SR144528 and WIN55212-3 had no measurable effects on IL-6 or IL-10 production when applied alone. Open in a separate window Figure 6 Induction of cytokine secretion by PBMC. by concurrent Gs activation. Monitoring downstream signaling events showed that activation of p38 was mediated by Gi, whereas ERK1/2 and Akt phosphorylation were mediated by Gi-coupled . Activation of CREB integrated multiple components; Gs and mediated 85% of the response, while 15% was attributed to Gi. Responses to HU308 had an important functional outcomesecretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1, and TNF- were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB2. Additionally, signaling and functional effects were completely blocked by a CB2-selective inverse agonist, giving additional evidence for CB2 involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB2 ligands as immunomodulatory therapeutics. state of leukocytes.15 The limited literature regarding CB2 signaling via other types of mitogen-activated protein kinases (MAPKs) indicates context-dependence of activation. CB2 can induce p38 phosphorylation (p-p38)12,16,17 which has been linked to growth inhibition of cancer cells, while dendritic cells18 and LPS-stimulated monocytes12 were nonresponsive. Akt kinase (protein kinase B), an important mediator of immunomodulation19 can be activated,20 inhibited,21 or unaffected22 by CB2 activation. A stress-activated protein kinase JNK (c-Jun NH2-terminal kinase), involved in pro-inflammatory signaling,23 has been shown to be activated24,25 and inhibited12 via CB2. The ultimate outcomes of CB2 activation in immune cells include inhibition of proliferation, induction of apoptosis, influences on cytokine/chemokine networks, and regulation of adhesion and migration.26 In human immunocompetent cells CB2 activation inhibits IL-2,27 IL-17, INF-, TNF-28 secretion, and stimulates IL-429 and TGF-30 secretion. Murine and models indicate that cannabinoids inhibit IL-2,31 IL-12, and IFN-,32,33 AX-024 and induce IL-4 secretion.32,33 However, these studies stimulated leukocytes with mitogens or antibodies. Species differences in cannabinoid signaling notwithstanding,5 cytokine secretion is generally highly dependent on the cellular environment. Indeed, cannabinoid effects on the cytokine network are sensitive to the method of cell activation.34 Therefore, close to the entirety of our current understanding of CB2 signaling and influence on the cytokine secretome has been obtained from models which exhibit considerable limitations in modeling normal human physiology. We endeavored to carry out a comprehensive study of CB2 AX-024 signaling in unstimulated human primary leukocytes (peripheral blood mononuclear cells, PBMC) under conditions closely preserving their state. We have observed an unprecedented CB2-mediated signaling and functional profile, including coupling to Gs. Results CB2, Mouse monoclonal to Ki67 but Not CB1, Is Expressed in Unstimulated Human Primary PBMC We quantified expression of CB1 and CB2 protein by whole cell radioligand binding with high affinity CB1/CB2 ligand [3H]-CP55940 displaced by CB1- and CB2- selective ligands, ACEA35 and HU30836, respectively. Although CB2 was expected to be the primary cannabinoid-responsive receptor in human PBMC, CB1 transcripts have also been detected in some contexts.1 Whereas CB1 protein could not be detected in PBMC from any of three healthy human donors, CB2 was readily detectable (Figure ?Figure11). Intrasubject variability in CB2 expression was low (independent experiments are from three blood draws from the same donor taken over the course of up to 8 months), while intersubject expression was within the same magnitude, ranging from 704 to 1323 fmol/mg. PBMC samples from these same three donors were utilized in subsequent experiments. Open in a separate window Figure 1 CB1 and CB2 protein quantification by AX-024 whole cell radioligand binding. Cells were incubated with [3H]-CP55940 (5 nM) in the presence and absence of a CB1-selective displacer ACEA (1 M), or a CB2-selective displacer HU308 (1 M). Total binding sites ( 0.0001C0.0005; two-way repeated measures [RM] ANOVA with Holm-?dk posthoc test). This was followed by a steady reduction in cAMP concentration, reaching a maximum extent by 30 min (to 60.3 2.8% of forskolin-stimulated), with cAMP subsequently returning back to the forskolin-induced level by 60 min. Prior studies in cells overexpressing CB2 describe an immediate onset and earlier peak cAMP inhibition.37 Given that we cannot readily measure inhibition of cAMP production without forskolin being present, we hypothesized that low basal cAMP and/or slow response to forskolin in the primary leukocytes might mask the ability to detect inhibition at early time-points and hence produce the observed delay. However, preincubation with.Although CB2 was expected to be the primary cannabinoid-responsive receptor in human PBMC, CB1 transcripts have also been detected in some contexts.1 Whereas CB1 protein could not be detected in PBMC from any of three healthy human donors, CB2 was readily detectable (Figure ?Figure11). and TNF- were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB2. Additionally, signaling and functional effects were completely blocked by a CB2-selective inverse agonist, giving additional evidence for CB2 involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB2 ligands as immunomodulatory therapeutics. state of leukocytes.15 The limited literature regarding CB2 signaling via other types of mitogen-activated protein kinases (MAPKs) indicates context-dependence of activation. CB2 can induce p38 phosphorylation (p-p38)12,16,17 which has been linked to growth inhibition of cancer cells, while dendritic cells18 and LPS-stimulated monocytes12 were nonresponsive. Akt kinase (protein kinase B), an important mediator of immunomodulation19 can be activated,20 inhibited,21 or unaffected22 by CB2 activation. A stress-activated protein kinase JNK (c-Jun NH2-terminal kinase), involved in pro-inflammatory signaling,23 has been shown to be activated24,25 and inhibited12 via CB2. The ultimate outcomes of CB2 activation in immune cells include inhibition of proliferation, induction of apoptosis, influences on cytokine/chemokine networks, and regulation of adhesion and migration.26 In human immunocompetent cells CB2 activation inhibits IL-2,27 IL-17, INF-, TNF-28 secretion, and stimulates IL-429 and TGF-30 secretion. Murine and models indicate that cannabinoids inhibit IL-2,31 IL-12, and IFN-,32,33 and induce IL-4 secretion.32,33 However, these studies stimulated leukocytes with mitogens or antibodies. Species differences in cannabinoid signaling notwithstanding,5 cytokine secretion is generally highly dependent on the cellular environment. Indeed, cannabinoid effects on the cytokine network are sensitive to the method of cell activation.34 Therefore, close to the entirety of our current understanding of CB2 signaling and influence on the cytokine secretome AX-024 has been obtained from models which exhibit considerable limitations in modeling normal human physiology. We endeavored to carry out a comprehensive study of CB2 signaling in unstimulated human primary leukocytes (peripheral blood mononuclear cells, PBMC) under conditions closely preserving their state. We have observed an unprecedented CB2-mediated signaling and functional profile, including coupling to Gs. Results CB2, but Not CB1, Is Expressed in Unstimulated Human Primary PBMC We quantified expression of CB1 and CB2 protein by whole cell radioligand binding with high affinity CB1/CB2 ligand [3H]-CP55940 displaced by CB1- and CB2- selective ligands, ACEA35 and HU30836, respectively. Although CB2 was expected to be the primary cannabinoid-responsive receptor in human PBMC, CB1 transcripts have also been detected in some contexts.1 Whereas CB1 protein could not be detected in PBMC from any of three healthy human donors, CB2 was readily detectable (Figure ?Figure11). Intrasubject variability in CB2 expression was low (independent experiments are from three blood draws from the same donor taken over the course of up to 8 months), while intersubject expression was within the same magnitude, ranging from 704 to 1323 fmol/mg. PBMC samples from these same three donors were utilized in subsequent experiments. Open in a separate window Figure 1 CB1 and CB2 protein quantification by whole cell radioligand binding. Cells were incubated with [3H]-CP55940 (5 nM) in the presence and absence of a CB1-selective displacer ACEA (1 M), or a CB2-selective displacer HU308 (1 M). Total binding sites ( 0.0001C0.0005; two-way repeated measures [RM] ANOVA with Holm-?dk posthoc test). This was followed by a steady reduction in cAMP concentration,.