Induction of lymphocyte apoptosis in rat liver allograft with ongoing rejection by FTY720

Abstract

SUMMARYThe action mechanism of FTY720, a novel immunosuppressant, is completely different from conventional immunosuppressants. The drug, which triggers apoptosis in murine and human lymphocytes, has a potent immunosuppressive activity to prevent allograft rejection without any severe side-effect. The present study was designed to determine whether FTY720 induces apoptotic cell death in activated lymphocytes infiltrated into liver grafts with ongoing rejection. FTY720 was orally administered at 5 mg/kg to the recipients on day 3 and day 4 after grafting, when the graft rejection was histologically confirmed. The intragraft patterns of IL-2, interferon-gamma (IFN-g), perforin, and granzyme B gene expression were detected by reverse transcriptase-polymerase chain reaction. The treatment reversed ongoing rejection and significantly prolonged recipient survival time compared with the control group. Light microscopic observation of the graft sections stained with the DNA nick-end labelling method showed that the apoptosis in the control allografts was mainly induced in hepatocytes, while that in the FTY720-treated allografts was in infiltrated lymphocytes. The rejection therapy with FTY720 did not alter the expression of IL-2, IFN-g, and perforin mRNAs, but slightly decreased granzyme B expression. Our results suggest that FTY720 does not alter the intrinsic lymphocyte function to produce the rejection-related cytokines, but strongly induces apoptotic cell death in the activated lymphocytes. Thus, FTY720 affords new insight into the mechanisms underlying improvements in immunosuppressive treatments.

Amelioration of Experimental Autoimmune Encephalomyelitis in Lewis Rats by FTY720 Treatment

“…As previously described (Li et al, 2001), we used Apop Tag Plus Kit (Oncor, Gaithersburg, MD), which uses certain reagents for nonisotopic DNA end-extension in situ and other reagents for immunohistochemical staining of the extended DNA technique, to detect DNA fragmentation. Briefly, we cut cryosections (6 m), fixed them in 10% neutral buffered formalin in a Coplin jar, and quenched them in 0.5 to 1% hydrogen peroxide in PBS for 5 min at room temperature.…”

“…We extracted total cellular RNA from frozen spinal cord and spleen tissue by using ISOGEN (Nippon Gene, Tokyo, Japan), as described previously (Li et al, 2001), and confirmed the RNA quality on formaldehyde-agarose gels. One microgram of total RNA was used for first-strand cDNA synthesis in 20 l of 100 mM Tris-HCl, 500 mM KCl, 5 mM MgCl 2 , 1 mM dNTP, 1 U/l RNase inhibitor, random 9-mer primer, and 0.25 U/l avian myeloblastosis virus reverse transcriptase (Takara, Shiga, Japan).…”

“…PCR products (10 l) were analyzed on 1 to 1.8% agarose gels. We visualized prominent bands of the correct size with ethidium bromide staining and measured the intensity of each band by a compact digital camera (DC40) with analysis software (BioMax 1D image analysis software; Eastman Kodak Co., Rochester, NY), as described in a previous article (Li et al, 2001). The relative quantities of genes are presented as the ratio between the intensity of IL-2, IL-6, or INF-␥ band and that of the housekeeping gene ␤-actin.…”

Induction of Lymphocyte Apoptosis in Rat Liver Allograft by Adenoviral Gene Transfection of Human Interleukin-10

“…Several in vitro studies have shown that the Fas/FasL pathway is not only involved in cytotoxicity of the target cell but also downregulates ongoing immune responses by acting on the T cell itself. This pathway is mainly responsible for activation-induced cell death to maintain the balance between T cell activation and inactivation [21,28,29] . T cell apoptosis plays a pivotal role in the allograft acceptance by inactivation and deletion of cytotoxic T cell clones, which result in transplant tolerance [30] .…”

“…To analyze the characterization of the isolated GIL, as described [21] , the cells were suspended in PBS containing 2% FCS and 0.1% sodium azide, and incubated for 1 h. 2 ! 10 6 cells were further incubated at 4 ° C for 30 min with a saturating concentration of FITC-conjugated mouse anti-rat R73 antibody and PE-conjugated mouse anti-rat CD45 antibody (BD Pharmingen, USA).…”