Cannabinoid Receptor 2-Selective Ligands as Immunosuppressive Compounds: Utility in Graft Rejection

Abstract

Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. The capacity of delta-9-tetrahydrocannabinol (delta-9-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation was tested. Both CB2-selective agonists and delta-9-THC significantly suppressed the MLR in a dose dependent fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2- selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3+ cells to these compounds completely inhibited their action in a reconstituted MLR and proliferation of purified T-cells by anti-CD3 and anti-CD28 antibodies was inhibited. Treatment of both CD4+ and CD8+ T-cells with a CB2-selective agonist inhibited the MLR, though significantly less than when both cell types were treated. T-cell function was decreased by CB2 agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly reduced in the cannabinoid treated cells. Further, treatment with O-1966 dose- dependently decreased levels of the active nuclear forms of the transcription factors NF- kappa-B and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures, generated using a PCR T-cell activation array, showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. An increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures and pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-dependent decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. The ability of O-1966 treatment to block rejection of skin grafts in vivo was also tested. Mice received skin grafts from a histoincompatible donor, and the time to graft rejection was analyzed. Compared to mice that received the vehicle, mice that received O-1966 treatment had significantly prolonged graft survival and increased Tregs in the spleen. The spleen cells from O-1966-treated mice had reduced proliferation in an MLR and an increased percentage of Tregs. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients and possibly as a new class of immunosuppressive drugs.