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We read with interest the recent review by Wiest et al 1 on spontaneous bacterial peritonitis (SBP), and would like to point out a new mechanism involved in the pathogenesis of this phenomenon. Cirrhotic patients have altered host-defence response mechanisms and increased susceptibility to bacterial infections, which seem to be related to alterations in the intestinal barrier and/or bacterial translocation from the mucosa to the mesenteric lymph nodes and the intestinal circulation. For this reason, a better understanding on the causes underlying this infection is important for effective future therapies. Both liver cirrhosis and lipopolyshaccaride (LPS)-induced sepsis have been associated with increased activity of endogenous cannabinoids.2 ,3 Our study aims to expand the knowledge on the relationship between Cannabinoid receptor 2 (CB2) receptors and SBP. We analysed the mRNA expression of CB2 in cirrhotic patients with or without SBP, with a mean age of 61±12 years. The aetiology of cirrhosis was alcohol-induced in 5 subjects, 3 with hepatitis B or C, 3 with both alcohol-induced and viral hepatitis, 1 with chronic biliary cirrhosis and 4 of unknown aetiology. We observed that CB2 mRNA expression was reduced in isolated circulating monocytes from cirrhotic patients and peritoneal macrophages, and that this reduction was even more marked in patients with SBP. Interestingly, although the CB1 receptor expression in immune cells was much lower, it was also found to be downregulated (figure 1). In order to mimic the situation occurring in cirrhotic patients, we assessed the effect of LPS on the mRNA and protein expression in U937 cells (a human monocytic cell line) after 6 h of stimulation at both supraphysiological (10 ng/ml) and pharmacological (1 μg/ml) doses. In concordance with what we observed in patients, both mRNA and protein expression were markedly downregulated in LPS-treated cells compared with non-treated cells. Table 1 shows the results obtained after a PCR array analysis of 84 antibacterial response genes and CB receptors. At 6 h, LPS treatment induced the expression of proinflammatory genes, mainly cytokines and chemokine receptors. However, it is important to note that the only significantly downregulated genes in LPS-treated cells were the CB1 and CB2 receptors.
Interestingly, the effects produced by LPS were almost completely suppressed when the cells were coincubated with 10 μg/ml of polymixin B (an antibiotic that prevents LPS cell binding), demonstrating the specificity of the effect triggered by LPS. This indicates that endotoxin-induced suppression of CB2 receptors is a highly specific effect for endocannabinoid signalling. Additionally, to determine if LPS-induced downregulation of the CB2 mRNA expression correlates with functionally significant changes, we evaluated the chemotactic activity in U937 cells. In fact, the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) have been shown to be powerful inducers of chemotaxis in macrophages and monocytes, including U937 cells.3 Cells pretreated with LPS (1 μg/ml) showed a significantly attenuated endocannabinoid-induced chemotactic activity. To further assess whether these effects are directly mediated by CB2 receptors, cells were exposed to AEA or 2-AG in the presence of SR144528 (1 μM), a specific antagonist of CB2 receptors. CB2 receptor blockade fully prevented the chemotactic-induced effect of the endocannabinoid.
Leukocytes are a major source of endocannabinoids. Under a framework of increased bacterial wall products, such as in advanced liver disease, LPS stimulation results in 2-AG and AEA secretion.4 Since CB2 receptors are abundantly expressed in monocytic cell lineage, the anticipated response would be enhanced mobility toward the site of infection, among others.5 However, as demonstrated in the present investigation, LPS would also suppress CB2 receptor expression and, therefore, the leukocyte ability to respond against bacterial infection becomes seriously compromised. This, in turn, provides a further explanation for the impaired defence response mechanisms in cirrhotic patients with liver disease.
Contributors VR Carried out the main part of the experimental work and designed most of the experiments. JML participated in the experimental procedures with human samples. JR contributed in the cell experimental culture designs and procedures, GC participated in the data analysis. MN kindly provided the human serum samples. GFV aided with the molecular experiments. MMR gave conceptual advice and revised the manuscript. WJ directed the research and wrote the manuscript.
Funding This work was supported by grants from Dirección General de Investigación Científica y Técnica (SAF09-08839) and from the Agència de Gestió d'Ajuts Universitaris i de Recerca (SGR 2009/1496). CIBERehd is funded by the Instituto de Salud Carlos III (Spain).
Competing interests None.
Patient consent Obtained.
Ethics approval The study was performed according to the criteria of the Investigation and Ethics Committee of the Hospital Clínic Universitari.
Provenance and peer review Not commissioned; externally peer reviewed.
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