A Cannabinoid 2-Selective Agonist Inhibits Allogeneic Skin Graft Rejection In Vivo

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.

Keywords: CB2 agonist; T reg cells; graft rejection; immunosuppresion; mixed lymphocyte reaction (MLR).

FIGURE 1

O-1966 treatment prolongs skin graft viability. Donor C3HeB/FeJ flank skin was transplanted to the back of a recipient C57BL/6J mice, sutured, and bandaged. Doses of O-1966 or vehicle (0.03% ethanol and 0.03% cremophor in saline) were injected i.p. every other day, from 1 h pre-op to 14 days post-op. On day 7 bandages were removed and grafts were monitored for rejection. Percent graft survival of mice treated with (A) 1 mg/kg O-1966 (), or vehicle (), (B) 5 mg/kg O-1966 () or vehicle (), or (C) 10 mg/kg O-1966 () or vehicle (). Panels (A,C) are results of a single experiment (n = 8 per group), and data in Panel (B) are the mean of two experiments (n = 17 per group). Median survival time of vehicle vs. O-1966 treatment at 5 mg/kg, **p < 0.001 by the Log rank [Mantel-Cox] test.

FIGURE 2

O-1966 decreases spleen weights in skin graft recipient mice. Donor C3HeB/FeJ flank skin was transplanted to the back of a recipient C57BL/6J mice, sutured, and bandaged. Doses of O-1966 (5 mg/kg) or vehicle (0.03% ethanol and 0.03% cremophor in saline) were injected i.p. every other day from 1 h pre-op to 14 days post-op. On post-op day 14, animals were sacrificed and spleens were removed. The spleen weight to body weight ratio was calculated and is expressed as a percentage, of control mice (▲) (n = 9) and grafted mice treated with 5 mg/kg O-1966 (■) (n = 8) or vehicle (●) (n = 10). **p < 0.001 for O-1966 vs. vehicle as determined by one-way ANOVA followed by Tukey’s multiple comparison test.

FIGURE 3

O-1966 treatment increases percentage of splenic Tregs in skin graft recipient mice. Donor C3HeB/FeJ flank skin was transplanted to the back of recipient C57BL/6J mice, sutured, and bandaged. Doses of O-1966 (5 mg/kg) or vehicle (0.03% ethanol and 0.03% cremophor in saline) were injected i.p. every other day from 1 h pre-op to 14 days post-op. Splenocytes were harvested from grafted mice treated with O-1966 () or vehicle () on day 14 and were analyzed by flow cytometry for CD4⁺CD25⁺FoxP3⁺ Tregs (n = 17 for both groups). Data show number of Tregs as a percentage of total live CD4⁺ cells (LIVE/DEAD^® dead cell stain negative). Data are mean of two separate experiments. **p < 0.01 for O-1966 vs. vehicle as determined by one-way ANOVA followed by Tukey’s multiple comparison test.

FIGURE 4

O-1966 treatment decreases CD4 expression in skin graft recipient mice. Donor C3HeB/FeJ flank skin was transplanted to the back of recipient C57BL/6J mice, sutured, and bandaged. Doses of O-1966 (5 mg/kg) or vehicle (0.03% ethanol and 0.03% cremophor in saline) were injected i.p. every other day from 1 h pre-op to 14 days post-op. Splenocytes were harvested on day 14, stained for CD4, and analyzed by flow cytometry. (A) Representative histograms of CD4 expression on CD3⁺ cells from mice treated with O-1966 (gray filled) or vehicle (white filled). (B) Mean fluorescence intensity (MFI) of CD4 in CD3⁺CD4⁺ populations from mice treated with O-1966 () or vehicle (). Data are mean of two experiments (n = 17 for both groups). **p < 0.01 for O-1966 vs. vehicle by one-way ANOVA followed by Tukey’s multiple comparison test).

FIGURE 5

In vivo O-1966 treatment decreases proliferation and increases the percentage of Tregs following ex vivo stimulation. Donor C3HeB/FeJ flank skin was transplanted to the back of recipient C57BL/6J mice, sutured, and bandaged. Doses of O-1966 (5 mg/kg) or vehicle (0.03% ethanol and 0.03% cremophor in saline) were injected i.p. every other day from 1 h pre-op to 14 days post-op. On post-op day 14, animals were sacrificed and spleens were aseptically removed, restimulated with C3HeB/FeJ splenocytes and put into culture for MLR (A) or harvested at 48 h and analyzed by flow cytometry (B). (A) Proliferation of cultures with splenocytes from O-1966 treated mice () or vehicle treated mice (). (B) Cultures harvested at 48 h from mice treated with O-1966 () or vehicle () and analyzed by flow cytometry for CD25⁺Foxp3⁺ Tregs (n = 17 for both groups). Data show number of Tregs as a percentage of total live CD4⁺ cells (LIVE/DEAD^® dead cell stain negative). Data are mean of two separate experiments. *p < 0.05, **p < 0.01 for O-1966 vs. vehicle by one-way ANOVA followed by Tukey’s multiple comparison test).

FIGURE 6

In vivo O-1966 treatment decreases CD4 expression following ex vivo stimulation. Donor C3HeB/FeJ flank skin was transplanted to the back of recipient C57BL/6J mice, sutured, and bandaged. Doses of O-1966 (5 mg/kg) or vehicle (0.03% ethanol and 0.03% cremophor in saline) were injected i.p. every other day from 1 h pre-op to 14 days post-op. On post-op day 14, animals were sacrificed and spleens were aseptically removed, restimulated with C3HeB/FeJ splenocytes and harvested at 48 h and analyzed by flow cytometry for CD4 expression. (A) Representative histogram of CD4 expression on CD3⁺ cells in cultures of splenocytes from skin graft recipient mice treated with O-1966 (gray filled) or vehicle (white filled). (B) Mean fluorescence intensity (MFI) of CD4 in CD3⁺ populations in cultures from O-1966 treated mice () or vehicle treated mice (). Data are representative of two experiments (A) or mean of two separate experiments (B). *p < 0.05 for O-1966 vs. vehicle by one-way ANOVA followed by Tukey’s multiple comparison test).