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

Abstract

The 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-gamma), 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-gamma, 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.

Fig. 1

Immunohistochemical studies in the grafts on day 5 after grafting. (A) R73⁺ cells in the syngeneic graft (a,b), control allograft (c,d) and FTY720-treated allograft (e,f). (b,d,f) are enlargements of the boxed areas in (a,c,e), respectively. There were many mononuclear cells around the portal area on day 5 in the control allografts (c,d). In these allografts, we observed a large number of R73⁺ T cells (d). A slightly decreased number of R73⁺ cells were seen in the FTY720-treated allograft (e,f). Magnifications: (a,c,e) × 100; (b,d,f) × 400. (B) The ratio of R73⁺ cells was markedly low in the syngeneic grafts on day 5 (0·035 ± 0·008), whereas it was significantly higher (*P < 0·01) in the control allografts (0·725 ± 0·028) and the FTY720-treated allografts (0·675 ± 0·041). There was no significant difference between the two allografts groups.

Fig. 2

TUNEL staining study in the grafts on day 5 after grafting. (A) TUNEL stainings in the syngeneic graft (a,b), control allograft (c,d) and FTY720-treated allograft (e,f). (b,d,f) are enlargements of the boxed areas in (a,c,e), respectively. Apoptotic cells observed in the control allograft were seen in many hepatocytes, and those in the FTY720-treated allograft were mainly in the lymphocytes (d,f). Magnifications: (a,c,e) × 100; (b,d,f) × 400. (B) Ratio of apoptotic cells calculated in each sample (n = 3 in each group) day 5 after transplantation. The ratio was low in syngeneic grafts (0·025 ± 0·008), while it was significantly higher (*P < 0·01) in the control allografts (0·255 ± 0·026) and the FTY720-treated allografts (0·385 ± 0·051). There was significant difference (P < 0·01) of the ratios between control allografts and FTY720-treated allografts.

Fig. 3

Detection of apoptotic hepatocytes and graft-infiltrating lymphocytes (GIL) 5 days after grafting in the control and FTY720-treated allografted liver. A number of apoptotic cells were seen in the hepatic cells of the control allograft (arrows in (a,b)). The infiltration of mononuclear cells was prominent around the portal area in the FTY720-treated allografts. The double staining with R73 and TUNEL showed that the infiltrated R73⁺ cells had induced apoptosis in the FTY720-treated graft (arrows in (c,d)). (b,d) are enlargements of the boxed areas in (a,c) respectively. Magnifications: (a,b) × 100; (c,d) × 1000.

Fig. 4

Apoptotic cell death of the graft-infiltrating lymphocytes (GIL) detected by flow cytometry. (a) GIL isolated from the control allografts showed >70% of the cells were double-stained with R73 and CD45. (b) GIL from each group were stained with both Annexin V and propidium iodide (PI). The ratios of apoptotic cells at an early stage (Annexin V-positive but PI-negative cells) were 10·1% in the syngeneic grafts, 49·2% in the control allograft and 66·2% in the FTY720-treated allograft. Data are representative of two separate experiments.

Fig. 5

Reverse transcriptase-polymerase chain reaction (RT-PCR) for IL-2, IFN-γ, perforin, and granzyme B mRNAs in the grafts. (a) Representative data show the intensity of RT-PCR products obtained from liver samples in each group. The syngeneic graft showed no or low expression of mRNAs for IL-2, IFN-γ, perforin, and granzyme B during the observation period. mRNA expression for IL-2, IFN-γ, and perforin showed a similar up-regulation pattern in the allografts treated with or without FTY720, while granzyme B mRNA expression was slightly decreased in the FTY720-treated graft. (b) The intensity of each band was calculated using Kodak Digital Science 1D Image Analysis Software. The relative quantities of the genes are presented as the ratio of intensities of IL-2, IFN-γ, perforin, and granzyme B bands against those of housekeeping gene, β-actin. Data are representative of three independent experiments and indicate the mean ratio of triplicate results in each experiment.