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GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils | killexams.com Real Questions and Pass4sure dumps

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

It has up to now been advised that there's a site distinctive from CB1R and CB2R, which impacts CB2R-mediated migration in human blood neutrophils and that this site can be GPR55 31. in addition, one other contemporary analyze confirmed that the highly metastatic MDA-MB231 breast cancer phone line expresses GPR55 and migrates effectively against LPI 40. hence, they set out to check the results of LPI on the migration of neutrophils. As figure 1Ai shows, neutrophils with no trouble migrated against a gradient of LPI (determine 1Ai, ▪). Likewise, the extra good artificial cannabinoid GPR55 agonist and CB1R antagonist AM251 21 brought on a awareness-based migration of neutrophils (determine 1Ai, •). The chemotactic homes of these ligands have been comparable to that of the CB2R agonist 2-AG (figure 1Ai, ♦). next, they tested even if the migration of neutrophils against both LPI or 2-AG can be blocked by using pretreating the cells with selective receptor antagonists. Pre-incubation of neutrophils with both the GPR55 antagonist cannabidiol (5 μM) forty one (determine 1Aii, CBD) or the selective CB2R antagonist AM630 (figure 1Aii, 5 μM) for 10 min significantly diminished the migratory houses of neutrophils against LPI (3 μM) or 2-AG (1 μM), respectively. In distinction, neutrophil migration turned into now not impaired when cells had been pretreated with the 'wrong' antagonists, i.e., 5 μM of CBD adopted by 1 μM 2-AG stimulation or 5 μM AM630 adopted by way of 3 μM LPI stimulation, respectively (statistics no longer proven).

determine 1 Figure 1

GPR55 and CB2R agonists set off the directional migration and polarization of neutrophils. (A) Human blood neutrophils were placed to the upper wells of a microBoyden chamber and (i) had been allowed emigrate in opposition t increasing concentrations of LPI (▪), AM251 (•) or 2-AG (♦) within the bottom wells for 1 h. Migrated cells within the backside wells had been counted by means of a circulate cytometer. (ii) Neutrophils have been pre-incubated with DMSO (0.05%), CBD (5 μM) or AM630 (5 μM) for 10 min at 37 °C and their migration in opposition t LPI (3 μM) or 2-AG (1 μM) become assessed as in panel Ai. (iii) Chemotaxis of neutrophils towards DMSO (0.01%, white bar), LPI (3 μM, gentle gray bar), 2-AG (1 μM, darkish grey bar) or LPI and 2-AG mixed (three μM and 1 μM, respectively, black bar) changed into assessed as in panel Ai. (iv) Chemotaxis of neutrophils become assessed as in panel iii, except that AM251 (three μM) changed into used as an alternative of LPI. (v) Neutrophils have been pre-incubated with DMSO (0.05%), CBD (5 μM) and/or AM630 (5 μM) for 10 min at 37 °C and their migration in opposition t DMSO (0.01%) and LPI (three μM) + 2-AG (1 μM) was assessed as in panel Ai. Representatives of 3-6 unbiased experiments, carried out in quadruplicates, are proven for all subpanels. records are mean±SEM (*P< 0.05; **P< 0.01; ***P< 0.001). (B) Neutrophils have been seeded on fibronectin-lined glass coverslips and handled with a gradient of 0.01% DMSO (manage; i) or ligands for 5 min at 37 °C and stained with methanolic Texas-pink Phalloidin (crimson) and DAPI (blue). (ii) LPI remedy (3 μM) brought about fuzzy protrusions (arrows), whereas (iii) 2-AG (1 μM) precipitated an elongation of the neutrophils (arrows). (iv) Extending head (arrow) and tail formation (dashed arrow) point out a polarization of neutrophils in response to a mix of LPI (3 μM) and a pair of-AG (1 μM). Cells have been analyzed the usage of a Zeiss LSM510 META Axioplan confocal microscope (fashioned magnification: one hundred×). Scale bars: 10 μm. consultant pictures of three impartial experiments are proven. (C) Neutrophils were seeded on fibronectin-coated glass coverslips and handled with LPI (three μM) and 2-AG (1 μM) for 5 min at 37 °C and stained with Texas-red Phalloidin (crimson) and DAPI (blue). (i) Cells displayed a transparent directional and polarized constitution when migrating towards a native supply of LPI/2-AG (white dot). (ii) In contrast, no cytoskeleton home improvement befell after a uniform addition of agonists to the medium. Cells were analyzed as in panel B (usual magnification: 63×). consultant photographs of three unbiased experiments are shown. Scale bars: 10 μm.

In many of the outdated reports, 2-AG has been used alone 12, 35 or in combination with chemokines to set off neutrophil migration 14, 42. on the grounds that each 2-AG and LPI are endogenous lipid mediators released by using stimulated macrophages 7, 29, 30, 43, they investigated the concomitant impact of two-AG and LPI on the migration of neutrophils. The combination of LPI (3 μM) and 2-AG (1 μM) showed a major impact on neutrophil migration, when compared to LPI or 2-AG alone (determine 1Aiii). more prominently, the migration of neutrophils against AM251 (three μM), when mixed with 2-AG (1 μM), was synergistically better compared to neutrophils migrating in opposition t AM251 or 2-AG alone (figure 1Aiv). These results can be drastically diminished via pretreating the cells with the GPR55 antagonist CBD (5 μM), the CB2R antagonist AM630 (5 μM) or a combination of both for 10 min (determine 1Av).

in keeping with chemokines, neutrophils undergo cytoskeletal rearrangement and form change, which culminates in cellular polarization and thereby allows for the cells emigrate efficiently 44. They therefore next assessed the cytoskeletal rearrangement of neutrophils in accordance with a 5-min publicity to a gradient of LPI or 2-AG alone, or a gradient of their aggregate thereof. Cytoskeletal rearrangement become assessed with the aid of F-actin phalloidin staining. In neutrophils handled with automobile, actin turned into found on the periphery of the cells, which had a circular spherical shape (determine 1Bi). LPI (three μM) precipitated random non-directional protrusions within the neutrophils (figure 1Bii, arrows). in line with outdated experiences 14, remedy of neutrophils with 2-AG (1 μM) resulted in an elongation of cells, although, with out displaying the attribute polarity of migrating leukocytes (determine 1Biii, arrows). most effective when neutrophils have been concomitantly incubated with a gradient of LPI (three μM) and a pair of-AG (1 μM), the typical polarity of migrating neutrophils – i.e. extending head (arrow) and retracting tail (dashed arrow) – may well be detected (figure 1Biv). This effect became no longer due to the better attention of the combined ligands per se, considering that neither LPI nor 2-AG alone, up to 5 μM, might evoke the equal cytoskeletal rearrangement (records not shown), nevertheless it changed into based on the gradient of compounds (see figure 1C).

When LPI (3 μM) and 2-AG (1 μM) had been concurrently applied to the media the usage of the excellent of a slender tip (determine 1Ci, white dot), a directional circulate against the supply of ligands was followed. The uniform addition of ligands into the culture medium, however, didn't evoke any form alternate in the neutrophils (figure 1Cii). Likewise, when verified in a Boyden-migration assay, no migration of neutrophils can be followed in the absence of a ligand gradient (Supplementary tips, figure S1).

Taken collectively, these statistics reveal that LPI, AM251 or 2-AG by myself might every evoke neutrophil migration – albeit to a lesser extent than when utilized in aggregate – and devoid of inducing the commonplace morphology of migrating neutrophils. in fact, only the mixture 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

subsequent, they 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 prior to now been stated that the sample of expression tiers of CB1 and CB2 receptors in neutrophils depends upon the isolation procedure forty five, 46, forty seven. right here they used an untouched, non-column primarily based gadget at room temperature in the absence of Ca2+ and Mg2+ ions to evade a stimulation of the cells. GPR55 mRNA replica numbers (figure 2Ai, black bar) were drastically larger than those of the CB2R (figure 2Ai, white bar). indeed, each CB2R and GPR55 mRNA might also be detected with the aid of RT-PCR (figure 2Aii). The expression of CB1R was no longer detectable in both RT-PCR or precise time PCR (data not shown). in addition, they verified the expression of GPR55 in differentiated neutrophil-like HL60 cells (dHL60), which expressed the differentiation marker CD11b at degrees akin to these of neutrophils (Supplementary advice, figure S2). real time PCR analysis confirmed a 5.5-fold raise in GPR55 mRNA tiers in dHL60 cells (determine 2Aiii, black bar) in comparison to undifferentiated HL60 cells (uHL60) (determine 2Aiii, white bar).

determine 2 Figure 2

GPR55 is incredibly expressed in human blood neutrophils and neutrophil-like HL60 cells. (A) GPR55 and CB2R mRNA expression in freshly remoted human blood neutrophils was assessed by means of (i) quantitative actual-time PCR and (ii) RT-PCR. PCR items have been analyzed on a three% agarose gel. (iii) Relative expression of GPR55 mRNA in undifferentiated (uHL60) and HL60 cells differentiated with 1.seventy five% DMSO for 4 days (dHL60) was measured with the aid of actual-time PCR. Representatives of three independent experiments are shown for all subpanels. statistics are imply±SEM (**P< 0.01). (B) (i) GPR55 and CB2R protein expression in neutrophils, uHL60 and dHL60 cells was assessed via western blotting using rat anti-GPR55 and rabbit anti-CB2R antibodies. Lysates have been probed for β-actin as a loading manage. A representative blot of three unbiased experiments is proven. (ii) Western blotting of GPR55 and CB2R in lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells changed into performed as in panel Bi. A representative blot of three impartial experiments is shown. (C) GPR55 expression in human blood neutrophils, uHL60 and dHL60 cells become validated with the rat anti-GPR55 antibody (1:250) and an Alexa Fluor-594 goat anti-rat secondary antibody (1:250). The dHL60 cells demonstrate multilobular nuclei (arrows). Cells had been analyzed using a Zeiss LSM510 META Axioplan confocal microscope (fashioned magnification: a hundred×). Scale bars: 10 μm. representative photos of two-three impartial experiments are shown.

We subsequent assessed the protein tiers and mobile expression of each GPR55 and CB2 receptors in neutrophils, as well as in uHL60 and dHL60 cells. HEK293 cell traces stably expressing the CB2 receptor (HEK-CB2R), the GPR55 receptor (HEK-GPR55) 18 alone or in mixture with the CB2 receptor (HEK-CB2R/GPR55) served as controls. the usage of antibodies especially targeting the respective receptors, they discovered that GPR55 and CB2R proteins had been expressed in neutrophils, uHL60 and dHL60 cells on the applicable protein sizes (determine 2Bi, ∼37 kDa for GPR55 and ∼forty five kDa for CB2R). The specificity of the antibodies become confirmed through western blot using the lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells. As determine 2Bii shows, the antibodies reacted with their respective goals only. in addition, they demonstrated the expression of GPR55 in freshly isolated neutrophils and HL60 cells by way of immunofluorescence using the rat anti-GPR55 antibody (determine 2C). As followed in different fundamental cells forty eight, GPR55 became predominantly found intracellulary in both neutrophils and HL60 cells. This staining became particular, considering most effective HEK-GPR55 cells, but now not untransfected HEK293 cells confirmed a positive immunoreactivity with the rat anti-GPR55 antibody (Supplementary suggestions, determine S3).

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

It has been validated that GPR55 mediates its downstream signaling movements by means of Gα13 and the RhoA small GTPase in HEK293 cells 15, 18, 19. moreover, they currently confirmed that – amongst all Gα subunits – GPR55 couples entirely to Gα13 in HEK293 cells forty nine. so as to check which Gα protein subunits are concerned within the GPR55 and CB2R mediated signaling consequences in neutrophils, they used the toxins C3 and pertussis toxin to inhibit the recreation of Gα13/RhoA and Gαi, respectively. Pre-incubation of neutrophils with C3 toxin (3 μg/ml, 2 h) vastly inhibited the LPI-prompted migration of neutrophils, however confirmed no impact on the two-AG-stimulated migration (figure 3Ai and 3Aii). nonetheless, pertussis toxin (three μg/ml, 2 h) averted the migration of neutrophils towards 2-AG, however had no large effect on LPI-triggered migration (figure 3Ai and 3Aii). moreover, migration of neutrophils against a mixture of LPI and a pair of-AG become enormously inhibited when the cells have been pre-incubated with both C3 or pertussis toxin (figure 3Aiii).

determine 3 Figure 3

GPR55 and CB2R mediate chemotaxis and cytoskeletal remodeling via coupling to Gα13/RhoA and Gαi proteins. Neutrophils had been pre-incubated with cell-permeable C3 toxin (C3, three μg/ml) or pertussis toxin (PTX, three μg/ml) for 2 h at 37 °C in PBG buffer. (A) Cells have been allowed to migrate against (i) LPI (3 μM), (ii) 2-AG (1 μM) or (iii) a mixture of LPI (3 μM) and a pair of-AG (1 μM) for 1 h. Migrated cells within the backside wells have been counted through a movement cytometer. The chemotactic index become calculated as variety of cells migrated towards agonists divided by using the number of cells migrated in opposition t vehicle (DMSO 0.01%). Representatives of two unbiased experiments, carried out in quadruplicates, are shown. records are imply±SEM (*P< 0.05; **P< 0.01). n.s.: no longer huge. (B) Cells had been seeded on fibronectin-covered glass coverslips and handled with 0.01% DMSO (handle), LPI (three μM), 2-AG (1 μM) or a mix of LPI (three μM) and 2-AG (1 μM) for 5 min at 37 °C and stained with Texas-purple Phalloidin (pink) and DAPI (blue). Cells were analyzed the use of an OLYMPUS fluorescence microscope geared up with a Hamamatsu ORCA CCD digital camera (original magnification: 60×). Scale bars: 10 μm. representative images from 2 impartial experiments are proven.

next, they demonstrated no matter if the have an impact on of these inhibitors on neutrophil migration turned into because of an impairment of cytoskeletal home improvement. C3 toxin (3 μg/ml, 2 h) inhibited the formation of protrusions caused with the aid of LPI (compare determine 3Bvi and 3Bii), however had no impact on the elongated morphology of two-AG-treated neutrophils (compare figure 3Bvii and 3Biii). in addition, inhibition of RhoA prevented the polarization of neutrophils in line with a combination of LPI and 2-AG (compare figure 3Bviii and 3Biv). Pertussis toxin (three μg/ml, 2 h) inhibited the elongation of two-AG-encouraged cells (compare figure 3Bxi and 3Biii), however didn't have an effect on the non-directional protrusions influenced by LPI (evaluate determine 3Bx and 3Bii). Inhibition of Gαi signaling abrogated the polarized head/tail morphology of neutrophils upon treatment with a mixture of LPI and a pair of-AG (examine figure 3Bxii and 3Biv).

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

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

The migration of leukocytes in opposition t chemotactic agents occurs through a coordinated collection of movements, typically including a cytoskeletal rearrangement that depends on the characteristic of the Rho household of small GTPases 50. for instance, neutrophil-like dHL60 cells have been reported to elongate in line with CB2R agonists (i.e. JWH015 and a pair of-AG), thereby activating Rac1 and Cdc42, whereas repressing RhoA 14. however, below these situations, dHL60 cells didn't display the general rear/entrance polarity of chemotaxing neutrophils.

We demonstrated no matter if Rac1 and Cdc42 have been differentially activated by way of both LPI, 2-AG or the mixed software of each ligands in neutrophils. per outdated stories of GPR55-mediated Rac1 activation in HEK293 cells 15, LPI (1 μM) prompted a quick – albeit modest – activation of Rac1 in neutrophils (determine 4Ai, lane 2). the two-AG (1 μM) brought about a rapid and important increase in activated Rac1 (figure 4Aii, lane three), whereby a optimum of Rac1 exercise became reached inside 60 s (Supplementary information, determine S4). This time body of activation is consistent with CB2R-mediated Rac1 activation in dHL60 cells 14. although, the incubation of neutrophils with both LPI and a pair of-AG didn't result in a synergistic activation of Rac1 (figure 4Ai, lane 4).

determine 4 Figure 4

Cytoskeletal rearrangement of (A) neutrophils and (B) HEK293 cells requires the concomitant activation of GPR55 and CB2R. (A) Neutrophils have been motivated with LPI (1 μM), 2-AG (1 μM) and LPI (1 μM) + 2-AG (1 μM) for 1 min at 37 °C. energetic GTP-certain Rac1 and Cdc42 GTPases have been extracted from the lysates with PAK domain-gluthatione agarose beads. GTP-sure and total GTPase stages have been visualized by using western blotting the usage of mouse anti-Rac1 (i) and rabbit anti-Cdc42 (ii) antibodies. The β-actin served as a loading manage. The ratio of GTP-sure vs complete GTPase tiers turned into assessed with ImageJ application (graphs). representative blots from three-four impartial experiments are shown. statistics are suggest±SEM. (*P< 0.05; ***P< 0.001). (B) HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells have been 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 have been stained for F-actin by way of methanolic Texas-purple Phalloidin (purple) and with DAPI (blue). Cells were analyzed the use of an OLYMPUS fluorescence microscope outfitted with a Hamamatsu ORCA CCD digital camera (long-established magnification: 60×). Scale bars: 20 μm. representative pictures from 3-four experiments are proven. (C) (i) HEK-CB2R/GPR55 cells have been transfected with 200 ng of NFAT-luciferase reporter plasmid and 24 h later cells had been influenced with agonists (1 μM) for three h in a serum-free medium. The luciferase activity become visualized the usage of a steadylite plus kit (PerkinElmer). Luminescence (relative easy devices (RLU)) become measured in a TopCounter (excellent count NXT; Packard) for 5 s. data are suggest±SEM from three impartial experiments carried out in quadruplicate (**P< 0.01) (ii) HEK-CB2R/GPR55 cells had been challenged with ligands (1 μM) and the ensuing picometer shifts of mirrored mild wavelength in opposition t the time (s) had been monitored. Transformation of optical signatures have been made through the use of the area below the curve (AUC) values between the 1 200 and 3 600 s time aspects. records were normalized and expressed as p.c of optimum activation precipitated by using LPI + 2-AG. information are suggest ±SEM from three unbiased experiments carried out in quadruplicate (***P< 0.001).

Activated Cdc42 became suggested to be concerned in the polarization and directional migration of neutrophils fifty one. again, 1 μM LPI brought about a modest activation of Cdc42 (figure 4Aii, lane 2), whereas 1 μM 2-AG changed into able to promote GTP-binding to Cdc42 in neutrophils within 1 min (figure 4Aii, lane three). apparently, incubation of neutrophils with each agonists led to an extra boost in Cdc42 recreation compared to 2-AG by myself (determine 4Aii, lane four).

In summary, the combined application of both the GPR55 agonist LPI and the CB2R agonist 2-AG additional potentiated Cdc42 undertaking. This manner can also for this reason underlie the polarized morphology (see determine 1Biv and 4A, top of lane 4) and the trafficking of neutrophils in opposition t a gradient of both ligands (see figure 1Aiii).

RhoA-based F-actin formation and activation of the downstream transcription component NFAT are mediated by using GPR55 and superior within the presence of activated CB2R

next, they wanted to verify the extent to which every of the respective receptors – i.e. GPR55 or CB2R – is involved in the formation of RhoA-based F-actin. due to the fact that the short existence-time of purified neutrophils does not enable for manipulations similar to siRNA knockdown, they took capabilities of HEK293 cells stably expressing these receptors on my own, or a mixture thereof. They and others have up to now shown that in HEK293 cells, GPR55 stimulation ends up in the activation of RhoA, Rac1 and Cdc42 15, 18.

The GPR55 agonist LPI (1 μM, 10 min) induced in demand F-actin fibers in HEK-GPR55 (figure 4Biv) however not in HEK-CB2R cells (determine 4Bv). This impact turned into dependent on the undertaking of the Gα13/RhoA axis, since the transient transfection of HEK-GPR55 cells with dominant poor mutants of Gα13 (Supplementary information, determine S5i) and RhoA (Supplementary assistance, determine S5ii) or a 10-min pretreatment with 10 μM ROCK inhibitor Y27632 (Supplementary tips, figure S5iii) avoided actin polymerization in response to 1 μM LPI. extraordinarily, GPR55-mediated F-actin formation changed into modestly attenuated in the presence of non-activated CB2R in HEK-CB2R/GPR55 cells (determine 4Bvi).

The CB2R agonist 2-AG (1 μM) could not induce actin rearrangement within the HEK-GPR55 cells (determine 4Bvii), but led to some accumulation of polymerized actin in the periphery of HEK-CB2R cells (figure 4Bviii, arrows). This effect could even be observed in HEK-CB2R/GPR55 cells (determine 4Bix, arrow). medicine of HEK-CB2R/GPR55 cells with both LPI (1 μM) and a pair of-AG (1 μM) tremendously improved the formation of filamentous actins (figure 4Bxii) when in comparison with LPI (determine 4Bvi) or 2-AG (determine 4Bix) on my own. Co-administration of LPI and a couple of-AG didn't show a metamorphosis in actin formation in HEK-GPR55 (determine 4Bx) or HEK-CB2R (determine 4Bxi) cells in comparison to remedy of those cells with any of the agonists by myself (evaluate figure 4Bx and 4Biv and 4Bxi and 4Bviii, respectively).

we've recently shown that in HEK293 cells, the stimulation of GPR55 triggers dissimilar signaling pathways, ultimately resulting in the activation of transcription factors such because the nuclear aspect of activated T cells (NFAT), the nuclear aspect-κB and the cAMP responsive factor binding (CREB) protein 21. additionally, we've suggested that GPR55-mediated NFAT activation is crucially dependent on the function of RhoA 18. hence, they subsequent tested whether NFAT-transcription component undertaking changed into differentially regulated in the presence of activated GPR55 and/or CB2 receptors in their HEK-CB2R/GPR55 cellphone model. Concomitant activation of GPR55 and CB2R with LPI (1 μM) and 2-AG (1 μM) resulted in a major enhancement of NFAT-activation when in comparison to cells encouraged with 1 μM LPI simplest (figure 4Ci, evaluate mild grey and black bars). the two-AG (1 μM) didn't result in NFAT endeavor in HEK-CB2R/GPR55 cells (figure 4Ci, dark grey bar). This turned into expected, on the grounds that CB2 receptors customarily mediate their signaling pursuits predominantly through Gαi-pathways, which haven't been said to induce NFAT-exercise. in an effort to look at various the signaling events within the HEK-CB2R/GPR55 model on a greater 'international' scale, they subjected their cells to a Dynamic Mass Redistribution Assay (DMR, Epic®). they now have previously stated the suitability of this system for label-free measurement of signaling events of both GPR55 21 and Gαi-coupled 7TM/GPCRs forty nine. basically, similar to their findings in the NFAT-assay, concomitant activation of GPR55 and CB2R with LPI (1 μM) and a couple of-AG (1 μM) led to a significantly better DMR response in these cells when in comparison to cells inspired with 1 μM LPI most effective (figure 4Cii, evaluate light gray and black bars). once more, 2-AG (1 μM) alone did not set off any DMR in HEK-CB2R/GPR55 cells (determine 4Cii, dark grey bar).

In abstract, these records indicate that the LPI-prompted activation of GPR55 is important for the RhoA-stylish rearrangement of the actin cytoskeleton and downstream signaling movements akin to activation of the transcription ingredient NFAT. despite the fact, these results are greater within the presence of a 2-AG-activated CB2 receptor.

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

A dramatic boost in ROS stages – favourite as the 'respiratory burst' – is a mechanism used through neutrophils to unravel infection. This method is catalyzed by means of the NADPH oxidase advanced 52 and is regulated by the small GTPase Rac2 38. In neutrophils and HL60 cells, 2-AG and the complement component 5a (C5a) have been said to spark off Rac2 by the use of their cognate Gαi-coupled receptors, i.e., the CB2R and the C5aR 14, fifty three, fifty four. right here, they validated even if the activation of GPR55 and CB2 receptors had an impact on ROS construction in neutrophils.

GPR55 agonists LPI (300 nM) or AM251 (300 nM) didn't induce ROS construction in neutrophils per se (determine 5Ai and 5Aii, light gray bars). In contrast, 2-AG (10 μM) caused ROS construction in neutrophils (determine 5Ai and 5Aii, darkish gray bars), an impact that may be inhibited with 10 μM selective CB2R antagonist AM630 (Supplementary suggestions, figure S6). curiously, 2-AG-prompted ROS creation turned into tremendously diminished when neutrophils were concomitantly inspired with LPI (300 nM) or AM251 (300 nM) (determine 5Ai and 5Aii, black bars). This effect changed into dose based (figure 5Aiii, 100 nM-300 nM LPI) and could also be observed in neutrophils activated with C5a (determine 5Aiii). corresponding to neutrophils, they accompanied that ROS formation inspired by using 2-AG (10 μM) became significantly inhibited with the aid of co-administration of LPI (determine 5Aiv) in dHL60 cells, besides the fact that children greater concentrations of LPI (1 μM-10 μM) have been essential to look an effect. besides the fact that children, like in neutrophils, LPI alone could not result in ROS production in dHL60 cells (determine 5Aiv).

figure 5 Figure 5

GPR55 activation inhibits (A) CB2R-mediated respiratory burst and (B) C5a-induced degranulation in neutrophils. (A) ROS production in neutrophils become measured via movement 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 mix of LPI and 2-AG for 20 min at 37 °C. ROS creation become measured as a transformation in fluorescence within the FL1 channel. (ii) ROS production in neutrophils became measured as in panel Ai apart from that AM251 (300 nM) changed into used instead of LPI. (iii) Neutrophils were incubated with C5a (5 nM) or 2-AG (10 μM) and treated with buffer (manage) or LPI (100 nM or 300 nM) for 20 min. ROS creation changed into assessed as in panel Ai. (iv) Serum-starved dHL60 cells had been loaded with 5 μM 2′,7′-DCF-DA for 10 min at 37 °C after which incubated with 2-AG (10 μM) in mixture with assay buffer (handle) or LPI (1 or 10 μM). LPI (10 μM) used in aggregate with DMSO (0.1%) didn't result in changes in ROS levels. ROS production became recorded in a Flex-Station II equipment (Ex. 485nm, Em. 535 nm) 20 min after ligand addition. Representatives of 3-four unbiased experiments, carried out in quadruplicates are shown for all subpanels. information are imply± SEM (*P< 0.05; **P< 0.01; ***P< 0.001). (B) (i) Neutrophils were incubated with LPI (300 nM) or assay buffer (manage) for 1 h at 37 °C. MPO release was brought about by way of increasing concentrations of C5a for 30 min and measured because the alternate in absorbance at 630 nm in a colorimetric assay. (ii) Neutrophils were incubated with expanding 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. records are mean±SEM of three impartial experiments performed in triplicates (*P< 0.05; **P< 0.01; ***P< 0.001). The MPO liberate induced with the aid of 300 nM C5a was set to one hundred%.

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

Activated GPR55 inhibits the C5a-induced degranulation of neutrophils

so as to be able to wreck infectious agents, neutrophils store a large number of enzymes in azurophilic granules 3. Upon activation by way of C5a and/or different inflammatory mediators, these granules unencumber their enzymes – e.g., myeloperoxidase (MPO) – to the milieu three. when you consider that activated GPR55 may block C5a-mediated ROS production in neutrophils (determine 5Aiii), they next tested even if LPI could likewise modify C5a-brought about MPO free up. in reality, pretreatment of neutrophils with 300 nM LPI for 1 h (figure 5Bi, black bars) drastically inhibited the MPO free up brought on by using distinct concentrations of C5a (determine 5Bi, white bars). expanding doses of LPI decreased MPO unencumber mediated via 300 nM C5a up to a maximum of seventy five% (figure 5Bii). No MPO free up turned into observed when neutrophils had been incubated with LPI alone (determine 5Bi, black bar at 0 attention of C5a). as a consequence, activated GPR55 can stay away from C5a-mediated degranulation of neutrophils.

Activated GPR55 inhibits ROS construction and degranulation in neutrophils via repression of Rac2 endeavor

It has generally been reported that the small GTPase Rac2 regulates degranulation and the NADPH oxidase pastime in neutrophils by the use of its translocation to the plasma membrane and incorporation in the NADPH oxidase complicated 53, 55. moreover, JWH015, an artificial CB2R-certain agonist, was shown to prompt Rac2 in dHL60 cells and first neutrophils 14. to peer even if the GPR55-mediated inhibition of ROS production and degranulation is based on Rac2, they investigated the activation and translocation of Rac2 in response to each GPR55 and CB2R agonists in neutrophils and dHL60 cells.

remedy of neutrophils with LPI (1 μM) for 1 min reduced the exercise of Rac2 when in comparison to car (figure 6A, lanes 1 and a pair of). In contrast, 2-AG (1 μM) resulted in a major activation of Rac2 (determine 6A, lane three), which reached the highest activity at 1 min and back to its basal undertaking after 2 min (Supplementary suggestions, determine S7). besides the fact that children, concomitant stimulation of neutrophils with 2-AG (1 μM) and LPI (1 μM) resulted in a significant inhibition of Rac2 exercise when in comparison to cells treated with 2-AG by myself (determine 6A, examine lanes three and four). Likewise, in dHL60 cells Rac2 exercise become reduced within the presence of 1 μM LPI (determine 6B, lane 2) and more suitable in the presence of 1 μM 2-AG (determine 6B, lane 3). once again, within the presence of each ligands, the Rac2 exercise become reduced when compared to dHL60 cells handled with 2-AG by myself (figure 6B, compare lanes 3 and four).

figure 6 Figure 6

GPR55 activation suppresses the CB2R-mediated activation and translocation of Rac2. (A) Neutrophils have been stimulated with agonists (1 μM) for 1 min at 37 °C. The active GTP-certain Rac2 was extracted from the lysates with PAK area-gluthatione agarose beads. GTP-certain and complete GTPase stages were visualized by means of western blotting the use of a rabbit anti-Rac2 antibody, β-actin served as a loading handle. The ratio of GTP-sure vs complete GTPase tiers changed into assessed with ImageJ software (graph). statistics are mean±SEM of three impartial 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 lively GTP-bound Rac2 become performed as in panel (A). facts are mean±SEM of three independent experiments (**P< 0.01; ***P< 0.001). (C) Neutrophils have been seeded on fibronectin-coated glass coverslips and handled with 0.01% DMSO (manage; i) or ligands for five min at 37 °C. mounted cells were incubated with rabbit anti-Rac2 antibody and stained with Alexa Fluor-488 goat anti-rabbit antibody (green), Texas-pink Phalloidin (red), and DAPI (blue). handle (i) and LPI (3 μM, ii) handled cells reveal a nuclear/perinuclear localization of Rac2 (arrows). Upon 2-AG stimulation (1 μM, iii), Rac2 disbursed evenly in the cytosol (arrow) and in part colocalized with actin at the plasma membrane (yellow, arrowhead). (iv) medicine with a mix of LPI (three μM) and 2-AG (1 μM) confirmed a nuclear localization of Rac2 in polarized neutrophils. Cells were analyzed using an OLYMPUS fluorescence microscope outfitted with a Hamamatsu ORCA CCD camera (original magnification: 60×). Scale bars: 10 μm. consultant photographs from 2-three experiments are shown.

The function of Rac2 in ROS creation and degranulation is based on its translocation from the nuclear and/or perinuclear zones to the plasma membrane. In neutrophils, Rac2 became above all discovered in or carefully around the nucleus (determine 6Ci, arrow, Rac2 in green, DAPI/nuclear staining in blue). medication of neutrophils with 3 μM LPI didn't alter the perinuclear distribution of Rac2 (determine 6Cii, arrow). In distinction, stimulation of neutrophils with 2-AG (1 μM) brought about a redistribution of Rac2 to the cytosol (determine 6Ciii, eco-friendly, arrow) and partly resulted in a colocolization with the peripheral actin (figure 6Ciii, yellow, arrowhead). This effect, although, could be averted by means of concomitant application of three μM LPI with 1 μM 2-AG, resulting in a perinuclear distribution of Rac2 in these cells (figure 6Civ, arrow).

In summary, these information indicate that GPR55 regulates both ROS and MPO creation in neutrophils via suppressing the undertaking of the small GTPase Rac2.


LPI Capital 1Q net earnings inside expectations | killexams.com Real Questions and Pass4sure dumps

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LPI Capital Bhd

(April sixteen, RM15.68)

preserve market function with an improved goal expense (TP) of RM16.50:

LPI Capital Bhd’s first quarter of fiscal year 2019 (1QFY19) core web income of RM77.2 million is inside their and consensus expectations — 23% of both full-yr estimates. The absence of dividends is additionally within expectations. The neighborhood usually pronounces dividends twice a yr.

12 months-on-yr, 1QFY19 salary grew three% on more advantageous gross premiums from the community’s key hearth and motor coverage segments (+4%), offset by means of a poorer performance via the marine, aviation and transit (-18%) in addition to miscellaneous (-sixteen%) coverage segments. The net earned top rate grew 9% from a higher retention ratio (65.8%; +3.9 percentage points [ppts]), but operating earnings expanded simplest 5% following a better combined ratio incurred (seventy three.9%; +0.7 ppt). This become an accumulation of a far better claims incurred ratio (47.four%; +0.3 ppt as superior motor claims were negated by bigger miscellaneous claims) and net commission ratio (+5.2%; +1.6 ppts), but a lower management cost ratio (21.2%; -1.3 ppts) become registered. Core web profit for 1QFY19 changed into at RM77.2 million (+6%).

Quarter-on-quarter, 1QFY19 earnings became flattish as a 3% decline in normal insurance properly line — especially on lessen internet fireplace coverage premiums — become negated by using an about 87% leap in funding salary. despite the fact, a more robust combined ratio of 73.9% (+9.6 ppts) changed into led via improved net claims from the marine, aviation and transit in addition to miscellaneous assurance companies. This translated into a lower 1QFY19 internet income by means of eight%, supported through greater valuable taxes of 19.2% (-5 ppts).

We consider the community’s trajectory — chiefly its lion’s share of the fireplace coverage segment — could nonetheless be backed by way of Public bank’s loan growth rates, fuelled by their broad company distribution network. despite the fact, issues about protecting a doable mixed ratio — which they are expecting to hike closer in opposition t about 70% ranges — are looming ahead of an upcoming evaluate of fire type coverage in 2019, doubtless leading to a competition-pushed margin compression.

besides the fact that children, the motor segment nonetheless demonstrates encouraging transaction volumes, indicating the the neighborhood’s electricity during this phase following its detariffication. On miscellaneous items, the neighborhood is asking against deleveraging its exposure within the building and engineering sectors via venturing into other courses — clinical and workmen compensation.

After releasing the results, they maintained their earnings estimates for FY19 and FY20. They also maintained their “market operate” score with an improved TP of RM16.50 from RM16.30 up to now. Their valuation is in line with an unchanged blended fee-revenue ratio (PER) and price-to-booklet value (PBV) of 19 instances and thrice on a rolled-over FY20 estimate base yr. The valuations are in response to the stock’s respective +1 normal deviation over the three-12 months imply of PER and PBV.

at present, they consider sentiments for the inventory may well be helmed by using the big economic institution Public bank’s solid backing, which may supply comfort for the neighborhood’s operations’ sustainability. Dividend returns of four.6% and 4.7% for FY19 and FY20 may be decent sufficient for investors as smartly. The risks to their call consist of a better or reduce top rate underwritten, better- or lower-than-anticipated claims and an improved- or lower-than-anticipated administration rate ratio. — Kenanga research, April sixteen


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GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils | killexams.com real questions and Pass4sure dumps

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 31. In addition, another recent study showed that the highly metastatic MDA-MB231 breast cancer cell line expresses GPR55 and migrates efficiently towards LPI 40. Therefore, they set out to determine the effects of LPI on the migration of neutrophils. As 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 21 induced a concentration-dependent migration of neutrophils (Figure 1Ai, •). The chemotactic properties of these ligands were comparable to that of the CB2R agonist 2-AG (Figure 1Ai, ♦). Next, they 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) 41 (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 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 12, 35 or in combination with chemokines to induce neutrophil migration 14, 42. Since both 2-AG and LPI are endogenous lipid mediators released by stimulated macrophages 7, 29, 30, 43, they investigated the concomitant effect of 2-AG and LPI on the migration of neutrophils. The combination of LPI (3 μM) and 2-AG (1 μM) showed a significant impact on neutrophil migration, when compared to LPI or 2-AG 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 44. They hence next assessed the cytoskeletal rearrangement of neutrophils in response to a 5-min exposure to a gradient of LPI or 2-AG alone, or a gradient of their combination thereof. Cytoskeletal rearrangement was assessed by F-actin phalloidin staining. In neutrophils treated with vehicle, actin was found at the periphery of the cells, which had a round spherical shape (Figure 1Bi). LPI (3 μM) induced random non-directional protrusions in the neutrophils (Figure 1Bii, arrows). Consistent with previous reports 14, treatment of neutrophils with 2-AG (1 μM) resulted in an elongation of cells, however, without showing the characteristic polarity of migrating leukocytes (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, they 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 45, 46, 47. Here they used an untouched, non-column based system at room temperature in the absence of Ca2+ 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, they 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 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) 18 alone or in combination with the CB2 receptor (HEK-CB2R/GPR55) served as controls. Using antibodies specifically targeting the respective receptors, they 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, they 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 48, GPR55 was predominantly found intracellulary in both neutrophils and HL60 cells. This staining was specific, since only HEK-GPR55 cells, but not untransfected HEK293 cells showed a positive immunoreactivity with the rat anti-GPR55 antibody (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 15, 18, 19. In addition, they recently showed that – among all Gα subunits – GPR55 couples solely to Gα13 in HEK293 cells 49. In order to test which Gα protein subunits are involved in the GPR55 and CB2R mediated signaling effects in neutrophils, they 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 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, they 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 50. For instance, neutrophil-like dHL60 cells have been reported to elongate in response to CB2R agonists (i.e. JWH015 and 2-AG), thereby activating Rac1 and Cdc42, while repressing RhoA 14. However, under these conditions, dHL60 cells did not show the typical rear/front polarity of chemotaxing neutrophils.

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 15, LPI (1 μM) induced a rapid – albeit modest – activation of Rac1 in neutrophils (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 14. However, the incubation of neutrophils with both LPI and 2-AG did not result in a synergistic activation of Rac1 (Figure 4Ai, lane 4).

Figure 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 51. Again, 1 μM LPI induced a modest activation of Cdc42 (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, they 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, they took advantage of HEK293 cells stably expressing these receptors alone, or a combination thereof. They and others have previously shown that in HEK293 cells, GPR55 stimulation leads to the activation of RhoA, Rac1 and Cdc42 15, 18.

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 21. Moreover, they have reported that GPR55-mediated NFAT activation is crucially dependent on the function of RhoA 18. Hence, they next tested whether NFAT-transcription factor activity was differentially regulated in the presence of activated GPR55 and/or CB2 receptors in their 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, they subjected their cells to a Dynamic Mass Redistribution Assay (DMR, Epic®). They have previously reported the suitability of this system for label-free measurement of signaling events of both GPR55 21 and Gαi-coupled 7TM/GPCRs 49. In fact, similar to their findings in the NFAT-assay, concomitant activation of GPR55 and CB2R 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 52 and is regulated by the small GTPase Rac2 38. In neutrophils and HL60 cells, 2-AG and the complement component 5a (C5a) have been reported to activate Rac2 via their cognate Gαi-coupled receptors, i.e., the CB2R and the C5aR 14, 53, 54. Here, they tested whether the activation of GPR55 and CB2 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, they 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 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 3. Upon activation by C5a and/or other inflammatory mediators, these granules release their enzymes – e.g., myeloperoxidase (MPO) – to the milieu 3. Since activated GPR55 could block C5a-mediated ROS production in neutrophils (Figure 5Aiii), they 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 53, 55. In addition, JWH015, a synthetic CB2R-specific agonist, was shown to activate Rac2 in dHL60 cells and primary neutrophils 14. To see whether the GPR55-mediated inhibition of ROS production and degranulation is dependent on Rac2, they investigated the activation and translocation of Rac2 in response to both GPR55 and CB2R 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 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.



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