Rapamycin And Tacrolimus Differentially Modulate Acute Graft-Versus-Host Disease In Rats After Liver Transplantation

IntroductionAcute graft-versus-host disease(aGVHD) is an uncommon(1-2%) complication of liver transplantation(LTx),which is associated with a high mortality(85 to 90%). aGVHD poses major diagnostic and therapeutic challenges.Most patients with aGVHD after LTx develop overwhelming sepsis,marrow aplasia and multi-organ failure. Current therapeutic approachcs for aGVHD after LTx are controversial.They include: increasing the dosage of immunosuppressive drugs;decreasing the dosage of,or discontinuing,immunosuppressive drugs;administering anti-lymphocyte therapy,such as anti-thymocyte globulin,anti-lymphocyte globulin or the monoclonal anti-lymphocyte agent OKT3;administering anti-interleukin-2 receptor antibodies,such as daclizumab or basiliximab;and infusing host immune cells.The precise mechanisms responsible for the development of aGVHD after LTx are uncertain.Recent literature on this condition is limited to reports of individual patients or small groups of patients.The development of a stable,reproducible model of aGVHD after LTx would appear to be desirable to facilitate determining whether changes in the status of the immune system occur that may contribute to its pathogenesis and developing new approaches that may be effective in its treatment. Over a period of several years,we have developed a model of aGVHD following LTx in the donor-dominant one-way MHC matching rat.This model has been shown to be associated with a relative decrease in treg cell.The aim of this study was to investigate the effects of FK506 and RAPA on the rat model of aGVHD after LTx and to explore associated changes in the status of the regulatory T cells that may have pathologenic relevance.Materials and methods1.Animals and experimental groups:Donors were male Lewis rats.Recipients were male(Lewis♀X BN♂) F1(RT1^1/n) rats.Freshly prepared Lewis splenocytes were injected into(LewisXBN) F1 recipients via the dorsal penile vein immediately after LTx(within 30 minutes).4X10⁸ splenocytes were infused into each rat.Experimental animals were divided into three groups:(1) Control group of F1 rats that received no treatment;(2) FK506 group,in which FK506,1 mg/kg per day,was administered by gastric perfusion from days 8 to 15 after LTx to achieve whole-blood trough concentrations of approximately 10-12 ng/mL.FK506 was diluted in vehicle containing PBS.(3) RAPA group,in which RAPA,1 mg/kg per day,was administered by gastric perfusion from days 8 to 15 after LTx to achieve whole-blood trough concentrations of 2-3 ng/mL.2.Assessment of aGVHD(1) Clinical course and survival:All animals were assessed twice daily for signs typical of aGVHD,such as dermatitis,alopecia,weight loss,diarrhea,hunched posture and cachexia.The actuarial survival rate and mean time to death(mean survival time, MST) were calculated after a period of observation of 100 days.(2) Morphometric and histopathologic investigations:Tissue samples were taken at the time of death or at the time of sacrifice on day 16 after LTx.Samples of skin and intestine were subjected to microscopic evaluation.3.Analysis of CD4+CD25+ regulatory T cells in the peripheral PBMCsAfter the isolation of PBMCs from peripheral blood,the count of CD4+CD25+ regulatory T cell was analyzed by Flow Cytometry4.Immunohistochemical SABC methodImmunohistochemical SABC method was used to detect the Foxp3 expression level of monoclear cells in the intestin,moreover,to analysis the correlation between the expression level of Foxp3 and the survival rate.5.Statistical analysisAll data were analyzed using SPSS software(version 15.0 for Windows) and were expressed as means±standard deviations.Survival analysis and inter-group comparisons were evaluated by ANOVA followed by determination of the least significant difference(LSD).A P value of＜0.05 was considered to be statistically significant.ResultsClinical course and pathology of aGVHDIn the rat model all recipients of a liver allografts developed aGVHD.The first features of aGVHD occurred 7 to 10 days after LTx,mild dermatitis,diarrhea appeared on the rats.Subsequently,allograft recipients in the control group began to get worse until die.In the EK506 and RAPA groups,in rats that survived more than 100 days, signs of aGVHD gradually abated;dermatitis,alopecia,diarrhea and cachexia resolved and body weight stabilized.However,rats in the FK506 and RAPA groups that died exhibited signs of the disease and body weight changes similar to those of allograft recipients in the control group.Histologic examination of the skin revealed pathologic features of aGVHD in the control group.In the epidermis and dermis of the control group there was an intense infiltrate of mononuclear cells;in the FK506 group there was a sparse infiltration of mononuclear cells in the skin;in the RAPA group there was no obvious mononuclear cell infiltrate in the skin.Mortality and survival timeIn the control group,all recipients died 19 to 29 days after LTx.The mortality in the FK506 group,which was 50%(4/8),was significantly lower than that in the control group.In the RAPA group,in which only one allograft recipient died,the mortality was significantly lower than that in both the control and FK506 groups.Immunohistochemical expression of Foxp3Expression of Foxp3 in intestinal tissue of the control,FK506,and RAPA groups was 0.9±0.4%,1.0±0.5%and 3.8±1.5%,respectively.Expression was significantly higher in the RAPA group than in either the control group or the FK506 group.Frequency of regulatory T cellsRegulatory T cells in the control group decreased to 3.1±0.4%and 2.0±0.4%of PBMCs by days 12 and 16 after LTx,respectively.Differences between the control and FK506 groups were not significant,but the percentages for the RAPA group were significantly higher than corresponding percentages in either the control or FK506 group. Conclusions1,RAPA administration significantly reduces mortality in a rat model of aGVHD after LTx.2,This effect of RAPA is associated with a greater increase in the percentage of PBMCs in peripheral blood and tissues that are CD4+CD25+Foxp3+ treg cells than occurs after administration of FK506 to the model.