CB₂-deficient mice and LDLR-deficient mice, both on a pure C57/BL6 background, were obtained from Jackson Laboratories. Mice were crossbred to generate CB₂ ^−/−/LDLR^−/− mice. Genotyping of each mouse used polymerase chain reaction and employed the following primers: LDLR, 5′-CCA TAT gCA TCC CCA gTC TT-3′ (common primer), 5′-gCg ATg gAT ACA CTC ACT gC-3′ (wild-type primer), 5′-AAT CCA TCT TgT TCA ATg gCC gAT C-3′ (mutant primer); CB₂, 5′- gAC Tag AgC TTT gTA ggT Agg Cgg -3′ (common primer), 5′- ggA gTT CAA CCC CAT gAA ggA gTA-3′ (wild-type primer) and CB₂, 5′- ggg gAT CgA TCC gTC CTg TAA gTC T-3′ (mutant primer). Six-week-old male CB₂ ^+/+/LDLR^−/− and CB₂ ^−/−/LDLR^−/− mice consumed a high-cholesterol diet (HCD) for 16 weeks (Ssniff modified after Research Diets D12108 containing 21% total fat and 1.235% cholesterol, Soest, Germany). In parallel, JWH-133 (diluted in a water-soluble solution with Tocrisolve, from Tocris, Bristol, UK) or vehicle was injected intra-peritoneally (i.p.) into LDLR-deficient mice consuming HCD at a concentration of 5 mg/kg body weight three times a week for 16 weeks. Subsequently, mice were euthanized, hearts and aortas were removed, and tissue was prepared and analyzed histologically as described previously [18], [19], [20]. Total cholesterol and triglyceride levels were assayed in EDTA plasma from blood obtained by retro-orbital bleeding before feeding and drawn from the right ventricle upon harvest. The total wall area ( = intima + media), intimal lesion area (intima) and medial area (media) as well as the percentage of positively stained area for macrophages (anti-mouse Mac-3), lipids (Oil-Red-O), T cells (anti-CD4), collagen (Picrosirius red), smooth muscle cells (anti-α-actin), and apoptotic cells (TUNEL, Roche, Basel, Switzerland) were quantified by blinded investigators employing computer-assisted image analysis software (Image Pro, Media Cybernetics, Bethesda, MD). Abdominal aortas were fixed with 10% formalin, opened longitudinally, pinned, stained with Oil-red-O solution (Sigma-Aldrich, St. Louis, MO, 2.5 h, RT), washed with 85% propylene glycol, and lipid accumulation was quantified as described above. All mice were housed under specific pathogen-free conditions. All procedures were approved by the Animal Care Committee of the University of Freiburg and the local authorities (Regierungspräsidium Freiburg) with the permit number G-08/46.

Murine endothelial cells were isolated using Invitrogen Dynabeads® (Invitrogen, Paisley, UK) as previously described [21]. Endothelial cells were seeded in 6 or 24 wells until they reached 80% confluence. Flow cytometric analysis for PECAM-1 and ICAM-2 showed a purity of about 97% of isolated murine endothelial cells (data not shown). After incubation in FCS-free medium for 24 hours, cells were stimulated with indicated concentrations of JWH-133 or vehicle, followed by TNFα (20 ng/ml) after 30 minutes. After 24 hours of incubation at 37°C supernatents were removed for ELISA and endothelial cells were lysed and used for Western Blotting. Apoptosis and cytotoxicity was evaluated by Apo-ONE® and CytoTox-One™ Assay according to the instructions of the manufacturer (Promega, Madison, WI).

Mouse MCP-1 was quantified in the supernatants of cell cultures using commercially available ELISA Kits (R&D DuoSet, Minneapolis, MN) according to the manufacturer's instructions.

Murine endothelial cells were stimulated with TNFα (20 ng/ml) for 24 hours. After incubation, murine endothelial cells were lysed, separated by SDS-PAGE under reducing conditions, and blotted to polyvinylidene difluoride membranes as described previously [21]. An anti-mouse ICAM-1 antibody (Santa Cruz, Santa Cruz, CA) was used as primary antibody, followed by an anti-peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson Laboratories, West Grove, PA) as secondary antibody.

To induce inflammation, mice were subjected to intraperitoneal injection of TNFα (200 ng/ml) as indicated. Blood was collected by cardiac puncture and serum separation. For analysis of inflammatory markers in mice the cytometric bead assay for Murine inflammation detecting IL-6, MCP-1, IFNγ, IL-10, and IL-12p70 was used according to manufacturer's instructions (BD Biosciences, San Diego, CA) optimized for higher sensitivity. Results were analyzed using the corresponding FCAP software (BD Franklin Lakes, NJ). The lower detection limits were in the range of 5–10 pg/ml.

Dynamic adhesion assays in the flow chamber were performed as described previously [19]. Murine endothelial cells were grown in 35 mm dishes (Costar, Bethesda, MD) and were subjected to the flow chamber. In brief, the Glycotech flow chamber (Gaithersburg, MD) was assembled with the dish as the bottom of the resulting parallel flow chamber. The chamber and tubes were filled with PBS without serum prior to the experiment. Subsequently, Murine leukocytes were applied with a syringe pump (Harvard apparatus PHD2000, Holliston, MA) with flow rates of 0.04 dyne/cm² (venous flow; a total of 10 min). Adherent cells were quantified under the microscope.

Flow cytometry was performed as previously described [22]. Cells were pre-incubated with mouse Fc-Block (αCD16/32, ebioscience, San Diego, CA). Antibodies included CD11b-FITC, CD115-PE, Ly6C/G (Gr1)-APC, CD4-Alexa488, CD8-PE, CD3-APC, CD20-PE, ICAM-1-FITC (all from ebioscience, San Diego, CA), ICAM-2-FITC, and PECAM-1-PE (PharMingen, San Diego, CA). The mean fluorescence indices (MFI) were quantified employing the FlowJo software (Tree Star Inc, Ashland, OR).

CB₂ ^+/+/LDLR^−/− and CB₂ ^−/−/LDLR^−/− mice were treated intraperitoneally with 4% thioglycollate. After 4 and 72 hours, mice were euthanized with CO₂, the peritoneal cavity was flushed with 6 ml of RPMI for 3 min. Leukocytes were quantified in a CASY counter. Similarly, wild-type (Bl6) mice received JWH-133 one hour before injection of 4% thioglycollate. The resulting leukocyte migration was measured after 4 and 72 hours.

Mice were subjected to i.p. injection of JWH-133 (5 mg/kg body weight) on day 1, 3, 6, 8 and 10. On day 10 retro-orbital blood was taken at 2, 12, 24, and 48 hours after the last injection of JHW-133. Mouse sera (100 µl, N = 6) were added to 5 µl internal standard (d₃-THC; 5 µg/ml in ethanol), followed by 2.9 ml acetic acid 0.1 M, followed by automated solid phase extraction using Aspec GX-274 (Gilson, Middleton, USA), reversed phase C₁₈ SPE-cartouche (type chromabond), and a 500 mg column bed (Macherey-Nagel, Düren, Germany). Samples were eluted with 1 ml acetonitrile, evaporated in a stream of nitrogen at 40°C, and incorporated in 25 µl ethyl acetate.

Frozen aortic tissue was incubated in 500 µl ethanol in an ultrasound bath twice for 15 min, pooled, 25 ng internal standard was added (d₃-THC), samples were evaporated in a stream of nitrogen at 40°C, reconstituted in ethanol, acetic acid was added, and automated solid phase extraction and elutriation was performed as described above.

1 µl of samples was analyzed using the Agilent 5973 GC-MS system (temperature gradient 100°C for 1 min, increase to 290°C for 2.5 min, increase to 310°C for 4 min, fragmentation energy 70 eV). The following fragments were chosen. For JWH-133: m/z 269 (quantifier), m/z 312 and m/z 229 (qualifier), retention time 6.5 min. For d₃-THC: m/z 302 (quantifier), m/z 317 and m/z 234 (qualifier), retention time 7.4 min. The detection limit was 25 ng/ml.

Data are expressed as means ± SEM of absolute or normalized values. Groups were compared employing the Student's t-test. A value of P<0.05 was considered significant. Data sets were analysed using GraphPad Prism® (GraphPad Software Inc, La Jolla, CA).

To explore the contribution of direct CB₂ receptor stimulation LDLR^−/− mice consuming a high-cholesterol diet (HCD) for 16 weeks were treated with intraperitoneal injections of the selective CB₂ receptor agonist JWH-133 or vehicle three times a week. JWH-133 was detectable in mouse serum after 2, 12, and 24 hours by mass spectrometry, proving bioavailability in vivo (Fig. 1). More importantly, JWH-133 could also be detected directly in aortic tissue of treated animals 48 hours after administration at a level of 2.2±0.67 ng/mg while it was undetectable in vehicle control-treated animals. Weights, cholesterol levels, and total leukocyte numbers did not differ between the study groups at baseline and end of feeding. Both groups also showed no difference in visceral fat mass, blood pressure, heart rate, and leukocyte subtypes as quantified at the end of the study (Table 1). Surprisingly, intimal lesion size in aortic roots was similar between JWH-133-treated mice and those receiving vehicle control (0.316±0.038 mm², N = 10 vs. 0.312±0.044 mm², N = 8, P = 0.94; Fig. 2A). Similar results were obtained in aortic arches (0.091±0.024 mm², N = 11 vs. 0.066±0.013 mm², N = 8, P = 0.41, Fig. 2B). Also, lipid deposition in en face analysis of abdominal aortas did not differ between both groups (Fig. 2C), demonstrating that CB₂ receptor stimulation does not attenuate atherogenesis in mice. Similarly, JWH-133 treatment did not modulate the content of lipids, macrophages, collagen, T cells, smooth muscle cells, and the cellular apoptosis rates in atherosclerotic plaques (Fig. 2D).

Consistent with our results for selective CB₂ receptor stimulation, CB₂ ^−/−/LDLR^−/− mice consuming HCD for 16 weeks developed lesions of similar size as respective CB₂ ^+/+/LDLR^−/− control animals in aortic roots (0.261±0.038 mm², N = 12 vs. 0.223±0.023 mm², N = 13, P = 0.40, Figure 3A), aortic arches (0.095±0.022 mm², N = 12 vs. 0.075±0.022 mm², N = 13, P = 0.54, Figure 3B), and abdominal aortas (Fig. 3C). Again, there was no change in the degree of apoptosis, the content of collagen, T cells, and smooth muscle cells within the atherosclerotic plaque. However, we could detect increased lipid and macrophage content (Fig. 3D). CB₁ expression quantified by RT-PCR did not differ between both groups rendering a CB₁-driven bias unlikely (0.0013±0.0002 vs. 0.0018±0.0004, P = 0.34, N = 3). Study characteristics were similar between both groups at baseline and end of feeding (Table 1).

Since previous reports implicated the CB₂ receptor in the recruitment of inflammatory cells, we investigated a potential role of CB₂ receptor signaling in Murine peritonitis [12], [23], [24]. 72 hours after intraperitoneal injection of thioglycollate peritoneal macrophage numbers were significantly reduced in JWH-133-treated mice compared with vehicle controls (Fig. 4A N = 5 per group). In contrast, JWH-133 treatment did not affect short term (4 h) thioglycollate-induced peritonitis predominated by neutrophils (Fig. 4A, N = 9 per group). Similar amounts of leukocytes accumulated in the peritoneal cavity of CB₂ ^−/−/LDLR^−/− mice and CB₂ ^+/+/LDLR^−/− control animals after 72 and 4 hours (N = 5 and N = 13 per group, respectively, Fig. 4B). In accord, CB₂ receptor signaling did not affect adhesion of inflammatory cells in the flow chamber (Fig. 4C), expression of ICAM-1 as assessed by Western blotting (Fig. 5A) and FACS (Fig. 5B), as well as chemokine expression in cultured endothelial cells (Fig. 5 C). JWH-133 did not modulate apoptosis (Fig. 5D) and cytotoxicity of the cells tested (Fig. 5E).

Since we did not observe an effect of CB₂ receptor signaling on atherosclerosis, a chronic inflammatory disease, we sought to explore its role in an acute model of inflammation. Interestingly, neither genetic deficiency nor selective stimulation of CB₂ by JWH-133 modulated the expression of IL-6, MCP-1, IL-10, IFNγ, or IL-12p70 in mice challenged intraperitoneally with TNFα (Fig. 6A). However, JWH-133-treated mice recruited lower numbers of monocytes with an inflammatory, GR1^high subtype to the blood pool upon stimulation with TNFα. Of note, no difference in numbers of this cellular subtype could be measured between CB₂ ^−/−/LDLR^−/− and CB₂ ^+/+/LDLR^−/− mice.