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

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

It has prior to now been cautioned that there's a web site diverse from CB1R and CB2R, which impacts CB2R-mediated migration in human blood neutrophils and that this web page can live GPR55 31. furthermore, an additional recent examine confirmed that the extremely metastatic MDA-MB231 breast melanoma mobilephone line expresses GPR55 and migrates correctly against LPI forty. for this reason, they got down to investigate the results of LPI on the migration of neutrophils. As figure 1Ai indicates, neutrophils with no grief migrated towards a gradient of LPI (figure 1Ai, ▪). Likewise, the extra superior synthetic cannabinoid GPR55 agonist and CB1R antagonist AM251 21 prompted a attention-dependent migration of neutrophils (figure 1Ai, •). The chemotactic properties of those ligands were comparable to that of the CB2R agonist 2-AG (determine 1Ai, ♦). subsequent, they confirmed whether the migration of neutrophils against either LPI or 2-AG may well live blocked by pretreating the cells with selective receptor antagonists. Pre-incubation of neutrophils with both the GPR55 antagonist cannabidiol (5 μM) 41 (figure 1Aii, CBD) or the selective CB2R antagonist AM630 (determine 1Aii, 5 μM) for 10 min drastically diminished the migratory residences of neutrophils towards LPI (3 μM) or 2-AG (1 μM), respectively. In distinction, neutrophil migration became now not impaired when cells abide been pretreated with the 'incorrect' antagonists, i.e., 5 μM of CBD followed with the aid of 1 μM 2-AG stimulation or 5 μM AM630 adopted by way of 3 μM LPI stimulation, respectively (records no longer proven).

figure 1 Figure 1

GPR55 and CB2R agonists induce the directional migration and polarization of neutrophils. (A) Human blood neutrophils abide been positioned to the higher wells of a microBoyden chamber and (i) had been allowed emigrate against expanding concentrations of LPI (▪), AM251 (•) or 2-AG (♦) in the bottom wells for 1 h. Migrated cells in the backside wells had been counted by using a circulation cytometer. (ii) Neutrophils had been pre-incubated with DMSO (0.05%), CBD (5 μM) or AM630 (5 μM) for 10 min at 37 °C and their migration towards LPI (three μM) or 2-AG (1 μM) become assessed as in panel Ai. (iii) Chemotaxis of neutrophils against DMSO (0.01%, white bar), LPI (3 μM, light grey bar), 2-AG (1 μM, black gray bar) or LPI and a pair of-AG combined (3 μM and 1 μM, respectively, black bar) turned into assessed as in panel Ai. (iv) Chemotaxis of neutrophils turned into assessed as in panel iii, apart from that AM251 (3 μM) become used as an alternative of LPI. (v) Neutrophils abide been 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 three-6 impartial experiments, performed in quadruplicates, are proven for every bit of subpanels. information are mean±SEM (*P< 0.05; **P< 0.01; ***P< 0.001). (B) Neutrophils had been seeded on fibronectin-covered glass coverslips and treated with a gradient of 0.01% DMSO (manage; i) or ligands for five min at 37 °C and stained with methanolic Texas-red Phalloidin (red) and DAPI (blue). (ii) LPI medicine (three μM) brought on fuzzy protrusions (arrows), whereas (iii) 2-AG (1 μM) brought on an elongation of the neutrophils (arrows). (iv) Extending head (arrow) and tail formation (dashed arrow) witness a polarization of neutrophils in line with a mix of LPI (three μM) and 2-AG (1 μM). Cells abide been analyzed the exercise of a Zeiss LSM510 META Axioplan confocal microscope (customary magnification: 100×). Scale bars: 10 μm. representative images of three independent experiments are proven. (C) Neutrophils had been seeded on fibronectin-covered glass coverslips and handled with LPI (three μM) and a pair of-AG (1 μM) for five min at 37 °C and stained with Texas-red Phalloidin (purple) and DAPI (blue). (i) Cells displayed a limpid directional and polarized constitution when migrating against a local source of LPI/2-AG (white dot). (ii) In contrast, no cytoskeleton home improvement happened after a uniform addition of agonists to the medium. Cells had been analyzed as in panel B (fashioned magnification: sixty three×). consultant photos of three independent experiments are shown. Scale bars: 10 μm.

In most of the previous stories, 2-AG has been used lonely 12, 35 or in compund with chemokines to set off neutrophil migration 14, 42. in view that each 2-AG and LPI are endogenous lipid mediators released by way of motivated macrophages 7, 29, 30, forty three, they investigated the concomitant consequence of 2-AG and LPI on the migration of neutrophils. The compund of LPI (3 μM) and a pair of-AG (1 μM) showed a major repercussion on neutrophil migration, when in comparison to LPI or 2-AG lonely (figure 1Aiii). greater prominently, the migration of neutrophils towards AM251 (3 μM), when mixed with 2-AG (1 μM), became synergistically superior in comparison to neutrophils migrating against AM251 or 2-AG on my own (determine 1Aiv). These results can live drastically diminished by pretreating the cells with the GPR55 antagonist CBD (5 μM), the CB2R antagonist AM630 (5 μM) or a mix of both for 10 min (determine 1Av).

in keeping with chemokines, neutrophils endure cytoskeletal rearrangement and configuration alternate, which culminates in cellular polarization and thereby allows for the cells emigrate effectively forty four. They hence subsequent assessed the cytoskeletal rearrangement of neutrophils according to a 5-min publicity to a gradient of LPI or 2-AG alone, or a gradient of their aggregate thereof. Cytoskeletal rearrangement was assessed via F-actin phalloidin staining. In neutrophils handled with vehicle, actin turned into discovered on the periphery of the cells, which had a round spherical configuration (determine 1Bi). LPI (three μM) caused random non-directional protrusions within the neutrophils (figure 1Bii, arrows). per outdated experiences 14, medication of neutrophils with 2-AG (1 μM) resulted in an elongation of cells, youngsters, with out displaying the characteristic polarity of migrating leukocytes (figure 1Biii, arrows). only when neutrophils had been concomitantly incubated with a gradient of LPI (3 μM) and a pair of-AG (1 μM), the touchstone polarity of migrating neutrophils – i.e. extending head (arrow) and retracting tail (dashed arrow) – may live detected (figure 1Biv). This consequence become no longer as a result of the larger attention of the combined ligands per se, because neither LPI nor 2-AG by myself, up to five μM, could elicit the selfsame cytoskeletal rearrangement (statistics not shown), however changed into elegant on the gradient of compounds (see determine 1C).

When LPI (three μM) and a pair of-AG (1 μM) had been concurrently utilized to the media the usage of the precise of a slender tip (determine 1Ci, white dot), a directional movement against the source of ligands become followed. The uniform addition of ligands into the way of life medium, youngsters, did not elicit any shape trade within the neutrophils (figure 1Cii). Likewise, when confirmed in a Boyden-migration assay, no migration of neutrophils may live observed in the absence of a ligand gradient (Supplementary counsel, determine S1).

Taken collectively, these information exhibit that LPI, AM251 or 2-AG lonely might each elicit neutrophil migration – albeit to a lesser extent than when utilized in combination – and with out inducing the habitual morphology of migrating neutrophils. truly, simplest the aggregate of LPI and a pair of-AG evoked a robust migratory response in human blood neutrophils by way of the institution of a rear-entrance uneven 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 up to now been mentioned that the sample of expression levels of CB1 and CB2 receptors in neutrophils depends on the isolation routine 45, forty six, forty seven. birthright here they used an untouched, non-column based mostly device at margin temperature in the absence of Ca2+ and Mg2+ ions to support away from a stimulation of the cells. GPR55 mRNA copy numbers (determine 2Ai, black bar) were enormously higher than these of the CB2R (figure 2Ai, white bar). certainly, both CB2R and GPR55 mRNA may even live detected by using RT-PCR (determine 2Aii). The expression of CB1R turned into not detectable in both RT-PCR or precise time PCR (facts now not proven). additionally, they proven the expression of GPR55 in differentiated neutrophil-like HL60 cells (dHL60), which expressed the differentiation marker CD11b at stages corresponding to these of neutrophils (Supplementary tips, determine S2). precise time PCR evaluation showed a 5.5-fold augment in GPR55 mRNA ranges in dHL60 cells (determine 2Aiii, black bar) compared to undifferentiated HL60 cells (uHL60) (determine 2Aiii, white bar).

determine 2 Figure 2

GPR55 is enormously expressed in human blood neutrophils and neutrophil-like HL60 cells. (A) GPR55 and CB2R mRNA expression in freshly remoted human blood neutrophils became assessed through (i) quantitative real-time PCR and (ii) RT-PCR. PCR products were 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) changed into measured by way of actual-time PCR. Representatives of three independent experiments are shown for every bit of subpanels. facts are mean±SEM (**P< 0.01). (B) (i) GPR55 and CB2R protein expression in neutrophils, uHL60 and dHL60 cells turned into assessed by using western blotting the exercise of rat anti-GPR55 and rabbit anti-CB2R antibodies. Lysates were probed for β-actin as a loading handle. A representative blot of three independent experiments is proven. (ii) Western blotting of GPR55 and CB2R in lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells turned into performed as in panel Bi. A consultant blot of three impartial experiments is shown. (C) GPR55 expression in human blood neutrophils, uHL60 and dHL60 cells turned into tested 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 abide been analyzed the exercise of a Zeiss LSM510 META Axioplan confocal microscope (fashioned magnification: a hundred×). Scale bars: 10 μm. representative photographs of two-three unbiased experiments are proven.

We next assessed the protein degrees and cellular expression of both GPR55 and CB2 receptors in neutrophils, as well as in uHL60 and dHL60 cells. HEK293 mobile lines stably expressing the CB2 receptor (HEK-CB2R), the GPR55 receptor (HEK-GPR55) 18 lonely or in combination with the CB2 receptor (HEK-CB2R/GPR55) served as controls. the exercise of antibodies particularly focused on the respective receptors, they institute that GPR55 and CB2R proteins had been expressed in neutrophils, uHL60 and dHL60 cells on the usurp protein sizes (determine 2Bi, ∼37 kDa for GPR55 and ∼forty five kDa for CB2R). The specificity of the antibodies turned into tested by means of western blot the usage of the lysates from HEK293, HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells. As determine 2Bii suggests, the antibodies reacted with their respective aims most effective. moreover, they validated the expression of GPR55 in freshly remoted neutrophils and HL60 cells by means of immunofluorescence the exercise of the rat anti-GPR55 antibody (figure 2C). As accompanied in different primary cells forty eight, GPR55 became predominantly institute intracellulary in each neutrophils and HL60 cells. This staining became particular, considering that most efficient HEK-GPR55 cells, but now not untransfected HEK293 cells confirmed a superior immunoreactivity with the rat anti-GPR55 antibody (Supplementary assistance, determine S3).

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

It has been demonstrated that GPR55 mediates its downstream signaling movements by means of Gα13 and the RhoA wee GTPase in HEK293 cells 15, 18, 19. furthermore, they currently confirmed that – amongst every bit of Gα subunits – GPR55 couples completely to Gα13 in HEK293 cells 49. with the objective to examine which Gα protein subunits are worried in the GPR55 and CB2R mediated signaling outcomes in neutrophils, they used the toxins C3 and pertussis toxin to inhibit the endeavor of Gα13/RhoA and Gαi, respectively. Pre-incubation of neutrophils with C3 toxin (three μg/ml, 2 h) vastly inhibited the LPI-caused migration of neutrophils, but showed no consequence on the 2-AG-inspired migration (determine 3Ai and 3Aii). in spite of this, pertussis toxin (3 μg/ml, 2 h) avoided the migration of neutrophils towards 2-AG, however had no significant consequence on LPI-caused migration (figure 3Ai and 3Aii). in addition, migration of neutrophils in opposition t a combination of LPI and 2-AG turned into drastically inhibited when the cells had been pre-incubated with either C3 or pertussis toxin (determine 3Aiii).

figure 3 Figure 3

GPR55 and CB2R mediate chemotaxis and cytoskeletal remodeling by way of coupling to Gα13/RhoA and Gαi proteins. Neutrophils were pre-incubated with cellphone-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 abide been allowed emigrate in opposition t (i) LPI (3 μM), (ii) 2-AG (1 μM) or (iii) a combination of LPI (3 μM) and a pair of-AG (1 μM) for 1 h. Migrated cells in the bottom wells had been counted by using a mosey cytometer. The chemotactic index became calculated as variety of cells migrated against agonists divided through the variety of cells migrated towards car (DMSO 0.01%). Representatives of two impartial experiments, carried out in quadruplicates, are shown. information are mean±SEM (*P< 0.05; **P< 0.01). n.s.: no longer colossal. (B) Cells had been seeded on fibronectin-coated glass coverslips and handled with 0.01% DMSO (manage), LPI (3 μM), 2-AG (1 μM) or a compund of LPI (3 μM) and a pair of-AG (1 μM) for 5 min at 37 °C and stained with Texas-pink Phalloidin (purple) and DAPI (blue). Cells abide been analyzed using an OLYMPUS fluorescence microscope equipped with a Hamamatsu ORCA CCD digital camera (customary magnification: 60×). Scale bars: 10 μm. consultant photos from 2 unbiased experiments are proven.

subsequent, they validated even if the abide an repercussion on of these inhibitors on neutrophil migration was because of an impairment of cytoskeletal remodeling. C3 toxin (three μg/ml, 2 h) inhibited the formation of protrusions triggered by means of LPI (examine determine 3Bvi and 3Bii), however had no repercussion on the elongated morphology of 2-AG-treated neutrophils (evaluate figure 3Bvii and 3Biii). moreover, inhibition of RhoA averted the polarization of neutrophils in response to a compund of LPI and 2-AG (compare determine 3Bviii and 3Biv). Pertussis toxin (3 μg/ml, 2 h) inhibited the elongation of 2-AG-influenced cells (compare figure 3Bxi and 3Biii), but did not abide an consequence on the non-directional protrusions stimulated by LPI (examine figure 3Bx and 3Bii). Inhibition of Gαi signaling abrogated the polarized head/tail morphology of neutrophils upon medication with a combination of LPI and 2-AG (examine figure 3Bxii and 3Biv).

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

Rac1 and Cdc42 are worried within the cytoskeletal rearrangement of neutrophils after concomitant activation of GPR55 and CB2R

The migration of leukocytes against chemotactic brokers happens via a coordinated sequence of events, customarily together with a cytoskeletal rearrangement that depends on the duty of the Rho household of wee GTPases 50. for example, neutrophil-like dHL60 cells had been said to elongate according to CB2R agonists (i.e. JWH015 and a pair of-AG), thereby activating Rac1 and Cdc42, while repressing RhoA 14. although, beneath these circumstances, dHL60 cells did not demonstrate the common rear/entrance polarity of chemotaxing neutrophils.

We established whether Rac1 and Cdc42 abide been differentially activated through either LPI, 2-AG or the combined application of both ligands in neutrophils. consistent with previous reviews of GPR55-mediated Rac1 activation in HEK293 cells 15, LPI (1 μM) brought on a hastily – albeit modest – activation of Rac1 in neutrophils (figure 4Ai, lane 2). the 2-AG (1 μM) brought about a quick and essential enhance in activated Rac1 (determine 4Aii, lane 3), whereby a optimum of Rac1 activity changed into reached inside 60 s (Supplementary tips, figure S4). This time frame of activation is per CB2R-mediated Rac1 activation in dHL60 cells 14. besides the fact that children, the incubation of neutrophils with both LPI and 2-AG did not influence in a synergistic activation of Rac1 (figure 4Ai, lane four).

figure four Figure 4

Cytoskeletal rearrangement of (A) neutrophils and (B) HEK293 cells requires the concomitant activation of GPR55 and CB2R. (A) Neutrophils abide been inspired 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 were extracted from the lysates with PAK area-gluthatione agarose beads. GTP-sure and complete GTPase tiers had been visualized by western blotting the exercise of mouse anti-Rac1 (i) and rabbit anti-Cdc42 (ii) antibodies. The β-actin served as a loading handle. The ratio of GTP-bound vs complete GTPase tiers became assessed with ImageJ application (graphs). consultant blots from three-four independent experiments are shown. records are imply±SEM. (*P< 0.05; ***P< 0.001). (B) HEK-GPR55, HEK-CB2R and HEK-CB2R/GPR55 cells were seeded on 1% PDL-lined glass coverslips. Serum-starved cells abide been incubated with agonists (1 μM) for 10 min in a serum-free medium. The fixed cells abide been stained for F-actin with the aid of methanolic Texas-purple Phalloidin (crimson) and with DAPI (blue). Cells abide been analyzed using an OLYMPUS fluorescence microscope geared up with a Hamamatsu ORCA CCD digital camera (normal magnification: 60×). Scale bars: 20 μm. representative photos from 3-4 experiments are proven. (C) (i) HEK-CB2R/GPR55 cells abide been transfected with 200 ng of NFAT-luciferase reporter plasmid and 24 h later cells had been influenced with agonists (1 μM) for 3 h in a serum-free medium. The luciferase activity changed into visualized the usage of a steadylite plus kit (PerkinElmer). Luminescence (relative simple instruments (RLU)) turned into measured in a TopCounter (appropriate weigh NXT; Packard) for 5 s. facts are mean±SEM from three impartial experiments performed in quadruplicate (**P< 0.01) (ii) HEK-CB2R/GPR55 cells abide been challenged with ligands (1 μM) and the ensuing picometer shifts of mirrored simple wavelength in opposition t the time (s) were monitored. Transformation of optical signatures had been made through the exercise of the enviornment below the curve (AUC) values between the 1 200 and three 600 s time facets. information were normalized and expressed as p.c of optimum activation brought on by LPI + 2-AG. information are connote ±SEM from three unbiased experiments performed in quadruplicate (***P< 0.001).

Activated Cdc42 changed into pronounced to live worried within the polarization and directional migration of neutrophils 51. once more, 1 μM LPI prompted a modest activation of Cdc42 (figure 4Aii, lane 2), whereas 1 μM 2-AG become able to promote GTP-binding to Cdc42 in neutrophils within 1 min (figure 4Aii, lane 3). curiously, incubation of neutrophils with each agonists ended in an additional augment in Cdc42 undertaking in comparison to 2-AG lonely (figure 4Aii, lane four).

In abstract, the mixed software of both the GPR55 agonist LPI and the CB2R agonist 2-AG further potentiated Cdc42 recreation. This manner may additionally for that understanding underlie the polarized morphology (see determine 1Biv and 4A, proper of lane 4) and the trafficking of neutrophils against a gradient of both ligands (see determine 1Aiii).

RhoA-based F-actin formation and activation of the downstream transcription factor NFAT are mediated with the aid of GPR55 and improved within the presence of activated CB2R

subsequent, they wanted to examine the extent to which every of the respective receptors – i.e. GPR55 or CB2R – is involved in the formation of RhoA-elegant F-actin. because the brief lifestyles-time of purified neutrophils does not allow for manipulations such as siRNA knockdown, they took skills of HEK293 cells stably expressing these receptors alone, or a combination thereof. They and others abide 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 trendy F-actin fibers in HEK-GPR55 (determine 4Biv) but not in HEK-CB2R cells (figure 4Bv). This consequence changed into elegant on the activity of the Gα13/RhoA axis, in view that the momentary transfection of HEK-GPR55 cells with paramount negative mutants of Gα13 (Supplementary assistance, determine S5i) and RhoA (Supplementary counsel, figure S5ii) or a ten-min pretreatment with 10 μM ROCK inhibitor Y27632 (Supplementary suggestions, determine S5iii) averted actin polymerization based on 1 μM LPI. pretty, GPR55-mediated F-actin formation turned into modestly attenuated within the presence of non-activated CB2R in HEK-CB2R/GPR55 cells (determine 4Bvi).

The CB2R agonist 2-AG (1 μM) couldn't result in actin rearrangement in the HEK-GPR55 cells (determine 4Bvii), but led to some accumulation of polymerized actin within the periphery of HEK-CB2R cells (determine 4Bviii, arrows). This repercussion may besides live accompanied in HEK-CB2R/GPR55 cells (determine 4Bix, arrow). remedy of HEK-CB2R/GPR55 cells with both LPI (1 μM) and a pair of-AG (1 μM) greatly accelerated the formation of filamentous actins (determine 4Bxii) when in comparison with LPI (determine 4Bvi) or 2-AG (determine 4Bix) by myself. Co-administration of LPI and 2-AG didn't divulge a change in actin formation in HEK-GPR55 (figure 4Bx) or HEK-CB2R (figure 4Bxi) cells in comparison to treatment of those cells with any of the agonists lonely (examine determine 4Bx and 4Biv and 4Bxi and 4Bviii, respectively).

we've these days shown that in HEK293 cells, the stimulation of GPR55 triggers assorted signaling pathways, eventually resulting in the activation of transcription components such as the nuclear ingredient of activated T cells (NFAT), the nuclear aspect-κB and the cAMP responsive factor binding (CREB) protein 21. moreover, they now abide reported that GPR55-mediated NFAT activation is crucially stylish on the feature of RhoA 18. hence, they subsequent confirmed no matter if NFAT-transcription factor recreation turned into differentially regulated in the presence of activated GPR55 and/or CB2 receptors in their HEK-CB2R/GPR55 phone mannequin. Concomitant activation of GPR55 and CB2R with LPI (1 μM) and a pair of-AG (1 μM) led to a significant enhancement of NFAT-activation when compared to cells stimulated with 1 μM LPI only (figure 4Ci, evaluate simple gray and black bars). the 2-AG (1 μM) didn't result in NFAT exercise in HEK-CB2R/GPR55 cells (determine 4Ci, black grey bar). This was anticipated, because CB2 receptors usually mediate their signaling events predominantly through Gαi-pathways, which abide not been stated to result in NFAT-recreation. to live able to test the signaling pursuits in the HEK-CB2R/GPR55 mannequin on a more 'world' scale, they subjected their cells to a Dynamic Mass Redistribution Assay (DMR, Epic®). we've up to now mentioned the suitability of this rig for label-free dimension of signaling pursuits of each GPR55 21 and Gαi-coupled 7TM/GPCRs forty nine. truly, similar to their findings within the NFAT-assay, concomitant activation of GPR55 and CB2R with LPI (1 μM) and a pair of-AG (1 μM) ended in a greatly greater DMR response in these cells when in comparison to cells motivated with 1 μM LPI handiest (figure 4Cii, examine mild gray and black bars). once more, 2-AG (1 μM) on my own did not induce any DMR in HEK-CB2R/GPR55 cells (figure 4Cii, darkish grey bar).

In summary, these records witness that the LPI-precipitated activation of GPR55 is essential for the RhoA-elegant rearrangement of the actin cytoskeleton and downstream signaling routine akin to activation of the transcription factor NFAT. however, these results are better in the presence of a 2-AG-activated CB2 receptor.

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

A stagy raise in ROS ranges – accepted as the 'respiratory burst' – is a mechanism used via neutrophils to obtain to the bottom of an infection. This process is catalyzed by the NADPH oxidase tangled fifty two and is regulated by the wee GTPase Rac2 38. In neutrophils and HL60 cells, 2-AG and the complement piece 5a (C5a) were stated to spark off Rac2 by way of their cognate Gαi-coupled receptors, i.e., the CB2R and the C5aR 14, fifty three, 54. here, they confirmed even if the activation of GPR55 and CB2 receptors had an consequence on ROS construction in neutrophils.

GPR55 agonists LPI (300 nM) or AM251 (300 nM) did not result in ROS construction in neutrophils per se (determine 5Ai and 5Aii, simple grey bars). In contrast, 2-AG (10 μM) precipitated ROS construction in neutrophils (determine 5Ai and 5Aii, darkish gray bars), an consequence that may well live inhibited with 10 μM selective CB2R antagonist AM630 (Supplementary advice, determine S6). interestingly, 2-AG-induced ROS construction changed into enormously diminished when neutrophils abide been concomitantly stimulated with LPI (300 nM) or AM251 (300 nM) (figure 5Ai and 5Aii, black bars). This repercussion turned into dose elegant (determine 5Aiii, a hundred nM-300 nM LPI) and could besides live accompanied in neutrophils activated with C5a (figure 5Aiii). similar to neutrophils, they observed that ROS formation stimulated by way of 2-AG (10 μM) changed into tremendously inhibited through co-administration of LPI (determine 5Aiv) in dHL60 cells, besides the fact that children larger concentrations of LPI (1 μM-10 μM) had been crucial to glance an impact. besides the fact that children, relish in neutrophils, LPI on my own could not induce ROS construction in dHL60 cells (figure 5Aiv).

figure 5 Figure 5

GPR55 activation inhibits (A) CB2R-mediated respiratory burst and (B) C5a-triggered degranulation in neutrophils. (A) ROS creation in neutrophils turned into measured through mosey cytometry. (i) Cells abide been loaded with 1 μM 2′,7′-DCF-DA after which incubated with DMSO (0.1%), LPI (300 nM), 2-AG (10 μM) or a mix of LPI and a pair of-AG for 20 min at 37 °C. ROS creation became measured as a change in fluorescence in the FL1 channel. (ii) ROS production in neutrophils became measured as in panel Ai except that AM251 (300 nM) turned into used instead of LPI. (iii) Neutrophils abide been incubated with C5a (5 nM) or 2-AG (10 μM) and treated with buffer (handle) or LPI (a hundred nM or 300 nM) for 20 min. ROS creation was 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 and then incubated with 2-AG (10 μM) in combination with assay buffer (manage) or LPI (1 or 10 μM). LPI (10 μM) used in aggregate with DMSO (0.1%) didn't set off alterations in ROS tiers. ROS production become recorded in a Flex-Station II device (Ex. 485nm, Em. 535 nm) 20 min after ligand addition. Representatives of three-4 impartial experiments, carried out in quadruplicates are proven for every bit of subpanels. records are imply± SEM (*P< 0.05; **P< 0.01; ***P< 0.001). (B) (i) Neutrophils had been incubated with LPI (300 nM) or assay buffer (manage) for 1 h at 37 °C. MPO liberate was triggered through expanding concentrations of C5a for 30 min and measured because the trade in absorbance at 630 nm in a colorimetric assay. (ii) Neutrophils had been incubated with expanding concentrations of LPI for 1 h at 37 °C. MPO unencumber become triggered with C5a (300 nM) for 30 min and assessed as in panel Bi. data are suggest±SEM of three unbiased experiments carried out in triplicates (*P< 0.05; **P< 0.01; ***P< 0.001). The MPO unencumber prompted via 300 nM C5a become set to a hundred%.

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

Activated GPR55 inhibits the C5a-prompted degranulation of neutrophils

in order to live capable of wreck infectious agents, neutrophils support a large variety of enzymes in azurophilic granules three. Upon activation via C5a and/or different inflammatory mediators, these granules liberate their enzymes – e.g., myeloperoxidase (MPO) – to the milieu three. considering that activated GPR55 could obstruct C5a-mediated ROS production in neutrophils (determine 5Aiii), they subsequent verified even if LPI may likewise alter C5a-triggered MPO release. really, pretreatment of neutrophils with 300 nM LPI for 1 h (figure 5Bi, black bars) drastically inhibited the MPO unencumber brought on by using several concentrations of C5a (figure 5Bi, white bars). expanding doses of LPI reduced MPO unlock mediated through 300 nM C5a up to a optimum of seventy five% (figure 5Bii). No MPO liberate was observed when neutrophils abide been incubated with LPI lonely (determine 5Bi, black bar at 0 attention of C5a). accordingly, activated GPR55 can support away from C5a-mediated degranulation of neutrophils.

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

It has generally been pronounced that the wee GTPase Rac2 regulates degranulation and the NADPH oxidase pastime in neutrophils by the exercise of its translocation to the plasma membrane and incorporation within the NADPH oxidase tangled fifty three, fifty five. moreover, JWH015, a synthetic CB2R-selected agonist, became shown to set off Rac2 in dHL60 cells and first neutrophils 14. to peer even if the GPR55-mediated inhibition of ROS construction and degranulation is stylish on Rac2, they investigated the activation and translocation of Rac2 in keeping with both GPR55 and CB2R agonists in neutrophils and dHL60 cells.

treatment of neutrophils with LPI (1 μM) for 1 min decreased the activity of Rac2 when compared to vehicle (determine 6A, lanes 1 and a pair of). In distinction, 2-AG (1 μM) resulted in a ample activation of Rac2 (figure 6A, lane 3), which reached the optimum pastime at 1 min and lower back to its basal recreation after 2 min (Supplementary guidance, figure S7). besides the fact that children, concomitant stimulation of neutrophils with 2-AG (1 μM) and LPI (1 μM) resulted in a ample inhibition of Rac2 pastime when compared to cells handled with 2-AG on my own (determine 6A, examine lanes 3 and four). Likewise, in dHL60 cells Rac2 activity became decreased in the presence of 1 μM LPI (figure 6B, lane 2) and improved in the presence of 1 μM 2-AG (determine 6B, lane 3). once again, in the presence of both ligands, the Rac2 activity changed into decreased when in comparison to dHL60 cells handled with 2-AG lonely (figure 6B, evaluate lanes three and four).

determine 6 Figure 6

GPR55 activation suppresses the CB2R-mediated activation and translocation of Rac2. (A) Neutrophils abide been influenced with agonists (1 μM) for 1 min at 37 °C. The lively GTP-sure Rac2 turned into extracted from the lysates with PAK domain-gluthatione agarose beads. GTP-certain and total GTPase degrees had been visualized by means of western blotting the usage of a rabbit anti-Rac2 antibody, β-actin served as a loading manage. The ratio of GTP-certain vs total GTPase stages was assessed with ImageJ utility (graph). facts are suggest±SEM of three independent experiments. (*P< 0.05; ***P< 0.001). (B) Serum-starved dHL60 cells had been prompted with agonists (1 μM) for 1 min at 37 °C. The extraction of lively GTP-sure Rac2 changed into carried out as in panel (A). data are mean±SEM of three unbiased experiments (**P< 0.01; ***P< 0.001). (C) Neutrophils were seeded on fibronectin-covered glass coverslips and treated with 0.01% DMSO (handle; i) or ligands for 5 min at 37 °C. fixed cells had been incubated with rabbit anti-Rac2 antibody and stained with Alexa Fluor-488 goat anti-rabbit antibody (green), Texas-red Phalloidin (red), and DAPI (blue). handle (i) and LPI (3 μM, ii) treated cells divulge a nuclear/perinuclear localization of Rac2 (arrows). Upon 2-AG stimulation (1 μM, iii), Rac2 dispensed evenly within the cytosol (arrow) and partly colocalized with actin at the plasma membrane (yellow, arrowhead). (iv) medicine with a mix of LPI (three μM) and a pair of-AG (1 μM) confirmed a nuclear localization of Rac2 in polarized neutrophils. Cells abide been analyzed the exercise of an OLYMPUS fluorescence microscope equipped with a Hamamatsu ORCA CCD digital camera (normal magnification: 60×). Scale bars: 10 μm. consultant images from 2-3 experiments are proven.

The feature of Rac2 in ROS production and degranulation is based on its translocation from the nuclear and/or perinuclear zones to the plasma membrane. In neutrophils, Rac2 become primarily discovered in or closely around the nucleus (figure 6Ci, arrow, Rac2 in eco-friendly, DAPI/nuclear staining in blue). remedy of neutrophils with three μM LPI did not alter the perinuclear distribution of Rac2 (determine 6Cii, arrow). In contrast, stimulation of neutrophils with 2-AG (1 μM) triggered a redistribution of Rac2 to the cytosol (determine 6Ciii, eco-friendly, arrow) and partly resulted in a colocolization with the peripheral actin (determine 6Ciii, yellow, arrowhead). This effect, despite the fact, could live avoided via concomitant software of three μM LPI with 1 μM 2-AG, resulting in a perinuclear distribution of Rac2 in these cells (determine 6Civ, arrow).

In summary, these information imply that GPR55 regulates both ROS and MPO creation in neutrophils by the exercise of suppressing the pastime of the wee GTPase Rac2.


LPI Expands Certifications to recognize the significance of DevOps to the Open source neighborhood | killexams.com actual Questions and Pass4sure dumps

TORONTO, ONTARIO, Nov 23, 2016 (Marketwired by way of COMTEX) -- LPI is Launching a community-primarily based consultation process to develop a brand new exam and Certification for DevOps

TORONTO, ONTARIO--(Marketwired - Nov 23, 2016) -

Editors word: there is one picture linked to this press liberate.

Designed to replicate the turning out to live value of DevOps collaboration fashions and rig in each gadget administration and utility development, the deliberate certification will require candidates to mosey an examination to live awarded LPI certified Open know-how DevOps Engineer. the primary assessments will seize location in Autumn 2017.

"DevOps, at its core, is a collaboration model supported through a particular set of equipment. we've been watching this device chain maturing over the final few years and it's the birthright time to confer with LPI's open supply neighborhood about how they can supply proof of talent in handling these rig and enforcing DevOps solutions," states Fabian Thorns, LPI's Director of Certification building.

government Director of LPI, G. Matthew Rice, adds: "As further and further businesses insert DevOps methodologies to their workflows, expertise in the exercise of tools supporting DevOps develop into more and more censorious and are having an immense move on the drudgery of every bit of their candidates. It additionally makes a lot of LPI's simple issues crucial for brand spanking new audiences, corresponding to builders. the 1st step of their neighborhood-based exam construction routine is the survey of americans working within the industry. This survey can live crucial in picking out the abilities which are required to support the collaboration of IT administrators and software developers."

The consultation - referred to as a Job job evaluation (JTA) - contains a web survey to open source authorities international and consultation with LPI's exam construction volunteer group. because the examination construction procedure continues there can live ongoing communicate by means of the examination development mailing listing in addition to ordinary weblog posts on the LPI website.

"We invite every bit of and sundry concerned in DevOps and participants of their exam development neighborhood to participate during this Job assignment evaluation. each vote is a crucial contribution to create a certification that displays and endorses the capabilities that depend within the office," says Mr. Thorns.

how to obtain involved:

find out greater about their new certification and how to participate in the DevOps exam construction: http://www.lpi.org/devops

be piece of their examination development mailing checklist: http://www.lpi.org/examdev-record

be trained more about the LPI exam pile process: http://www.lpi.org/value-of-certification/our-certification-building-specifications

about the Linux knowledgeable Institute (LPI)

LPI is the international certification balanced and profession support corporation for open supply specialists. With greater than 500,000 checks delivered, it live the world's first and biggest vendor-impartial Linux and open source certification physique. LPI has certified specialists in 181 international locations, promises tests in 9 languages, and has over four hundred working towards companions.

we are dedicated to proposing the IT group with certifications of the highest quality, relevance, and accuracy. This dedication requires that their examination pile procedure live extremely detailed, participatory, and consultative. live piece of the LPI community and obtain concerned. connect with us on facebook, or live a piece of their exam pile neighborhood of volunteers, collaborating on certifications, examination construction, and latest technologies - http://www.lpi.org/examdev-list.

For additional info, tickle consult with their website at http://www.lpi.org.

To view the photograph linked to this press liberate, tickle consult with the following hyperlink: http://media3.marketwire.com/medical doctors/DevOpsEngBadge2.jpg

Linux expert Institute G. Matthew Rice executive Director mrice@lpi.org

� 2016 Nasdaq, Inc. every bit of rights reserved.


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

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

It has previously been suggested that there is a site several from CB1R and CB2R, which affects CB2R-mediated migration in human blood neutrophils and that this site could live 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 live 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 rush 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, black 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 every bit of 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) witness a polarization of neutrophils in response to a compund 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 limpid 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 lonely 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 consequence of 2-AG and LPI on the migration of neutrophils. The combination of LPI (3 μM) and 2-AG (1 μM) showed a significant repercussion on neutrophil migration, when compared to LPI or 2-AG lonely (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 lonely (Figure 1Aiv). These effects could live 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 institute 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 live detected (Figure 1Biv). This consequence 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 elicit the selfsame cytoskeletal rearrangement (data not shown), but it was contingent 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 elicit any shape change in the neutrophils (Figure 1Cii). Likewise, when tested in a Boyden-migration assay, no migration of neutrophils could live observed in the absence of a ligand gradient (Supplementary information, figure S1).

Taken together, these data demonstrate that LPI, AM251 or 2-AG lonely could each elicit 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 margin 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 besides live detected by RT-PCR (Figure 2Aii). The expression of CB1R was not detectable in either RT-PCR or actual 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). actual time PCR analysis showed a 5.5-fold augment 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 every bit of 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 demonstrate 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 lonely or in combination with the CB2 receptor (HEK-CB2R/GPR55) served as controls. Using antibodies specifically targeting the respective receptors, they institute that GPR55 and CB2R proteins were expressed in neutrophils, uHL60 and dHL60 cells at the usurp 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 institute 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 wee GTPase in HEK293 cells 15, 18, 19. In addition, they recently showed that – among every bit of 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 consequence 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 consequence 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 rush 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 compund 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 repercussion 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 consequence 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 move 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 sequence of events, typically including a cytoskeletal rearrangement that relies on the duty of the Rho family of wee GTPases 50. For instance, neutrophil-like dHL60 cells abide 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 demonstrate 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 augment 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. lively 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 weigh 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 belt 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 connote ±SEM from three independent experiments performed in quadruplicate (***P< 0.001).

Activated Cdc42 was reported to live 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 augment in Cdc42 activity compared to 2-AG lonely (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 odds of HEK293 cells stably expressing these receptors alone, or a combination thereof. They and others abide 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 consequence was contingent on the activity of the Gα13/RhoA axis, since the momentary transfection of HEK-GPR55 cells with paramount 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 consequence could besides live 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 demonstrate 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 lonely (compare figure 4Bx and 4Biv and 4Bxi and 4Bviii, respectively).

We abide 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 abide reported that GPR55-mediated NFAT activation is crucially contingent on the duty 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, black gray bar). This was expected, since CB2 receptors typically mediate their signaling events predominantly through Gαi-pathways, which abide 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 abide 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) lonely did not induce any DMR in HEK-CB2R/GPR55 cells (Figure 4Cii, black gray bar).

In summary, these data intimate that the LPI-induced activation of GPR55 is censorious 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 stagy augment 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 tangled 52 and is regulated by the wee GTPase Rac2 38. In neutrophils and HL60 cells, 2-AG and the complement component 5a (C5a) abide 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 consequence 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, black gray bars), an consequence that could live 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 consequence was dose contingent (Figure 5Aiii, 100 nM-300 nM LPI) and could besides live 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 descry an effect. However, relish in neutrophils, LPI lonely 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 rush 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 every bit of 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 demonstrate 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 live able to blow 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 obstruct 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 lonely (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 wee GTPase Rac2 regulates degranulation and the NADPH oxidase activity in neutrophils via its translocation to the plasma membrane and incorporation in the NADPH oxidase tangled 53, 55. In addition, JWH015, a synthetic CB2R-specific agonist, was shown to activate Rac2 in dHL60 cells and primary neutrophils 14. To descry whether the GPR55-mediated inhibition of ROS production and degranulation is contingent 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 lonely (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 lonely (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 lively 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 lively 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 demonstrate 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 duty of Rac2 in ROS production and degranulation is contingent 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 live 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 intimate that GPR55 regulates both ROS and MPO production in neutrophils via suppressing the activity of the wee GTPase Rac2.



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