A short course of methylprednisolone immunosuppression inhibits both rejection and spontaneous acceptance of rat liver allografts

BACKGROUND: The effects of immunosuppressive drugs on transplant tolerance have not been extensively studied, although their effect on rejection is well established. METHODS: We examined the effects of a short course of treatment with the immunosuppressive drug methylprednisolone (MP) on the survival of PVG liver allografts in Dark Agouti (DA) recipients that accepted the livers and in Lewis recipients that rejected the livers. Infiltration of liver allografts was examined by immunohistochemical staining of liver sections, and apoptosis was measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling. RESULTS: A 5-day course of MP (days 0 to 4) led to rejection of four of six livers (mean survival time [MST] 99 days) in DA recipients compared with long-term survival (MST >100 days) in untreated animals. Delayed administration of MP (days 3 to 7) exacerbated rejection in DA recipients, and all eight animals rejected the graft (MST 68.5 days). Treatment of Lewis recipients with MP did not significantly prolong survival when administered from days 0 to 4 (MST 13 days), although delay of administration improved the outcome. Treatment from days 3 to 7 resulted in an MST of 21 days, whereas treatment from days 7 to 11 resulted in an MST of 41.5 days. MP treatment from day 3 to day 7 reduced T cells and interleukin 2 receptor-expressing cells but increased the numbers of apoptotic cells infiltrating both DA and Lewis strain allografts. CONCLUSIONS: These results show that immunosuppression with MP inhibits both spontaneous tolerance and rejection of liver allografts in a rat model and question the efficacy of administering MP to all liver allograft recipients from the time of transplantation.     

Expression of CD44 in rat liver allografts during rejection

As CD44 is believed to be a homing receptor involved in lymphoid trafficking and inflammatory responses, it is expected to be closely linked to transplant rejection. In this study, the expression of CD44 during liver transplant rejection was compared with the expression of lymphocyte-function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), which play an essential role in cell interactions and the initiation of immune responses. Male Brown Norway (BN) and Lewis (LEW) rats were used as donors and recipients, respectively. Orthotopic liver transplantation (OLTX) was done using the cuff technique of Kamada and Calne. Animals were killed on days 3, 5, and 7 after OLTX, and a piece of tissue from each of the liver grafts was obtained. Immunohistochemical staining was used to investigate the expression of CD44, ICAM-1, and LFA-1. CD44 was strongly expressed in portal areas of the rejected liver, and LFA-1 and ICAM-1 were expressed mainly on sinusoids and hepatocytes. These findings indicate that CD44 is closely involved in lymphocyte infiltration, which is dominant in portal areas, and that lymphocyte infiltration during the rejection process may involve a homing mechanism. Received for publication on Oct. 17, 1997; accepted on Oct. 21, 1997     

Regulatory cells develop after the spontaneous acceptance of rat liver allografts

BACKGROUND: After donor-specific transfusion, tolerance to heart transplants is serially passed to naive rats by the adoptive transfer of long-term survivor (LTS)-tolerant splenocytes (SC). We examined whether regulatory cells similarly develop after the spontaneously accepted Lewis (LEW) to Dark Agouti (DA) liver transplants. METHODS: SC from a LTS DA rat with a LEW liver were adoptively transferred to a naive DA 1 day before transplantation of an irradiated (1000 rad) LEW liver. RESULTS: Untreated LEW to DA liver allografts were uniformly accepted; whereas all irradiated LEW liver grafts were rejected. In contrast, when 1.5 x 10 8 DA LTS SC were transferred to a naive DA recipient, all irradiated LEW liver grafts were accepted. When decreased to 1.0 x 10 8 LTS DA SC, only 1 of 4 irradiated LEW grafts was accepted. However, if 1.5 x 10 8 DA SC harvested only 30 days after liver transplantation were transferred, only 2 of 5 irradiated LEW liver grafts were accepted. The serial second and third adoptive transfers of 1.5 x 10 8 DA LTS SC also resulted in the uniform acceptance of irradiated LEW livers. CONCLUSION: Regulatory cells that develop after the spontaneous acceptance of a LEW to DA liver transplant can serially transfer tolerance to new naive LEW liver allograft DA recipients. This "infectious tolerance" is dependent on the time of cell harvest after transplantation and on the cell dose given.     

Long-term acceptance of porcine pulmonary allografts without chronic rejection

OBJECTIVES: The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. Using a strategy to induce tolerance across strong allogeneic barriers, we have employed a brief, intensive course of immunosuppression to determine whether the induction of donor-specific hyporesponsiveness would prevent allograft rejection in a preclinical model of lung transplantation using MHC-inbred miniature swine. METHODS: Orthotopic left lung allografts were performed using MHC class I-disparate donors. The recipients received a 12-day postoperative course of cyclosporine (n = 6) or a 12-day postoperative course of high-dose tacrolimus (n = 3) as their only immunosuppression. Control animals received no immunosuppression (n = 3). RESULTS: Cyclosporine-treated recipients exhibited graft survival ranging from 67 to >605 days. All six animals developed acute cellular rejection between postoperative days (PODs) 27 and 108. Two animals lost their grafts on PODs 67 and 69, before developing obliterative bronchiolitis (OB). The other four recipients developed OB between PODs 119 and 238. In contrast, all tacrolimus-treated recipients maintained their grafts long term, without developing chronic rejection (>339, >308, and >231). These recipients also exhibited donor-specific hyporesponsiveness in assays of cell-mediated lymphocytotoxity. All untreated control animals lost their grafts to acute rejection by POD 11. CONCLUSIONS: This study demonstrates the ability of a brief course of high-dose tacrolimus to induce long-term graft acceptance with donor-specific hyporesponsiveness in a class I-disparate preclinical lung transplant model.     

Cyclosporin, toxicity and efficacy in rejection of liver allografts in the rat

52 orthotopic liver transplants were performed in DA to lewis rat strain combination, in order to appreciate cyclosporine toxicity, and efficacy at doses of 10 mg/kg day (G II) and 20 mg/kg/day (GIII) compared to liver allografts in DA/lewis rats. The first signs of cyclosporine hepatotoxicity are biological (increased plasma level of bilirubine and transaminase) that were noticed at the dose of 20 mg/kg/day. Histological signs (cells inclusion, hepatocytic necrosis) appeared late and were less constant as well as difficult to assert creatinine plasma level was the best reflect of cyclosporine nephrotoxicity. Renal toxicity was practically constant at the dose of 20 mg/kg/day. In spite of renal and hepatic toxicity, cyclosporin by itself, allows the abolition of the acute rejection of liver allografts in the rat.     

Humoral immunity in allograft rejection. The role of cytotoxic alloantibody in hyperacute rejection and enhancement of rat cardiac allografts

The role of humoral immunity in graft rejection in the rat model remains controversial. Passive transfer of cytotoxic alloantibody (CAA) has resulted either in hyperacute rejection or in graft enhancement. This study examines the effect of transfer of CAA on cardiac allograft survival in three rat strain combinations that are fully mismatched at the major histocompatibility (MHC) loci. Strain-specific immune responsiveness in donor-recipient pairs varied from low (Lewis-to-ACI) to high (ACI-to-Lewis) as measured by mixed lymphocyte reactions. CAA was obtained from rats sensitized by three successive skin grafts at weekly intervals. Group 1 (high responder recipients), which consisted of Lewis rats presensitized to ACI and had a lymphocytotoxicity titer of 1:512 to 1:2048, rejected ACI cardiac allografts in 10.8 +/- 7.2 hr compared with 6.5 +/- 0.5 days in naive controls (p less than 0.001). Injection of 1 ml of high-titer CAA into naive Lewis rats immediately after ACI cardiac grafting led to hyperacute rejection of ACI hears in 2.1 +/- 0.8 hr while 1 ml of CAA followed by 2 ml of guinea pig complement (GPC) resulted in even faster rejection (mean survival time (MST) of 23.8 +/- 4.7 min). Injection of 2 ml GPC alone or in combination with 1 ml naive Lewis serum had no effect on graft survival. Multiple pretransplant injections of 1 ml of CAA on days -3, -2,-1, and 0 relative to transplantation resulted in significant prolongation of allograft survival (MST of 10.3 +/- 0.3 days; P less than 0.01). In group 2 (intermediate responder recipients), where Lewis rats were presensitized to WF strain and where cytotoxicity titer was 1:16 to 1:256, the recipients rejected WF hearts in 23.8 +/- 5.8 hr compared with 6.8 +/- 0.8 days in unsensitized control recipients (P less than 0.001). Injection of 1 ml of Lewis anti-WF CAA resulted in prolonged graft survival of 9.7 +/- 3.5 days, while injection of 1 ml of CAA followed by 2 ml of GPC caused hyperacute rejection in 104 +/- 61.7 min. Pretransplant injections of CAA on days -3, -2, -1, and 0 resulted in enhancement, with an MST of 16.3 +/- 1.3 days (P less than 0.001). In group 3 (low responder recipients), ACI presensitized to Lewis developed a cytotoxicity titer of 1:2 to 1:32 and rejected Lewis hearts in 5.3 +/- 0.4 days compared with 10.6 +/- 1.0 days in naive recipients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antiviral and immunomodulatory effects of desferrioxamine in cytomegalovirus-infected rat liver allografts with rejection

BACKGROUND: Cytomegalovirus (CMV) infection is associated with acute and chronic allograft rejection. We have recently shown that rat CMV increases portal inflammation and bile duct destruction in a model of rat liver allograft rejection. Desferrioxamine (DFO), an iron chelator and antioxidant, has recently been demonstrated to have antiviral as well as immunomodulatory effects in vitro. We therefore investigated whether DFO inhibits (a) CMV infection and (b) graft destruction in our rat model. METHOD: One day after liver transplantation, PVG (RT1c) into BN(RT1n), the rats were infected with rat CMV (RCMV, Maastricht strain; 10(5) plaque-forming units i.p.). The effects of 100 mg/kg body weight and 200 mg/kg body weight DFO were examined. RESULTS: In the untreated group, the grafts were uniformly RCMV culture-positive. In the group receiving 200 mg/kg DFO, RCMV replication was effectively inhibited. Inflammatory response in the graft, and especially the number of macrophages, was significantly reduced by DFO. Portal inflammation and bile duct destruction were also significantly reduced. In the untreated group, the bile duct epithelial cells were found to be strongly positive for tumor necrosis factor-alpha and this expression was clearly decreased by DFO. In addition, DFO significantly inhibited vascular cell adhesion molecule-1 expression on sinusoidal endothelial cells. CONCLUSIONS: Our in vivo transplant study strongly supports the inhibitory effects of metal chelators on CMV infection and their possible usefulness in the treatment of CMV-induced pathogenic changes.     

The potent role of graft-derived NKR-P1+TCRalphabeta+ T (NKT) cells in the spontaneous acceptance of rat liver allografts

BACKGROUND: The mechanism involved in the spontaneous acceptance of liver allografts in some rat strain combinations remains unclear. Immunoregulatory NKR-P1TCRalphabetaT (NKT) cells primarily produce IL-4 and IFN-gamma, and enhance the polarization of immune responses to Th2 and Th1, respectively. The aim of this study was to clarify the role of graft-derived NKT cells in inducing the spontaneous acceptance of rat orthotopic liver transplantation (OLTx) METHODS: The experimental groups were divided as follows: Group 1, BN to LEW "low responder (acceptor)" combination; Group 2, DA to LEW "high responder (rejector)" combination; na?ve BN (Group 3) or LEW recipients (Group 4) with liver allografts from irradiated BN donors. The recipients had liver allografts from irradiated donors reconstituted from the following cell populations 24 hr before harvesting, spleen cells (SPCs, Group 5), IgSPCs (Group 6), IgNKR-P1SPCs (Group 7), and IgTCRabSPCs (Group 8) RESULTS: In Group 1, the percent of graft-derived NKT cells harvested on day 7 posttransplant were significantly higher than in Group 2. In the case of BN liver allografts that had been irradiated and reconstituted with cell populations including NKT cells (Groups 5 and 6), the mean graft survival (MST) was extended to 39.2+/-5.7 and 38.8+/-8.0 days, respectively. In contrast, when NKT cells were excluded (Groups 7 and 8), the grafts were acutely rejected within MST of 17.8+/-4.0 and 18.8+/-7.7 days, respectively. The concentrations of IL-10 and TGF-beta, but not IL-4 in IgGICs culture supernatants were predominant in the acceptor, whereas those with IFN-gamma predominated in the rejector. CONCLUSIONS: Graft-derived NKT cells might be responsible for spontaneous acceptance in the rat OLTx.     

Vascular rejection and graft eosinophilia in rat lung allografts

Allogeneic lung transplantation was performed using a rat model in order to assess the pathologic changes that developed during the process of rejection. The left lungs of 38 BN rats (RT-1n) were orthotopically transplanted into LEW rats (RT-1). The allografts developed the well-known changes of perivascular, peribronchial, and interstitial lymphocytic infiltrates resulting in necrosis of the pulmonary parenchyma at 7-8 days after transplantation. In addition, we document two findings that have not been reported previously in lung transplantation: vasculitis and eosinophilic infiltrates. Vasculitis with swelling and vacuolization of the endothelial cells was observed in transplants as early as 3 days following transplantation. Vasculitis with fibrinoid necrosis of the vessel wall was prominent at 7-8 days after grafting. The allografts also exhibited eosinophilia at 2 to 4 days following transplantation. The density of eosinophils in the inflammatory infiltrate reached a peak of 20% on Day 3 post-transplantation. These findings suggest an important role of humoral immunity and a possible involvement of eosinophils in lung allograft rejection.     

Increased risk of antibody-mediated rejection of reduced-size liver allografts

Because of the shortage of liver allografts in children, transplantation of reduced-size liver allografts from adult cadaveric donors or living, related donors is being done more frequently. Reduced-size liver allografts may be used in cases of ABO incompatibility and T-cell warm cross-match positivity. This experimental study in inbred rats was undertaken to determine if reduced-size liver allografts are more sensitive to antibody-mediated rejection than full-size liver allografts. Brown-Norway (BN) (RT1(n)) rats were sensitized by three successive skin grafts at 10-day intervals. Then orthotopic Lewis (LEW) (RT1(1)) liver grafts were transplanted into these BN rats. Full-size liver allografts were compared with reduced-size liver allografts (70% of donor liver). Control groups were composed of full-size and/or reduced-size isografts. Titers of specific antibodies were assayed using a complement-dependent assay before and after orthotopic liver transplantation. Histological and immunofluorescence studies (IgG, IgM, C(3), and fibrinogen deposits) were assessed. Recipients of reduced-size liver allografts died of hyperacute rejection at 36.6 +/- 4.1 h, significantly earlier than recipients receiving full-size liver allografts, which died of accelerated acute rejection at 259.2 +/- 25.2 h (P < 0.001). Either full-size or reduced-size isograft recipients survived indefinitely. A decrease in the titers of donor-specific antibodies was observed in both groups of animals. Slight deposits of IgG, IgM, C(3), and fibrinogen were observed in recipients of reduced-size liver allografts, whereas larger deposits were observed in recipients of full-size liver allografts. Our data demonstrate that there is an increased risk of antibody-mediated rejection of reduced-size liver allografts in sensitized recipients. This may have important clinical implications for partial liver grafting in cases of ABO incompatibility and T-cell warm cross-match positivity.