Schrepfer S, Deuse T, Koch-Nolte F, Detter C, Reichenspurner H
Department of Cardiovascular Surgery, University Heart Center, Hamburg, Germany.
Transplant Proc. 2006 Apr;38(3):757-61. doi: 10.1016/j.transproceed.2006.01.058.
The new malononitrilamide FK778 is currently being evaluated as an immunosuppressant for organ transplantation. Its main mechanism is inhibition of a pivotal enzyme of pyrimidine biosynthesis. This report revealed new mechanisms of action on different cell types involved in acute and chronic allograft rejection.
Purified Brown-Norway rat aortic endothelial cell (EC) cultures were pretreated with several concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) stimulated with TNF-alpha was quantified by immunofluorescence. Purified Lewis rat lymphocytes (LC) incubated with FK778 were stimulated via TCR/CD28 signals, and CD25 expression was quantified using FACS analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte-EC interaction was assessed by micromanipulator-assisted single-cell adhesion assays. Finally, smooth muscle cell (SMC) proliferation and migration was analyzed. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade.
TNF-alpha stimulation and TCR/CD28 co-stimulation significantly increased EC ICAM-1/VCAM-1-expression and LC CD25 surface expression, respectively. These effects were dose-dependently inhibited by FK778 and were not reversed by the addition of uridine. FK778 dose-dependently attenuated LC adhesion to allogeneic EC. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation.
FK778 directly reduced endothelial adhesion molecule up-regulation, inhibited lymphocyte activation, and attenuated lymphocyte-endothelium interactions, critical early steps in graft rejection. These effects were separate from the blockade of pyrimidine synthesis. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection.
新型丙二腈酰胺FK778目前正作为一种用于器官移植的免疫抑制剂进行评估。其主要机制是抑制嘧啶生物合成的关键酶。本报告揭示了其对参与急性和慢性同种异体移植排斥反应的不同细胞类型的新作用机制。
用几种浓度的FK778预处理纯化的棕色挪威大鼠主动脉内皮细胞（EC）培养物。通过免疫荧光定量TNF-α刺激的内皮粘附分子表达（ICAM-1/VCAM-1）。用FK778孵育的纯化刘易斯大鼠淋巴细胞（LC）通过TCR/CD28信号进行刺激，并使用流式细胞术分析定量CD25表达。在所有试验中均添加尿苷以逆转嘧啶合成阻断。通过显微操作辅助单细胞粘附试验评估淋巴细胞与内皮细胞的相互作用。最后，分析平滑肌细胞（SMC）的增殖和迁移。在所有试验中均添加尿苷以逆转嘧啶合成阻断。
TNF-α刺激和TCR/CD28共刺激分别显著增加EC的ICAM-1/VCAM-1表达和LC的CD25表面表达。这些作用被FK778剂量依赖性抑制，并且添加尿苷不能逆转。FK778剂量依赖性减弱LC对同种异体EC的粘附。添加尿苷可消除FK778对SMC增殖的剂量依赖性抑制，而对SMC迁移的抑制作用不受尿苷补充的影响。
FK778直接降低内皮粘附分子上调，抑制淋巴细胞活化，并减弱淋巴细胞与内皮细胞的相互作用，这些是移植排斥反应中的关键早期步骤。这些作用与嘧啶合成阻断无关。FK778对SMC的抗增殖作用可能是抑制慢性器官排斥反应纤维增生性病变的重要机制。
