Cannabinoid receptor 2 activation reduces intestinal leukocyte recruitment and systemic inflammatory mediator release in acute experimental sepsis

Cannabinoids from the plant Cannabis sativa have been widely used in medicine for over a millennium as anticonvulsant, analgesic, anti-emetic, anti-inflammatory and immunosuppressive drugs [1]. Cannabinoids, including both phytocannabinoids and synthetic cannabinoids, mediate their effect through binding to specific receptors, members of the G-protein-coupled receptor superfamily [2, 3]. Cannabinoid-1 receptors are found throughout the central and peripheral nervous system, their major role being to modulate neurotransmitter release, whereas the cannabinoid-2 receptors (CB2Rs) are found mainly on immune cells and are known to play a role in immune responses and regulation of inflammatory processes [4, 5]. The endogenous ligands for cannabinoid receptors are bioactive lipids called endocannabinoids. Collectively, endocannabinoids, their receptors and biosynthetic and degradative enzymes are referred to as the endocannabinoid system [1].

Sepsis and septic shock are the most frequent causes of death in surgical ICU patients [6]. The endocannabinoid system is upregulated during sepsis [7, 8]. However, the role of the endocannabinoid system in the immune response is still not completely understood, particularly with specific regard to the complex disease progression of sepsis. To date, two studies have examined the impact of CB2R modulation on survival and other parameters in experimental sepsis induced by cecal ligation and puncture (CLP) using CB2R knockout mice. These studies reported opposing results. In one case, CB2R knockout mice following CLP-induced sepsis had a higher mortality and the administration of a selective CB2R agonist, GP1a, improved survival of wild-type mice [9]. In contrast, the second study demonstrated that CB2R knockout mice had a better survival from sepsis than did wild-type mice [10]. While these studies clearly demonstrate the importance of CB2R involvement in sepsis, they also underscore the requirement for further studies in order to clarify the mechanisms of CB2R-related modulation of inflammation in sepsis of varying severity and type.

The aim of our study was to investigate the effects of CB2R manipulation (activation or inhibition) in two different acute experimental sepsis models, given the conflicting studies of Tschöp and colleagues [9] and Csoka and colleagues [10]. We used 2 experimental sepsis models: endotoxemia and colon ascendens stent peritonitis (CASP)-induced sepsis. Endotoxemia was selected to study the effects of CB2R activation and inhibition in a standardized, acute model of systemic inflammation. CASP-induced sepsis was used to confirm the experimental results in a more clinically relevant model of sepsis.

Impairment of the intestinal microcirculation represents a key event in sepsis pathology [11, 12]. The focus of our study was therefore the intestinal microcirculation. We evaluated leukocyte recruitment and functional capillary density in the intestinal microvasculature by intravital microscopy (IVM). Furthermore, we studied intestinal histology and plasma release of inflammatory mediators during experimental CB2R modulation.

The main finding of our study was that CB2R activation reduced leukocyte recruitment within the intestinal microvasculature in two distinct acute experimental sepsis models.

In the first model (endotoxemia) we observed a significant increase in the number of adhering leukocytes in the intestinal microvasculature caused by LPS challenge. We found that the CB2R agonist HU308, given at a dosage of 2.5 mg/kg intravenously, significantly reduced the number of adherent leukocytes in submucosal V1 and V3 venules. The fluorescence dye Rhodamine 6G used for intravital imaging stains all leukocytes, so the specific cell types could not be further determined [16]. However, because of the high percentage of neutrophils in the bloodstream, this subpopulation is expected to be the most important cell fraction in this regard. In our LPS experiments, we observed no significant effects of the CB2R antagonist AM630 on LPS-induced leukocyte adhesion. LPS-induced leukocyte adhesion was probably already at its maximum and therefore could not be further exacerbated by CB2R block.

As CB2R inhibition by AM630 showed no impact on leukocyte recruitment in our (endotoxemia) model, we used only the agonist HU308 in the CASP-induced sepsis experiments. The CASP acute sepsis model showed stable macrohemodynamics (no septic shock). However, the agonist dosage used in the LPS endotoxemia experiments was not sufficient to produce significant effects on leukocyte adhesion in the CASP model; an increase in the dosage of HU308 to 10 mg/kg was required to obtain the same extent of reduction of intestinal leukocyte adhesion. In support of the results from our CASP model, Ni and colleagues also showed in a more clinical-related experimental inflammation model (autoimmune encephalomyelitis) that the cannabinoid 1/cannabinoid 2 receptor agonist WIN 55212-2 was only able to attenuate leukocyte-endothelial interactions in the cerebral microcirculation if given at a higher dosage (10 mg/kg) [17].

Taken together, these findings indicate that the effect of CB2R activation on inflammation is dose dependent and varies according to the experimental conditions. The conflicting survival results of the experimental sepsis studies reported in CB2R knockout mice [9, 10] may thus reflect differences in the severity of the CLP-induced sepsis model used in these studies. For example, CB2R activation was only beneficial in more severe sepsis, comparable with endotoxemia used in our study. However, in less severe CLP models, inefficient clearance of bacterial pathogens could lead to prolongation of the infectious process and resultant death. Attenuation of inflammation would thus prove more beneficial for survival in a severe model of CLP, but could lead to increased mortality in a moderate model of CLP [18].

In support of this hypothesis, our inflammatory mediator data provided evidence that the CASP-induced sepsis model generated an acute proinflammatory cytokine and adhesion molecule profile. Administration of (the higher dose of) the CB2R agonist HU308 decreased the levels of proinflammatory, soluble adhesion molecules and chemotactic factors, consistent with an anti-inflammatory effect of CB2R activation in this sepsis model.

TNFα is a key proinflammatory cytokine involved in mediating endotoxemia and sepsis [19]. Inhibition of TNFα production therefore has a high therapeutic potential in managing sepsis. Several studies have now reported that endocannabinoids can modulate the release of proinflammatory mediators via CB2R-related pathways. For example, the endocannabinoids anandamide and 2-arachidonylglycerol were observed in vitro to inhibit the production of TNFα released from LPS-treated rat microglial cells [20]. Another study showed that 2-arachidonylglycerol inhibits TNFα release from LPS-treated murine macrophages both in vitro and in vivo [21]. The involvement of CB2R in regulating the release of TNFα was reinforced in several studies using β-caryophyllene, a selective CB2R agonist. β-Caryophyllene significantly inhibited LPS-stimulated TNFα expression in human peripheral blood [22], and a recent in vitro study by Bento and colleagues showed that, in addition to TNFα, treatment with β-caryophyllene also reduced release of IL-1β and IFNγ by LPS-stimulated macrophages [23]. In keeping with these results, our study also found a significant reduction of the systemic IL-1β release in animals with CASP-induced sepsis treated with the CB2R agonist HU308. Furthermore, we also observed a reduction in the levels of soluble adhesion molecules by CB2R activation in CASP-induced sepsis. Several groups have shown that the levels of soluble adhesion molecules are correlating with sepsis severity and outcome [24]. The observed decrease in the levels of (soluble) adhesion molecules is in line with our IVM findings of attenuated leukocyte recruitment within the intestinal microvasculature by CB2R activation. Studies using other CB2R agonists (for example, JWH133 and HU309) during hepatic ischemia/reperfusion injury demonstrated that CB2R activation also resulted in a reduction of TNFα and macrophage inflammatory protein-2 levels, and tissue expression of adhesion molecule intercellular adhesion molecule-1 [25, 26]. These beneficial effects of CB2R agonists could be attenuated by CB2R genetic deletion or inhibition.

The expression of inflammatory mediators is regulated by various signaling pathways, including NF-κB activation, which are affected by CB2R modulation [27]. For example, Rajesh and colleagues demonstrated that CBR activation attenuates TNFα-triggered NF-κB and RhoA activation [28]. Furthermore, noladin ether, a stable analog of the endocannabinoid 2-arachidonylglycerol, inhibited the nuclear translocation of NF-κB that leads to the arrest of the cell cycle and inhibition of growth of prostate carcinoma cells [29]. Taken together, these studies suggest that endocannabinoid signaling, and specifically CB2R modulation, is able to alter signaling pathways that regulate production of inflammatory mediators.

The data reported in this study support the involvement of CB2R signaling in leukocyte activation during sepsis. We have shown that CB2R activation has anti-inflammatory actions in two acute experimental models of severe sepsis in rats. Drugs targeting the CB2R may have therapeutic potential in the treatment of inflammatory diseases, such as severe sepsis.