Primary Macrophage Chemotaxis Induced by Cannabinoid Receptor 2 Agonists Occurs Independently of the CB2 Receptor

Abstract

Activation of CB2 has been demonstrated to induce directed immune cell migration. However, the ability of CB2 to act as a chemoattractant receptor in macrophages remains largely unexplored. Using a real-time chemotaxis assay and a panel of chemically diverse and widely used CB2 agonists, we set out to examine whether CB2 modulates primary murine macrophage chemotaxis. We report that of 12 agonists tested, only JWH133, HU308, L-759,656 and L-759,633 acted as macrophage chemoattractants. Surprisingly, neither pharmacological inhibition nor genetic ablation of CB2 had any effect on CB2 agonist-induced macrophage chemotaxis. As chemotaxis was pertussis toxin sensitive in both WT and CB2-/- macrophages, we concluded that a non-CB1/CB2, Gi/o-coupled GPCR must be responsible for CB2 agonist-induced macrophage migration. The obvious candidate receptors GPR18 and GPR55 could not mediate JWH133 or HU308-induced cytoskeletal rearrangement or JWH133-induced β-arrestin recruitment in cells transfected with either receptor, demonstrating that neither are the unidentified GPCR. Taken together our results conclusively demonstrate that CB2 is not a chemoattractant receptor for murine macrophages. Furthermore we show for the first time that JWH133, HU308, L-759,656 and L-759,633 have off-target effects of functional consequence in primary cells and we believe that our findings have wide ranging implications for the entire cannabinoid field.

Cannabinoid 2 Receptor Agonist Improves Systemic Sensitivity to Insulin in High-Fat Diet/Streptozotocin-Induced Diabetic Mice

“…Similarly no differences of the all 4 tested cytokine concentrations were recorded in the pancreatic lysates of SER601-treated and control animals (data not shown). However, a recent publication reported that the CB 2 receptors are not involved in mediating macrophage migration in response to pharmacological agents that are traditionally known as CB 2 receptor agonists [38], which may offer a partial explanation to the lack of effect of SER601, a highly selective for the CB 2 receptors, on islet macrophage infiltration.…”

Cannabis: A Treasure Trove or Pandora's Box?

“…The previously detailed off-target actions at opioid receptors are not exceptional: phytocannabinoids, endocannabinoids and other synthetic cannabinoid ligands are notorious for producing diverse off-target effects in the micromolar range [381,390,391,509,719,[978][979][980][981][982][983] with unclear (patho)physiological significance. These must be taken into consideration, and whenever possible, cannabinoids should be used at the concentration not greater than 1 µM in vitro.…”

Cannabinoid Receptors Are Overexpressed in CLL but of Limited Potential for Therapeutic Exploitation

“…AM251 and AM630 are also ion channel activators [ 71 ], AM251 additionally acts as agonist at GPR55 and antagonist at μ-opioid receptors [ 40 , 72 ]. JHW133 not only acts on CB2 but also the TRPV1 vanilloid receptor plus showed off-target effects in chemotaxis experiments in macrophages [ 73 , 74 ]. Besides these two ligand pairs, two compounds were included which are being widely used in cytotoxic experiments and which have long been known for broader activity: R-(+)-methanandamide (RM) is CB1 agonist, shows activity at GPR and ion channels [ 1 , 25 ], and (-)-cannabidiol (CNB) is a weak CB1 antagonist/CB2 inverse agonist interacting also with GPR55, TRPVR1 vanilloid receptors and μ-opioid receptors [ 1 , 39 , 75 , 76 ].…”

“…They also suggest that the cytotoxic effects of cannabinoids very likely are mediated by more than one receptor and one mechanism even in cases where molecules are reportedly highly specific. As mentioned above, a certain degree of unspecific action seems to be inherent to the nature of cannabinoids, an aspect that is increasingly reflected by the literature reporting a broad range of cannabinoid activity on a variety of G-protein coupled and other receptors [ 1 , 39 – 41 , 66 , 71 – 74 , 81 – 83 ].…”