FK506对大鼠离体肾细胞凋亡及bcl-2蛋白、bcl-2 mRNA表达的影响

目的观察在含FK506的肾保存液保存的大鼠离体肾脏中bcl-2蛋白、bcl-2mRNA表达及肾细胞凋亡。探讨FK506对离体肾的保护的作用。方法建立离体大鼠肾脏模型，试验组以含FK506的4℃uw保存液保存大鼠离体肾脏，采用免疫组织化学、逆转录-聚合酶链反应（RT-PCR）和细胞凋亡原位末端标记技术，分别检测肾脏保存2、4、8、16h组bcl-2蛋白、bcl-2-mRNA表达。肾细胞凋亡，对照组以不含FK506的4℃ UW保存液保存离体肾脏。结果FK506保存组4、8、16h组bcl-2蛋白、bcl-2mRNA表达明显高于对照组（P〈0．05）；FK506保存组8、16h组肾细胞凋亡指数明显低于对照组（P〈0．05）。结论含FK506肾保存液保存的离体肾脏中bcl-2蛋白、bcl-2mRNA表达增强。肾细胞凋亡指数降低。FK506对肾脏保存过程中的损伤具有保护作用。     

Distribution characteristics of immunosuppressants FK506 and cyclosporin A in the blood compartment

Using two representative immunosuppressants, FK506 (FK) and cyclosporin A (CyA), of which the mechanism of pharmacological action is the same although there is a great difference in the pharmacological intensity, the distribution characteristics were studied in both in vivo and in vitro experiments using rat, dog, and human blood. Blood samples were fractionated by means of sedimentation in Ficoll-Paque®, and the drug contents in the diluted plasma fraction, erythrocyte fraction, and lymphocyte fraction were measured by an HPLC method. FK distributes to the lymphocyte fraction to a level about three times greater than that of CyA, while CyA distributes to the erythrocyte fraction to a level ten times that of FK. The distribution pattern of these fractions was independent of the drug concentration and species after correcting the drug concentration in each fraction with the blood drug concentration. The uptakes of FK and CyA in the isolated lymphocytes obtained from the rat spleen and human peripheral blood were also studied. The amount of FK taken up by the spleen lymphocytes is five times greater than that of CyA. In the case of the uptake study using human peripheral blood lymphocytes, the concentration of FK in the lymphocyte is 100-fold higher than that of CyA. This difference in the lymphocyte level between the two immunosuppressants is thought to be one of the reasons why FK is more potent than CyA, a difference of about 100-fold in the in vitro pharmacological study and about tenfold in the in vivo organ transplantation experiments.     

Differential Effect of Diarrhea on FK506 Versus Cyclosporine A Trough Levels and Resultant Prevention of Allograft Rejection in Renal Transplant Recipients

Diarrhea is the most frequently reported adverse event in patients treated with mycophenolate mofetil. Twenty-six renal transplant patients on a mycophenolate mofetil-based immunosuppressive regime with persistent afebrile diarrhea were examined. Diarrhea caused a significant rise in FK-506 trough levels despite intake of stable doses, necessitating FK-506 dose reductions of 30% to obtain pre-diarrhea trough levels. In contrast, trough levels of cyclosporine A remained stable without dose adjustments. This suggests that absorption and/or metabolism is differentially altered for FK506 compared with cyclosporine A in patients with diarrhea. In nine patients mycophenolate mofetil was reduced or stopped because of persistent diarrhea without identifiable cause. This resulted in end-stage renal disease because of chronic rejection in two patients, and in acute rejection in two patients, all taking FK506 and steroids. Therefore, dose adjustments of FK506 in patients with diarrhea must be carefully monitored, especially when doses of mycophenolate mofetil are also reduced.     

Activation of MAP kinases,pp90rsk and pp70-S6 kinases in mouse mast cells by signaling through the c-kit receptor tyrosine kinase or FcϵRI: rapamycin inhibits activation of pp70-S6 kinase and proliferation in mouse mast cells

The high-affinity receptor for IgE, Fc_?RI, represents the major cell surface structure through which mast cells express immunologically specific secretory function. By contrast, the stem cell factor receptor (SCFR), which is encoded by c-kit, is essential for normal mast cell development. The signaling pathways initiated by the stimulation of mast cells through the Fc_?RI, which lacks intrinsic kinase activity, and the SCFR, a member of the receptor tyrosine kinase family, generally have been regarded to be distinct. We report here that mouse mast cells stimulated either with SCF or with IgE and specific antigen exhibit a remarkably similar pattern of activation of mitogen-activated protein kinases (MAPK), 90 kDa-S6 kinases (pp90^rsk), and pp70-S6 kinases (pp70-S6K). These results indicate that all three families of protein kinases are associated with the cell surface receptor-dependent activation of secretion, as well as proliferation, in mast cells. We also show that the immunosuppressant rapamycin, but not FK506, can inhibit both SCF-dependent pp70-S6 kinase activation and SCF-dependent proliferation in mouse mast cells, without suppressing IgE- and antigen-dependent mediator release. These findings suggest that the activation of pp70-S6 kinase represents an important link in the stimulation of cell proliferation by SCF. Our results also indicate that the intracellular signaling pathways initiated by stimulation of mast cells through the Fc_?RI or the SCFR exhibit more overlap than has previously been appreciated.     

Reduction of FK-506 requirements by combination with polyethylene glycol superoxide dismutase in orthotopic rat liver transplantation

Reperfusion after ischemia results in endothelial cell injury and Kupffer cell activation. Inflammatory cytokines thus released can induce major histocompatibility complex antigens and increase the immunogenecity of the graft. An orthotopic rat liver allotransplant model was used to test the hypothesis that prevention of reperfusion injury by infusion of polyethylene glycol superoxide dismutase (PEG-SOD) would result in long-term allograft survival in the presence of subthreshold immunosuppressive dosages. ACI rats were used as donors, and Lewis strain rats as recipients. Orthotopic liver transplantation was initially performed to identify a subthreshold dose of the immunosuppressant FK-506, which would be unable to extend survival longer than control untreated rats with this strain combination. After testing three intramuscular FK-506 doses of 0.04, 0.08, and 0.16 mg/kg, it was observed that an FK-506 dose of 0.04 mg/kg/day for 14 days was unable to extend survival longer than in untreated recipients. This dose of FK-506 was used in combination with PEG-SOD at doses of 1000, 3000, 10,000, or 30,000 units. Recipient animals were treated intravenously with PEG-SOD as a loading dose to facilitate tissue penetration on day 1, and beginning on the day of transplantation, every 2 days for the duration of the study. Results of histologic studies and mean survival time were compared in untreated recipients and in rats treated with PEG-SOD plus 0.04 mg/kg/day FK-506. Mean survival time was increased significantly in these animals (p < 0.007) to 40.6 ± 25.6 days as compared with either untreated rats (10.0 ± 2.7 days) or rats treated with 0.04 mg/kg FK-506 alone (13.7 ± 4.2 days). Histologic examination demonstrated a significant reduction in the cellular infiltrate in rats treated with PEG-SOD plus FK-506, as compared with recipients treated with either agent alone or left untreated. Our results therefore suggest a potential approach to reducing immunosuppression in transplantation. (J ALLERGY CLIN IMMUNOL 1995;95:1276-81.)     

Anti-arthritic properties of FK506 on collagen-induced arthritis in rats

Objective and design:To determine the effect of FK506 (tacrolimus) on paw inflammation, TNF- expression in joint, and bone and cartilage destruction in type II collagen-induced arthritis (CIA) model in rats.Methods:CIA was induced by immunization of female Lewis rats with an emulsion of bovine type II collagen and incomplete Freunds adjuvant. Paw inflammation was assessed by the increase in paw volume. Tumor necrosis factor (TNF) - expression in hind knee joint was assessed by immunohistochemical analysis. Lesions of bone and cartilage were assessed on the basis of histological change in knee joint, radiographic analysis in hind paw, bone mineral density in femora and proteoglycan contents in the cartilage of femoral heads. FK506 at doses of 1, 1.8 and 3.2 mg/kg or its placebo formulation was orally administered to rats for 28 days from the day after immunization (n = 10). Effect of FK506 was compared with that of vehicle (distilled water).Results:FK506 at a dose of 1.8 mg/kg significantly suppressed paw swelling (p < 0.01) and histological change in knee joint (p < 0.05). Tumor necrosis factor (TNF)- was mainly expressed in the region with a marked infiltration of inflammatory cells in the hind knee joint. FK506 (3.2 mg/kg) markedly reduced TNF- expression. FK506 at a dose of 1.8 mg/kg suppressed radiographic changes in hind paw (p < 0.05) and also recovered the decrease in bone mineral density in the femora (p < 0.05). Proteoglycan contents in the cartilage of femoral heads were determined to evaluate the cartilage destruction more quantitatively and found to significantly decrease in CIA rats. FK506 at a dose of 1.8 mg/kg recovered the loss of proteoglycan contents (p < 0.01).Conclusion:These results show that FK506 is effective in suppressing inflammation, TNF- expression in joint, and damage to bone and cartilage in rat CIA, and may be useful in the treatment of rheumatoid arthritis.     

Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: Effect of FK506, an inhibitor of T cell activation

Objective and design: To characterize rat collagen-induced arthritis (CIA) on the basis of levels of inflammatory cytokines, tumor necrosis factor (TNF)-, interleukin (IL)-1 and IL-6 in paw tissues, and further investigate the effect of FK506 (tacrolimus), a potent inhibitor of T cell activation, on cytokine levels.Methods: CIA was induced in female Lewis rats. The volume of hindpaws was measured before and after collagen immunization. TNF-, IL-1 and IL-6 levels in paw tissue extracts were determined by ELISA. Proteoglycan contents of cartilage in femoral heads was measured as an indication of cartilage destruction. To assess the effect of FK506 on inflammatory cytokine levels, rats were orally treated with 5 mg/kg of FK506 from days 14–21.Results: TNF- a level in paw tissues did not significantly change compared to levels found before collagen immunization, throughout development of CIA. In contrast, IL-1 and IL-6 levels in paw tissues significantly increased between day 14 and day 28 after collagen imuninization, when the arthritis was at a developed stage. Therapeutic treatment with FK506 reduced the elevated level of IL-6, but not IL-1, in paw tissue. FK506 treatment was effective in suppressing paw swelling and also recovering the loss of proteoglycan contents in the cartilage.Conclusions: Levels of IL-1 and IL-6, but not TNF- , in paw tissue were upregulated in association with the development of arthritis in rat CIA. These results suggest that IL-1 and IL-6, rather than TNF- , may play important roles at local inflammatory sites in producing joint destruction in rat CIA. FK506 may improve arthritis in established stages of CIA, by reducing the elevated level of IL-6.Received 4 March 2004; returned for revision 2 April 2004; accepted by M. J. Parnham 9 April 2004     

Influence of FK506 and cyclosporin A on alloantibody production and lymphocyte activation following blood transfusion.

The effect of administration of cyclosporin A (CyA) or the novel macrolide FK506 was investigated in AO rats given DA blood transfusions. CyA (10 mg/kg, orally) or FK506 (1 mg/kg, intramuscularly) administered for 14 days from the time of transfusion effectively inhibited primary anti-MHC class I alloantibody production. This profound inhibitory effect persisted throughout the 2-month investigation period, with little increase in 'secondary' alloantibody production following a challenge injection 28 days after drug withdrawal. Flow cytometric analysis revealed no significant differences in the absolute numbers of W3/25+ (CD4+), OX-8+ (CD8+) or OX-12+ (B lymphocytes), in either the spleen or peripheral blood of transfused compared with normal, untreated animals. However, a small but significant increase in the numbers of splenocytes expressing the activation marker OX-40 (activated CD4+ cells) was observed in transfused animals. Either CyA or FK506 significantly reduced the number of cells expressing OX-39 (interleukin-2 receptors) and OX-40. Treatment of transfused animals with CyA, but not FK506 for 14 days resulted in minor, transient reduction in peripheral blood OX-19+ and W3/25+ cells, while 'sparing' the OX-8+ cells; these changes were not observed in spleens. In contrast, the absolute spleen cell numbers of OX-19+, W3/25+ and OX-8+ cells were significantly reduced in transfused animals given 14 days of FK506 treatment, while the corresponding blood cells were unaffected. Induction of splenic lymphoproliferative responses by the T cell mitogen concanavalin A remained normal in animals receiving transfusion alone or with CyA. In contrast, profound inhibition of mitogenic responses was observed in FK506-treated animals and this inhibitory effect declined gradually following drug withdrawal. No non-specific suppressor cell activity was detected in the spleens of rats given transfusion alone or in CyA or FK506-treated transfused animals.