DA Liver Research Articles

“Infectious tolerance” develops after the spontaneous acceptance of Lewis-to-Dark Agouti rat liver transplants

Background. After monoclonal antibody or donor-specific transfusion treatment, infectious tolerance to rat or mouse heart or skin transplants can be passed on to naive recipients by adoptive transfer of tolerant lymphocytes. We examined whether similar regulatory cells develop after the spontaneous acceptance of Lewis-to-Dark Agouti (DA) rat liver transplants without immunomodulating agents. Methods. After Lewis-to-DA rat liver transplantation, 100 × 106 splenocytes were harvested and adoptively transferred into a 450 rad-irradiated naive DA rat 24 hours before Lewis heart transplantation. Adoptive transfer of CD4+ or CD8+ T cells was also examined. In some experiments, splenocytes from recipients with long-term accepted Lewis hearts induced by adoptive transfer were serially transferred to multiple generations of recipients before Lewis rat heart transplantation. In vitro mixed lymphocyte culture response and cytotoxic T lymphocyte generation were measured. Results. When splenocytes from a DA rat recipient >60 days after Lewis rat liver acceptance were transferred into irradiated DA rat recipients, all Lewis rat hearts were accepted, whereas third-party Brown-Norway rat hearts were rejected. However, splenocytes from DA rat recipients 30 days after liver transplantation did not prolong Lewis rat heart survival. Adoptive transfer of 40 × 106 CD4+, 10 × 106 CD4+ or 10 × 106 CD8+ cells from a DA rat bearing Lewis rat liver >60 days resulted in acceptance of 88%, 80%, or 57% acceptance of Lewis rat hearts, respectively. Serial second and third adoptive transfer of long-term survivor splenocytes resulted in the acceptance of all Lewis rat hearts. In mixed lymphocyte culture, splenocytes from a naive DA rat and a DA rat accepting a Lewis rat liver transplant for >60 days showed similar proliferative responses to both Lewis and Brown-Norway rat stimulators. An equivalent level of indirect cytotoxic T lymphocyte activity was exhibited by splenocytes from both a naive DA and a DA rat accepting a Lewis rat liver transplant for >60 days. Conclusion. Regulatory cells developing after the spontaneous acceptance of a Lewis to DA liver transplant can serially adoptively transfer the acceptance of a Lewis rat cardiac graft in spite of the presence of in vitro antidonor reactivity. Both CD4+ and CD8+ populations have this regulatory activity, although the CD4+ population plays the dominant role. (Surgery 2003;134:227-34.)

The fas and fas ligand pathways in liver allograft tolerance.

The Fas and Fas ligand (Fas/FasL) pathways may play a central role in cytotoxicity or immunoregulation in liver transplantation. Here, in an attempt to examine the role of Fas/FasL on drug-free tolerance, we measured mRNA levels of Fas/FasL in livers by reverse transcriptase-polymerase chain reaction (RT-PCR), and also protein levels of Fas/FasL in livers by immunohistochemistry and in serum by dot blot assay. PVG recipients bearing DA livers showed serious rejection between post-operative (POD) days 7 and 14, but this rejection was naturally overcome without any immunosuppression. Fas gene and protein products were expressed on almost every cell in livers taken from naive rats, and at any time point in both syngeneic and allogeneic orthotopic liver transplantation (OLT) rats. In contrast, FasL mRNA in DA livers was detectable at POD 2, peaked at POD 14, and declined at POD 63 in allogeneic OLT (DA-PVG). Although the FasL gene was detectable in isografts at POD 14, its expression was much lower than in allografts. The time course and localization of FasL expression indicated that the expression of FasL gradually switched from infiltrating cells to hepatocytes when the rejection was naturally overcome and tolerance was induced in this OLT model. Soluble Fas could constitutively be detected at any time point in the serum of the tolerogenic OLT (DA-PVG) rats and was not diminished during the rejection phase. Soluble FasL peaked at POD 14 in allogeneic OLT, while sFasL was significantly lower in the serum of normal and syngeneic OLT rats. These findings suggest that the Fas and FasL pathways, including soluble forms, may contribute to the control of the immune response in this drug-free tolerance OLT model.

Expression of clusterin in a rat tolerogenic OLT model

IN RAT orthotopic liver transplantation (OLT), PVG recipients of DA livers (tolerogenic model) survive without immunosuppression and become tolerant of subsequent grafts of other DA organs such as skin, heart, and kidney. Many aspects of the mechanism regarding this naturally achieved tolerance have been discussed in our previous reports. However, the mechanism of liver allograft tolerance from the aspect of the complement system and its regulatory proteins has not yet been investigated. The complement system comprises a group of proteins that interact with other immune system molecules to provide many of the effector functions of humoral immunity and inflammation. Clusterin, which is a plasma glycoprotein, regulates the complement system by inhibiting the membrane attack complex (MAC) formation. Here, we studied the kinetics of clusterin expression after transplantation in the tolerogenic model (DA-PVG) compared with syngenic model (DA-DA) and acute rejector model (DA-LEW) by immunoblotting and Northern blotting.

NKR-P1+TCR????+ T (NKT) CELLS WERE INCREASED IN RAT HEPATIC ALLOGRAFT OF NATURAL ACCEPTOR AND REJECTOR

183 NKR-P1+TCRαβ+T (NKT) cells are mainly distributed at the sites such as bone marrow and liver. NKT cells play an important role in the development and regulation of immune responses. We analyzed the immunological characteristics of NKT cells in rat liver transplantation. Methods; In acutely rejected combination, LEW (RT11) rats rejected DA(RT1a) liver grafts within 11 days (REJ; n=5). In natural tolerant combination, LEW rats indefinitely accepted BN(RT1n) liver grafts, (TOL; MST>100 days, n=5). Also in this combination, 10 Gy irradiated BN rats were used for donors to deplete donor passenger leukocytes and 4 days after irradiation. BN livers were transplanted in the LEW recipients (IRR). Intragraft mononuclear cells (IGCs) were collected on day 7 and 10 after transplantation for flow cytometry with anti-NKR-P1(3.2.3), anti-TCRαβ(R73) and anti CD4/CD8 mAbs, and spleen T cells were suspended for MLR. Total RNA was extracted from liver allografts and RT-PCR was done to analyze cytokine gene expression in the grafts. Serum cytokines were measured by ELISA assay. Results;Flow cytometry; In phenotypic analysis of IGCs on day 7 after transplantation, populations of T cells and NKT cells were significantly higher in REJ than in TOL. The ratio of NKT/TCRαβ+T cells was significantly (p<0.05) lower in REJ than in TOL (Table). Donor type NKT cells remained in the graft with the ratio (donor NKT/recipient NKT) of 6.7±0.8% in TOL, and 3.9±0.5% in REJ on day 7. MLR; Spleen T cells obtained from TOL on day 7 after transplantation displayed to be less responsive (41146.4±4170.8 cpm) against donor antigens than those from naive LEW rats (101300.2±4979.2 cpm). RT-PCR; Th1 cytokines (IFN-γ) were strongly expressed in the REJ grafts. Th2 cytoknes (IL-4 and IL-10) were expressed both in the TOL and REJ grafts. Serum IL-4 and IFN-γ were higher in TOL (5.75pg/ml and 12.07pg/ml) than in REJ (1.65pg/ml and 4.52pg/ml) on day 7. By donor irradiation, liver mononuclear cells were depleted from 3.0×107 to 6.0×105 /liver, and the grafts were rejected in the MST of 24.3±3.8 days. And the ratio of NKT/αβT cells was lower than that of the TOL. Conclusions; Donor derived NKT cells may be responsible for the induction of tolerance by a deviation of Th2 reactivity.Table

IMMUNOLOGICAL CHARACTERISTICS OF INTRAGRAFT NKR-P1+TCRαβ+ T (NKT) CELLS IN RAT HEPATIC ALLOGRAFT OF NATURAL ACCEPTOR AND REJECTOR

269 NKR-P1+TCRαβ+T (NKT) cells are mainly distributed at the sites such as bone marrow and liver. NKT cells play an important role in the development and regulation of immune responses. We analyzed the immunological characteristics of NKT cells in rat liver transplantation. Methods; In acutely rejected combination, LEW (RT1l) rats rejected DA(RT1a) liver grafts within 11 days (REJ; n=5). In natural tolerant combination, LEW rats indefinitely accepted BN(RT1n) liver grafts, (TOL; MST>100 days, n=5). Also in this combination, 10 Gy irradiated BN rats were used for donors to deplete donor passenger leukocytes and 4 days after irradiation, BN livers were transplanted in the LEW recipients (IRR). Intragraft mononuclear cells (IGCs) were collected on day 7 and 10 after transplantation for flow cytometry with anti-NKR-P1(3.2.3), anti-TCRαβ(R73) and anti CD4/CD8 mAbs, and spleen T cells were suspended for MLR. Total RNA was extracted from liver allografts and RT-PCR was done to analyze cytokine gene expression in the grafts. Serum cytokines were measured by ELISA assay. Results;Flow cytometry; In phenotypic analysis of IGCs on day 7 after transplantation, populations of T cells and NKT cells were significantly higher in REJ than in TOL. The ratio of NKT/TCRαβ+T cells was significantly (p<0.05) lower in REJ than in TOL (Table). Donor type NKT cells remained in the graft with the ratio (donor NKT/recipient NKT) of 6.7±0.8% in TOL, and 3.9±0.5% in REJ on day 7. MLR; Spleen T cells obtained from TOL on day 7 after transplantation displayed to be less responsive (41146.4±4170.8 cpm) against donor antigens than those from naive LEW rats (101300.2±4979.2 cpm). RT-PCR; Th1 cytokines (IFN-γ) were strongly expressed in the REJ grafts. Th2 cytoknes (IL-4 and IL-10) were expressed both in the TOL and REJ grafts. Serum IL-4 and IFN-γ were higher in TOL (5.75pg/ml and 12.07pg/ml) than in REJ (1.65pg/ml and 4.52pg/ml) on day 7. By donor irradiation, the grafts were rejected in the MST of 24.3±3.8 days. And the ratio of NKT/αβT cells was similar to that of the TOL. Conclusions; Donor derived NKT cells may be responsible for the induction of tolerance by a deviation of Th2 reactivity.Table

Decreased expression of the interleukin 2 receptor on CD8 recipient lymphocytes in intestinal grafts rendered tolerant by liver transplantation in rats

Background—In a previous study, it was shown that a spontaneously tolerated DA (RT1a) liver allograft in a PVG (RT1c) recipient was able to induce tolerance of a DA small bowel...

THE ROLE OF EARLY IMMUNE ACTIVATION AND LEUCOCYTE APOPTOSIS IN INDUCTION OF SPONTANEOUS LIVER TRANSPLANT TOLERANCE

485 PVG → DA liver transplants (Tx) are spontaneously tolerated whilst kidney Tx in the same strains are rejected in 8.1±1.0 days (d). We have previously shown that the induction of tolerance in this model is consistent with a high dose or activation-associated mechanism. Peri-Tx administration of methylprednisolone (MPX) to rats receiving liver grafts interferes with the development of systemic tolerance. Grafted organs, spleens and draining lymph nodes were harvested from recipients of PVG → DA liver Tx, either unmodified or treated with MPX, from kidney recipients and from normal (N) and syngeneic (syn) controls at days 1-7 after Tx (n=3). We compared tempo of infiltration (using immunohistochemical staining to define cell subsets), cytokine mRNA expression (determined by quantitative RT-PCR) and apoptosis (TUNEL) between these groups. Liver allografts showed early but non-progressive cytokine upregulation with maximum intragraft expression of IL-2 and IFN-γ on d1 and d3 respectively. In contrast, renal Tx showed maximal expression of IL-2 on d3 and IFN-γ on d5. Similarly, early but abortive infiltration of liver grafts occurred. The ratio of IL-2R+ T cells to total T cells reached a peak of 54% on d3, declining rapidly by d5 (11%), whilst in renal Tx this ratio continued to increase and was 58% on d7. Apoptotic cells were significantly more numerous in liver (261±17) than in kidney (18±13 cells/mm2)(p<0.01), and were confirmed to be leucocytes in livers and tubular cells in kidneys by 2 color immunofluorescence. Cells of donor origin were present in much greater numbers in the spleen and LN of liver Tx recipients (548±119) than in those of kidney recipients (20±12 cells/mm2 in spleen)(p=0.01) on d1 and thereafter. At the same time there was strong, early upregulation of IL-2 and IFN-γ mRNA in lymphoid organs of liver but not kidney recipients. Significantly greater numbers of apoptotic cells were found in the splenic white pulp after liver Tx(247±6) compared with Kidney Tx (88±18)(P<0.01, d3). Double-staining showed many apoptotic cells in spleens of liver Tx recipients in close proximity to "migrant" donor leucocytes. This was not noted after renal Tx. Apoptosis of graft-infiltrating cells was significantly decreased in liver recipients treated with MPX (54±8 v/s 15±1 on d1, P<0.01) as was the number of apoptopic cells in spleen (216±7 v/s 77±2 on d1, P<0.01). In conclusion, induction of spontaneous liver Tx tolerance is associated with early immune activation in spleen and increased numbers of apoptotic cells in both splenic white pulp and graft infiltrate. Manipulation such as the administration of MPX, which interfere with activation of responding cells, both reduce apoptosis and break tolerance in this model, suggesting that activation and subsequent death of alloreactive cells may be important in the development of tolerance.

PROLONGED SURVIVAL OF RAT LIVER ALLOGRAFTS TRANSFECTED WITH FAS-LIGAND EXPRESSING PLASMID

524 The aim of this study is to evaluate the preventive effect of Fas-ligand transfected allogenic liver on graft rejection in rat. We constructed two expression vectors; pCAGGSFL to express rat Fas-ligand and pCAGGSLZ to express E. coli β-galactosidase (LacZ) under the control of chickenβ-actin promoter with CMV enhancer. These vectors were transfected into donor DA rat liver with an inactivated hemagglutinating virus of japan conjugated to liposome vesicles(HVJ-liposome). A couple of rats were sacrificed on 3,5,7 and 14 days after the HVJ-liposome injection, and analyzed the transduction efficiency with RT-PCR and X-gal staining. X-gal staining showed about 12% positive cells in liver sections obtained from the pCAGGSLZ transduced livers; Fas-ligand mRNA was detected by RT-PCR in pCAGGSFL transduced livers. These observations suggest that efficientin vivo gene transduction was achieved by using HVJ-liposome. Then, the genetic modified DA livers were transplanted into Lewis rats three days after HVJ-liposome injection, resulting in significant by (p<0.01) prolonged recipient survival time. Histological observation showed that pCAGGSFL-transfected liver allografts caused apoptotic cell death in infiltrating activated T cells. Based on these observations, we conclude that expression of Fas-ligand in donor liver probably acts to induce apoptotic cell death of recipient activated T cells, expressing a large amount of Fas antigens in response to donor graft antigens to reduce rejection induced in allogenic liver transplantation.

THE ROLE OF EARLY IMMUNE ACTIVATION AND LEUCOCYTE APOPTOSIS IN INDUCTION OF SPONTANEOUS LIVER TRANSPLANT TOLERANCE.

485 PVG → DA liver transplants (Tx) are spontaneously tolerated whilst kidney Tx in the same strains are rejected in 8.1±1.0 days (d). We have previously shown that the induction of tolerance in this model is consistent with a high dose or activation-associated mechanism. Peri-Tx administration of methylprednisolone (MPX) to rats receiving liver grafts interferes with the development of systemic tolerance. Grafted organs, spleens and draining lymph nodes were harvested from recipients of PVG → DA liver Tx, either unmodified or treated with MPX, from kidney recipients and from normal (N) and syngeneic (syn) controls at days 1-7 after Tx (n=3). We compared tempo of infiltration (using immunohistochemical staining to define cell subsets), cytokine mRNA expression (determined by quantitative RT-PCR) and apoptosis(TUNEL) between these groups. Liver allografts showed early but non-progressive cytokine upregulation with maximum intragraft expression of IL-2 and IFN-γ on d1 and d3 respectively. In contrast, renal Tx showed maximal expression of IL-2 on d3 and IFN-γ on d5. Similarly, early but abortive infiltration of liver grafts occurred. The ratio of IL-2R+ T cells to total T cells reached a peak of 54% on d3, declining rapidly by d5 (11%), whilst in renal Tx this ratio continued to increase and was 58% on d7. Apoptotic cells were significantly more numerous in liver (261±17) than in kidney (18±13 cells/mm2)(p<0.01), and were confirmed to be leucocytes in livers and tubular cells in kidneys by 2 color immunofluorescence. Cells of donor origin were present in much greater numbers in the spleen and LN of liver Tx recipients (548±119) than in those of kidney recipients (20±12 cells/mm2 in spleen)(p=0.01) on d1 and thereafter. At the same time there was strong, early upregulation of IL-2 and IFN-γ mRNA in lymphoid organs of liver but not kidney recipients. Significantly greater numbers of apoptotic cells were found in the splenic white pulp after liver Tx (247±6) compared with Kidney Tx(88±18)(p<0.01, d3). Double-staining showed many apoptotic cells in spleens of liver Tx recipients in close proximity to “migrant” donor leucocytes. This was not noted after renal Tx. Apoptosis of graft-infiltrating cells was significantly decreased in liver recipients treated with MPX (54±8 v/s 15±1 on d1, P<0.01) as was the number of apoptotic cells in spleen (216±7 v/s 77±2 on d1, P<0.01). In conclusion, induction of spontaneous liver Tx tolerance is associated with early immune activation in spleen and increased numbers of apoptotic cells in both splenic white pulp and graft infiltrate. Manipulations such as the administration of MPX, which interfere with activation of responding cells, both reduce apoptosis and break tolerance in this model, suggesting that activation and subsequent death of alloreactive cells may be important in the development of tolerance.

The prevention of graft-versus-host disease by the serum of liver retransplanted rats

The effect of serum from orthotopic liver retransplanted plants (re-OLT serum) on graft-versus-host disease (GVHD) was studied in rats. In the re-transplantation model of rat liver, orthotopic liver transplantation (OLT) was carried out in the DA (RT1 a) into PVG (RT1 c) combination; two days later the DA liver was removed and a new PVG liver implanted into the same recipient (re-OLT). In the in vivo GVHD model, male PVG rats were sublethally irradiated and injected intravenously with 3 × 10 8 DA or BN (RT1 n) spleen cells through the penial vein. Within 1 h of the inoculation, rats of the experimenta;l group were injected with 1 ml of re-OLT serum taken at postoperative day (POD) 7. Rats in the control group received 1 ml of normal PVG serum or syngeneic re-OLT inoculation of DA or BN spleen lymphocytes. However, when the animals were treated with re-OLT serum, 100% ( 6 6 ) of the rats survived more than 60 days, following inoculation with DA lymphocytes but now with BN lymphocytes. The POD 7 re-OLT serum showed a strong inhibition against DA anti-PVG mixed lymphocyte reaction (MLR), although re-OLT serum did not contain soluble DA class I antigens, anti-DA class I or II antibody. The potential GVHD inhibitory factors in re-OLT serum may be two unique immunosuppressive proteins, which have been detected by SDS PAGE and reported previously. We conclude that re-OLT serum has immunisuppressive factors, which, at least in part, prevented the induction of GVHD in rats.

Rejection of spontaneously accepted rat liver allografts with recipientinterleukin-2 treatment or donor irradiation.

While MHC incompatible DA (RTl(a)) to Lewis (RT1(1), LEW) rat liver allografts are acutely rejected, the reciprocal LEW to DA liver grafts are spontaneously accepted. The mechanism of this acceptance remains unclear. We evaluated the effect of donor treatment with total body irradiation (TBI) or gadolinium chloride (GdCl3), and recipient treatment with exogenous IL-2 after transplantation on the survival of the spontaneously accepted liver grafts. Male LEW and DA rats were used as donors and recipients for orthotopic liver allo- or iso-graft transplants. The LEW liver donor was treated by TBI (10 gray) 7 days before transplantation, or LEW donor Kupffer cell phagocytosis was blocked with GdCl3 (7 mg/kg) on days -2 and -1 pretransplant. In an attempt to reverse LEW liver graft acceptance, 180,000 units human IL-2 (hIL-2) were administered daily IP to the DA liver recipients from days 1 to 7 after liver grafting. While untreated LEW recipients rejected DA liver grafts within 13 days, DA recipients accepted LEW livers indefinitely (>302 days). In contrast, irradiation of the LEW liver donor prevented the spontaneous acceptance by DA recipients, and resulted in acute rejection of the liver grafts in 9-20 days. However, spontaneous graft tolerance was restored by parking the irradiated LEW donor liver in naive LEW rats for 48 hr before retransplantation to DA recipients (>50 days). When LEW donors were treated with GdCl3, which is known to block Kupffer cell phagocytosis and antigen processing, the spontaneous acceptance of the LEW liver grafts by DA recipients was unaffected. However, when exogenous rhIL-2 was given daily, LEW liver allografts were rejected by the DA recipients. The resulting liver failure correlated with a progressive increase in serum bilirubin and the development of a predominantly lymphocytic portal tract infiltration, bile duct epithelial damage, and portal vein endothelitis, which is consistent with acute allograft rejection. LEW and DA recipients of liver isografts developed no toxicity and survived indefinitely (>100 days) when treated with the same dose of IL-2. These results indicate that spontaneous rat liver allograft acceptance is associated with the presence of radiosensitive cells in the donor liver that may interact with recipient T cells to inhibit (Th1) production of IL-2.

Immunological study of unresponsive state in rat hepatic transplant model. 1. Phenotypic and functional analyses of infiltrating cells.

In order to elucidate the immunological characteristics of rat liver transplantation, graft-infiltrating cells (GIC) isolated from rat hepatic allografts were analyzed phenotypically and functionally. GIC from long-surviving recipients (Brown Norway livers into Lewis hosts) and acutely rejecting recipients (DA livers into Lewis hosts) were compared. The relative proportions of all T cells and activated T cells determined by flow cytometry were significantly higher in acutely rejecting Lewis recipients than in long-surviving recipients on day 6 after grafting. Phenotypic kinesis of GIC on days 6, 14, and 45 after transplantation from long-surviving Lewis hosts was analyzed. Each proportion of all T cells, OX8-positive cells (cytotoxic T and natural killer cells), and OX39-positive cells (IL-2 receptor), was greatest on day 6 and decreased by day 45. Cytotoxic activity of GIC toward donor lymphocytes on day 6 was greater in acutely rejecting versus long-surviving recipients. These results demonstrate that an immunosuppressive mechanism is already present on day 6 posttransplantation, and that infiltration or activation of cytotoxic T cells is inhibited in the long-surviving rat hepatic allografts.

Allograft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats

In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart grafts in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination. DA (RT1 a) donor into PVG (RT1 c) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosupressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-1) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 μg of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RT1 n) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-1), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In 5 6 cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the induction of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in vivo results, suggests a role for LSF-1 in the induction of tolerance.

Restoration of tolerance to rat hepatic allografts by spleen-derived passenger leukocytes.

The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchimerism detected in the recipients. When donor DA (RT1a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5 x 10(8) splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.

Restoration of tolerance to rat hepatic allografts by spleen-derived passenger leukocytes

Abstract The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchi-merism detected in the recipients. When donor DA (RT1a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5 times 108 splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.

New immunosuppressive proteins (KX-2, -4, and -5) induced by liver transplantation.

The presence in rat serum of three novel immunosuppressive proteins is associated with induction of tolerance after liver grafting. One of these, KX-5 (40 kilodalton molecular weight) is present 40-100 days after grafting of DA liver into PVG, a combination in which liver grafts are not rejected; two others, KX-2 (87 kilodaltons) and KX-4 (10 kilodaltons), are present in PVG sera in a retransplantation model. N-terminal amino acid sequencing indicated that none of the proteins had been described previously. Induction of KX-5 was inhibited by cyclosporin A. All three proteins inhibited mixed lymphocyte responses nonspecifically. Administration of a single dose of 300 micrograms purified KX-5 to PVG rats receiving heterotopic heart allografts led to prolonged graft acceptance. The results raise the possibility of clinical application of the immunosuppressive properties of liver grafts through administration of soluble proteins.

Novel immunosuppressive proteins purified from the serum of liver-retransplanted rats.

Liver grafts between certain rat strain combinations, such as DA (RT1a)-into-PVG (RT1c), are accepted without the use of immunosuppressive agents. To explore the nature and role of serum proteins in liver-induced immunosuppression, we have developed a retransplantation model of rat liver grafting. In this procedure, orthotopic liver transplantation (OLT) is carried out in the DA-into-PVG combination; two days later the DA liver is removed and a new PVG liver implanted into the same recipient (re-OLT). Serum from re-OLT rats was immunosuppressive when tested in mixed lymphocyte reactions (MLR). Three novel proteins were detected in re-OLT serum by SDS-PAGE, with sizes of 180 kD, 87 kD, and 10 kD. The N-terminal sequences of these were distinct and did not match protein sequences in the computer databases, although there was some homology between the 10 kD sequence and the beta-chain of rat hemoglobin. Purified 87 kD and 10 kD proteins were immunosuppressive in MLR; in both cases suppression was dose-dependent and nonstimulator-specific. Production of the 180 kD and 87 kD molecules required the presence of the recipient spleen. We conclude that re-OLT serum contains novel immunosuppressive proteins, which may be products of immune recognition and associated with the immediate termination of graft rejection.

Acute liver allograft rejection in the rat. An analysis of the immune response.

Liver allografts are vigorously rejected in 9-12 days in Lewis recipients of fully histoincompatible DA livers. The purpose of this study was to examine the initial events in this cascade, specifically the role of CD4+ T helper cells. Lewis recipients of DA or Lewis livers were killed at days 1, 2, 3, 4, and 7 days after transplant. Indicators of acute liver rejection, including a marked inflammatory infiltrate and decreased liver function, progressed in untreated recipients of allografts. Splenocytes taken from allogeneic recipients on days 1-4 and 7 proliferated in response to donor and third-party stimulators, whereas graft-infiltrating cells did not respond to donor and third-party antigens until day 3 after transplant, but thereafter maintained a good response. To further characterize the host T helper cell response to liver allografts, cytokine expression was analyzed in graft tissue and in the periphery. IL-4 mRNA was present in both syngeneic and allogeneic liver grafts, while message for IL-10 was present early in all liver grafts but persisted only in allografts. In contrast, IL-2 and IFN-gamma transcripts were specific to rejecting allografts. Similar patterns of cytokine expression were observed in the spleen, indicating the immune response to the graft involves the peripheral lymphoid organs. Thus, the cytokine profile detected during liver allograft rejection is extremely similar to that observed in other experimental models of transplantation.

INDUCTION OF NATURAL CHIMERISM AFTER RETRANSPLANTATION OF THE LIVER IN RATS

Immunological aspects after orthotopic rat liver retransplantation (re-OLT) were examined in association with cell migration and mixed chimerism. At day 2 after the first orthotopic liver transplantation (day 0) in the combination of DA (MHC haplotype, RT1a) donor into PVG (RT1c) recipient, the grafted DA liver was removed and a new PVG liver was implanted into the same PVG recipient (re-OLT). In the PVG recipient at various times after the re-OLT, DA-derived antigen and cells were detected using a DA-specific anti-class I mAb R3/13 in conjunction with ELISA, immunoblotting, and immunohistochemistry. The level of soluble class I antigen, which had risen to 270 ng/ml after the first OLT, substantially decreased within 24 hr after re-OLT. Using immunoblotting, DA class I antigen was detected in the PVG recipient's lymphoid organs at day 3 after DA liver grafting and persisted for up to 21 days after the DA liver was replaced by a new PVG liver. Immunohistochemistry on sections of spleen from re-OLT rats showed that the level of migratory cells expressing DA class I correlated with the findings obtained by immunoblotting. While the DA-derived antigen and cells were detected in the re-OLT recipient, the DA-specific inhibition of mixed lymphocyte reaction was observed in re-OLT serum. Our results suggest that the implanted DA liver graft was the source of DA soluble class I antigen, but DA-derived antigen and cells detected in the re-OLT recipient organs could persist for a relatively long time under immunosuppression after the implanted DA liver was removed by re-OLT.

Functional abnormalities of sinusoidal endothelial cells in rats with acute liver rejection.

The purpose of this study was to determine the changes of hepatic sinusoidal endothelial cell (SEC) function in acute liver rejection with respect to receptor-mediated endocytosis. Orthotopic rat liver transplantation was performed in Lewis rats grafted with DA livers and in Lewis rats grafted with Lewis livers as rejectors and controls, respectively. Animals were killed at 1, 3, 5, 7, and 10 days after the operation. Fc receptors (FcRs) were histochemically stained on frozen liver sections by applying peroxidase-antiperoxidase IgG complex as a ligand, and the FcR activity, i.e., capacity of binding the ligands represented by the FcR staining intensity, was semiquantitatively analyzed as an indicator of SEC function. The serum level of hyaluronic acid, which is specifically cleared from the circulation by receptor-mediated SEC endocytosis, was also assayed, along with the total serum bilirubin. Three days after the operation, the SECs of rejectors showed a significantly weaker FcR staining intensity of about half the value of that seen in the controls (P < 0.05), and staining disappeared after 5 days (P < 0.01). The decrease of FcR staining intensity, i.e., FcR activity, showed a correlation with elevation of the serum hyaluronic acid level (r = -0.77; P < 0.001). Histological evidence of endothelialitis and a significant elevation of total serum bilirubin (P < 0.01) were also present at 3 and 5 days, respectively. These results suggest that impairment of the endocytic function of SECs occurs at an earlier phase of acute liver rejection when compared with development of abnormalities of traditional indicators. Determination of receptor-mediated SEC endocytic functions may thus provide useful information for the early diagnosis of acute rejection.