Abstract
Previous work from our laboratory showed that a CB2 selective agonist, O-1966, blocked the proliferative response of C57BL/6 mouse spleen cells exposed to spleen cells of C3HeB/FeJ mice in vitro in the mixed lymphocyte reaction (MLR). The MLR is widely accepted as an in vitro correlate of in vivo grant rejection. Mechanisms of the immunosuppression induced by the cannabinoid were explored, and it was shown that O-1966 in this in vitro assay induced CD25+Foxp3+ Treg cells and IL-10, as well as down-regulated mRNA for CD40 and the nuclear form of the transcription factors NF-κB and NFAT in T-cells. The current studies tested the efficacy of O-1966 in prolonging skin grafts in vivo. Full thickness flank skin patches (1-cm2) from C3HeB/FeJ mice were grafted by suturing onto the back of C57BL/6 mice. O-1966 or vehicle was injected intraperitoneally into treated or control groups of animals beginning 1 h pre-op, and then every other day until 14 days post-op. Graft survival was scored based on necrosis and rejection. Treatment with 5 mg/kg of O-1966 prolonged mean graft survival time from 9 to 11 days. Spleens harvested from O-1966 treated mice were significantly smaller than those of vehicle control animals based on weight. Flow cytometry analysis of CD4+ spleen cells showed that O-1966 treated animals had almost a 3-fold increase in CD25+Foxp3+ Treg cells compared to controls. When dissociated spleen cells were placed in culture ex vivo and stimulated with C3HeB/FeJ cells in an MLR, the cells from the O-1966 treated mice were significantly suppressed in their proliferative response to the allogeneic cells. These results support CB2 selective agonists as a new class of compounds to prolong graft survival in transplant patients.
Highlights
The discovery of the CB2 receptor and its abundance and fairly selective expression on cells of the immune system (Munro et al, 1993; Galiegue et al, 1995) has posed the question of its function on immune responses
Δ9-THC has been shown to inhibit macrophage presentation of antigen to T cells, which occurred through effects at the CB2 receptor, as macrophages taken from CB2 k/o mice were not suppressed (Buckley et al, 2000)
Splenic weight was determined in mice that had received skin grafts, with or without treatment with O-1966
Summary
The discovery of the CB2 receptor and its abundance and fairly selective expression on cells of the immune system (Munro et al, 1993; Galiegue et al, 1995) has posed the question of its function on immune responses. Many studies investigating beneficial or detrimental effects of cannabinoids on immune responses and resistance to infection have focused on Δ9-THC or on the endogenous cannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide. All three of these ligands bind to both CB1 and CB2. To prove that an effect of these agonists occurs via the CB2 receptor, investigators have used selective CB1 and CB2 antagonists, or used CB1 or CB2 receptor knock-out mice. Δ9-THC has been shown to inhibit macrophage presentation of antigen to T cells, which occurred through effects at the CB2 receptor, as macrophages taken from CB2 k/o mice were not suppressed (Buckley et al, 2000). CB2 selective agonists have been shown to ameliorate autoimmune reactions in a variety of mouse models that include experimental autoimmune encephalitis (EAE) (a model for multiple sclerosis) (Ni et al, 2004; Maresz et al, 2007), systemic sclerosis (Akhmetshina et al, 2009; Servettaz et al, 2010), autoimmune uveoretinitis (Xu et al, 2007), murine colitis and inflammatory bowel disease (Storr et al, 2009; Singh et al, 2012; Fichna et al, 2014; Leinwand et al, 2017)
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