. Surprisingly, GPR55-mediated F-actin formation was modestly attenuated in the presence of non-activated CB2R in HEK-CB2R/GPR55 cells (Figure 4Bvi).

The CB2R agonist 2-AG (1 μM) could not induce actin rearrangement in the HEK-GPR55 cells (Figure 4Bvii), but led to some accumulation of polymerized actin in the periphery of HEK-CB2R cells (Figure 4Bviii, arrows). This effect could also be observed in HEK-CB2R/GPR55 cells (Figure 4Bix, arrow). Treatment of HEK-CB2R/GPR55 cells with both LPI (1 μM) and 2-AG (1 μM) drastically increased the formation of filamentous actins (Figure 4Bxii) when compared with LPI (Figure 4Bvi) or 2-AG (Figure 4Bix) alone. Co-administration of LPI and 2-AG did not show a change in actin formation in HEK-GPR55 (Figure 4Bx) or HEK-CB2R (Figure 4Bxi) cells compared to treatment of these cells with any of the agonists alone (compare Figure 4Bx and 4Biv and 4Bxi and 4Bviii, respectively).

We have recently shown that in HEK293 cells, the stimulation of GPR55 triggers multiple signaling pathways, eventually leading to the activation of transcription factors such as the nuclear factor of activated T cells (NFAT), the nuclear factor-κB and the cAMP responsive element binding (CREB) protein 2 receptors in our HEK-CB2R/GPR55 cell model. Concomitant activation of GPR55 and CB2R with LPI (1 μM) and 2-AG (1 μM) led to a significant enhancement of NFAT-activation when compared to cells stimulated with 1 μM LPI only (Figure 4Ci, compare light gray and black bars). The 2-AG (1 μM) did not induce NFAT activity in HEK-CB2R/GPR55 cells (Figure 4Ci, dark gray bar). This was expected, since CB2 receptors typically mediate their signaling events predominantly through Gαi-pathways, which have not been reported to induce NFAT-activity. In order to test the signaling events in the HEK-CB2R/GPR55 model on a more 'global' scale, we subjected our cells to a Dynamic Mass Redistribution Assay (DMR, Epic®). We have previously reported the suitability of this system for label-free measurement of signaling events of both GPR55 i-coupled 7TM/GPCRs 2R with LPI (1 μM) and 2-AG (1 μM) led to a significantly higher DMR response in these cells when compared to cells stimulated with 1 μM LPI only (Figure 4Cii, compare light gray and black bars). Again, 2-AG (1 μM) alone did not induce any DMR in HEK-CB2R/GPR55 cells (Figure 4Cii, dark gray bar).

In summary, these data suggest that the LPI-induced activation of GPR55 is critical for the RhoA-dependent rearrangement of the actin cytoskeleton and downstream signaling events such as activation of the transcription factor NFAT. However, these effects are enhanced in the presence of a 2-AG-activated CB2 receptor.

Activated GPR55 inhibits CB2R- and C5aR-mediated respiratory burst in neutrophils

A dramatic increase in ROS levels – known as the 'respiratory burst' – is a mechanism used by neutrophils to resolve infection. This process is catalyzed by the NADPH oxidase complex i-coupled receptors, i.e., the CB2R and the C5aR 2 receptors had an effect on ROS production in neutrophils.

GPR55 agonists LPI (300 nM) or AM251 (300 nM) did not induce ROS production in neutrophils per se (Figure 5Ai and 5Aii, light gray bars). In contrast, 2-AG (10 μM) induced ROS production in neutrophils (Figure 5Ai and 5Aii, dark gray bars), an effect that could be inhibited with 10 μM selective CB2R antagonist AM630 (Supplementary information, Figure S6). Interestingly, 2-AG-induced ROS production was significantly diminished when neutrophils were concomitantly stimulated with LPI (300 nM) or AM251 (300 nM) (Figure 5Ai and 5Aii, black bars). This effect was dose dependent (Figure 5Aiii, 100 nM-300 nM LPI) and could also be observed in neutrophils activated with C5a (Figure 5Aiii). Similar to neutrophils, we observed that ROS formation stimulated by 2-AG (10 μM) was significantly inhibited by co-administration of LPI (Figure 5Aiv) in dHL60 cells, although higher concentrations of LPI (1 μM-10 μM) were needed to see an effect. However, like in neutrophils, LPI alone could not induce ROS production in dHL60 cells (Figure 5Aiv).

Figure 5

GPR55 activation inhibits (A) CB2R-mediated respiratory burst and (B) C5a-induced degranulation in neutrophils. (A) ROS production in neutrophils was measured by flow cytometry. (i) Cells were loaded with 1 μM 2′,7′-DCF-DA and then incubated with DMSO (0.1%), LPI (300 nM), 2-AG (10 μM) or a combination of LPI and 2-AG for 20 min at 37 °C. ROS production was measured as a change in fluorescence in the FL1 channel. (ii) ROS production in neutrophils was measured as in panel Ai except that AM251 (300 nM) was used instead of LPI. (iii) Neutrophils were incubated with C5a (5 nM) or 2-AG (10 μM) and treated with buffer (control) or LPI (100 nM or 300 nM) for 20 min. ROS production was assessed as in panel Ai. (iv) Serum-starved dHL60 cells were loaded with 5 μM 2′,7′-DCF-DA for 10 min at 37 °C and then incubated with 2-AG (10 μM) in combination with assay buffer (control) or LPI (1 or 10 μM). LPI (10 μM) used in combination with DMSO (0.1%) did not induce changes in ROS levels. ROS production was recorded in a Flex-Station II device (Ex. 485nm, Em. 535 nm) 20 min after ligand addition. Representatives of 3-4 independent experiments, performed in quadruplicates are shown for all subpanels. Data are mean± SEM (*P< 0.05; **P< 0.01; ***P< 0.001). (B) (i) Neutrophils were incubated with LPI (300 nM) or assay buffer (control) for 1 h at 37 °C. MPO release was induced by increasing concentrations of C5a for 30 min and measured as the change in absorbance at 630 nm in a colorimetric assay. (ii) Neutrophils were incubated with increasing concentrations of LPI for 1 h at 37 °C. MPO release was induced with C5a (300 nM) for 30 min and assessed as in panel Bi. Data are mean±SEM of three independent experiments performed in triplicates (*P< 0.05; **P< 0.01; ***P< 0.001). The MPO release induced by 300 nM C5a was set to 100%.

In summary, these data show that – once activated – GPR55 inhibited the CB2R-mediated ROS production in both neutrophils and dHL60 cells.

Activated GPR55 inhibits the C5a-induced degranulation of neutrophils

In order to be able to destroy infectious agents, neutrophils store a large number of enzymes in azurophilic granules Figure 5Aiii), we next tested whether LPI could likewise modify C5a-induced MPO release. In fact, pretreatment of neutrophils with 300 nM LPI for 1 h (Figure 5Bi, black bars) significantly inhibited the MPO release triggered by different concentrations of C5a (Figure 5Bi, white bars). Increasing doses of LPI reduced MPO release mediated by 300 nM C5a up to a maximum of 75% (Figure 5Bii). No MPO release was observed when neutrophils were incubated with LPI alone (Figure 5Bi, black bar at 0 concentration of C5a). Thus, activated GPR55 can prevent C5a-mediated degranulation of neutrophils.

Activated GPR55 inhibits ROS production and degranulation in neutrophils via repression of Rac2 activity

It has frequently been reported that the small GTPase Rac2 regulates degranulation and the NADPH oxidase activity in neutrophils via its translocation to the plasma membrane and incorporation in the NADPH oxidase complex 2R-specific agonist, was shown to activate Rac2 in dHL60 cells and primary neutrophils 2R agonists in neutrophils and dHL60 cells.

Treatment of neutrophils with LPI (1 μM) for 1 min reduced the activity of Rac2 when compared to vehicle (Figure 6A, lanes 1 and 2). In contrast, 2-AG (1 μM) resulted in a significant activation of Rac2 (Figure 6A, lane 3), which reached the highest activity at 1 min and returned to its basal activity after 2 min (Supplementary information, Figure S7). However, concomitant stimulation of neutrophils with 2-AG (1 μM) and LPI (1 μM) resulted in a significant inhibition of Rac2 activity when compared to cells treated with 2-AG alone (Figure 6A, compare lanes 3 and 4). Likewise, in dHL60 cells Rac2 activity was reduced in the presence of 1 μM LPI (Figure 6B, lane 2) and enhanced in the presence of 1 μM 2-AG (Figure 6B, lane 3). Again, in the presence of both ligands, the Rac2 activity was reduced when compared to dHL60 cells treated with 2-AG alone (Figure 6B, compare lanes 3 and 4).

Figure 6

GPR55 activation suppresses the CB2R-mediated activation and translocation of Rac2. (A) Neutrophils were stimulated with agonists (1 μM) for 1 min at 37 °C. The active GTP-bound Rac2 was extracted from the lysates with PAK domain-gluthatione agarose beads. GTP-bound and total GTPase levels were visualized by western blotting using a rabbit anti-Rac2 antibody, β-actin served as a loading control. The ratio of GTP-bound vs total GTPase levels was assessed with ImageJ software (graph). Data are mean±SEM of three independent experiments. (*P< 0.05; ***P< 0.001). (B) Serum-starved dHL60 cells were stimulated with agonists (1 μM) for 1 min at 37 °C. The extraction of active GTP-bound Rac2 was performed as in panel (A). Data are mean±SEM of three independent experiments (**P< 0.01; ***P< 0.001). (C) Neutrophils were seeded on fibronectin-coated glass coverslips and treated with 0.01% DMSO (control; i) or ligands for 5 min at 37 °C. Fixed cells were incubated with rabbit anti-Rac2 antibody and stained with Alexa Fluor-488 goat anti-rabbit antibody (green), Texas-Red Phalloidin (red), and DAPI (blue). Control (i) and LPI (3 μM, ii) treated cells show a nuclear/perinuclear localization of Rac2 (arrows). Upon 2-AG stimulation (1 μM, iii), Rac2 distributed evenly in the cytosol (arrow) and partially colocalized with actin at the plasma membrane (yellow, arrowhead). (iv) Treatment with a combination of LPI (3 μM) and 2-AG (1 μM) showed a nuclear localization of Rac2 in polarized neutrophils. Cells were analyzed using an OLYMPUS fluorescence microscope equipped with a Hamamatsu ORCA CCD camera (original magnification: 60×). Scale bars: 10 μm. Representative images from 2-3 experiments are shown.

The function of Rac2 in ROS production and degranulation is dependent on its translocation from the nuclear and/or perinuclear zones to the plasma membrane. In neutrophils, Rac2 was mainly located in or closely around the nucleus (Figure 6Ci, arrow, Rac2 in green, DAPI/nuclear staining in blue). Treatment of neutrophils with 3 μM LPI did not alter the perinuclear distribution of Rac2 (Figure 6Cii, arrow). In contrast, stimulation of neutrophils with 2-AG (1 μM) induced a redistribution of Rac2 to the cytosol (Figure 6Ciii, green, arrow) and partly resulted in a colocolization with the peripheral actin (Figure 6Ciii, yellow, arrowhead). This effect, however, could be prevented by concomitant application of 3 μM LPI with 1 μM 2-AG, resulting in a perinuclear distribution of Rac2 in these cells (Figure 6Civ, arrow).

In summary, these data suggest that GPR55 regulates both ROS and MPO production in neutrophils via suppressing the activity of the small GTPase Rac2.