Donor IL-4-treatment induces alternatively activated liver macrophages and IDO-expressing NK cells and promotes rat liver allograft acceptance

Most approaches to transplant tolerance involve treatment of the recipient to prevent rejection. This study investigates donor treatment with IL-4 for its effect on subsequent rat liver allograft survival. Rat orthotopic liver transplants were performed in rejecting (PVG donor to Lewis recipient) or spontaneously tolerant (PVG to DA) strain combinations. Donors were untreated or injected intraperitoneally with IL-4 (30,000 U/day) for 5 days. Tissue infiltrates and gene expression were examined by immunohistochemistry and real-time quantitative PCR. IL-4 induced a marked leukocyte infiltrate in donor livers prior to transplant. Macrophages comprised the major population, although B cells, T cells and natural killer (NK) cells also increased. IL-4-induced liver macrophages had an alternatively activated phenotype with increased expression of mannose receptor but not inducible nitric oxide synthase (NOS2). IL-4 also induced IDO and IFN-gamma expression by NK cells. Donor IL-4-treatment converted rejection to acceptance in the majority of Lewis recipients (median survival time > 96 days) and did not prevent acceptance in DA recipients. Acceptance in Lewis recipients was associated with increased donor cell migration to recipient spleens and increased splenic IL-2, IFN-gamma and IDO expression 24 h after transplantation. Donor IL-4-treatment increased leukocytes in the donor liver including potentially immunosuppressive populations of alternatively activated macrophages and IDO-expressing NK cells. Donor treatment led to long-term acceptance of most livers in association with early immune activation in recipient lymphoid tissues.     

The persistence of regulatory cells developing after rat spontaneous liver acceptance

BACKGROUND: We have previously reported that the spontaneous acceptance of Lewis (LEW, RT1(l)) to Dark Agouti (DA, RT1(a)) rat orthotopic liver transplant (OLT) is eliminated by donor gamma-irradiation. The acceptance of the irradiated LEW liver is also reestablished in a na?ve rat after the adoptive transfer of T regulatory (T-reg) cells from a LEW to a DA liver-tolerant long-term (>60 days) survivor (LTS) into a na?ve DA rat. However, little is known about the growth conditions required to maintain T-reg function. In this study, we examined the need for continued donor-specific alloantigen stimulation for the maintenance and function of T-reg cells. METHODS: Splenocytes (SCs; 1.5 x 10(8) cells) from a LEW liver allograft-tolerant LTS DA recipient were adoptively transferred fresh or after in vitro stimulation into another naive DA rat on day 1, 4, or 7 before an irradiated (1000R) LEW liver transplant. For in vitro alloantigen stimulation, SCs from LEW to DA LTS were co-cultured with mitomycin-C (MMC)-treated na?ve LEW (donor alloantigen-specific) or Brown Norway (BN) (RT1(n); third party) SCs for 72 hours. Graft rejection, as defined by death of the recipient, was confirmed histologically. RESULTS: All LEW liver grafts were accepted spontaneously by DA recipients for more than 60 days (n=32), while all irradiated LEW livers were acutely rejected (n=9; mean survival time [MST]=12.8 +/- 4.0 days). When LTS DA SCs were adoptively transferred into a naive DA rat 1 day before OLT, all irradiated LEW grafts were accepted greater than 60 days (n=9). However, when fresh LTS DA SCs were transferred to a new na?ve DA rat on 4 or 7 days before OLT, all irradiated LEW liver grafts were acutely rejected (MST=10.2 +/- 0.5 days [n=4] and MST=13.5 +/- 5.0 days [n=4], respectively). When LTS DA SCs were stimulated in vitro before adoptive transfer, irradiated LEW liver grafts after 4 days (n=5) were then accepted. In vitro culture of LTS DA SCs with MMC-treated BN SCs (third-party) for 72 hours before adoptive transfer resulted in 3 of 5 irradiated LEW livers at day 4 being accepted (n=5). CONCLUSIONS: The maintenance of T-reg function requires continuous LEW donor-specific alloantigen stimulation.     

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.     

Induction of long-term graft acceptance by a combination treatment of donor splenocytes and CTLA4Ig in a high responder rat liver transplantation model.

The CTLA4Ig has led to an improved survival rate in various allograft transplantation models. We investigated in a high responder rat model (Dark Agouti to Lewis) of orthotopic liver transplantation (ORLT), whether an additional adoptive cell transfer can enhance the effect of CTLA4Ig. After transplantation, recipients (n = 13/group) were treated with donor or third-party splenocytes alone or in combination with CTLA4Ig. Administration of splenocytes alone had no significant effect on survival (median 13 days, range 9-14) compared with untreated controls (median 10 days, range 8-12). CTLA4Ig monotherapy prolonged survival to a median of 30 days (range 11-150) but resulted in long-term graft rejection. The additional administration of third-party splenocytes showed no significant improvement over CTLA4Ig monotherapy. Only the combination of donor splenocytes with CTLA4Ig led to long-term graft acceptance (>150 days) without clinical and/or histological signs of rejection. A higher rate of apoptosis could be detected in livers at early time-points in long-term survivors receiving CTLA4Ig and donor splenocytes. Analysis of cytokine mRNA expression revealed a decrease of interleukin-2 at early time-points in all groups receiving CTLA4Ig; whereas, interferon-gamma was increased in long-term survivors receiving CTLA4Ig and donor cells or donor cells alone. The combination of CTLA4Ig and donor derived splenocytes is potent to induce long-term survival and graft acceptance. The mechanisms appear to involve the induction of an early inflammatory impulse and apoptosis.     

Combination of donor-specific blood transfusion with anti-CD28 antibody synergizes to prolong graft survival in rat liver transplantation

Donor-specific blood transfusion (DST) has been shown to effectively induce tolerance to certain allografts. In addition, it is well known that blockade of costimulatory signals reduces the ability of T cells to respond to alloantigens, prolonging allograft survival in some transplant models. We assessed the effects of single or multiple DSTs in the absence or presence of anti-CD28 monoclonal antibodies (mAbs) on graft function and host survival in rat liver transplantation (LTx). Fully MHC-mismatched adult male Dark Agouti (DA) and Lewis (LEW) rats were used as donors and recipients, respectively. Heparinized DA blood was administered to na?ve LEW rats 7 days before LTx [DST(-7d)], 14 and 7 days before LTx [DST(1 x 2)], twice a week for 2 weeks prior to LTx [DST(2 x 2)] and once a week for 4 weeks prior to LTx [DST(1 x 4)]. For some experiments, two different monoclonal antibodies (mAb) to rat CD28 (JJ316 and JJ319) were administered in combination with some DST treatments. We found that DST administration induced a time- and dose-dependent increase in host survival. Treatment of LEW rats with JJ316 or JJ319 mAb alone failed to prolong graft survival over untreated rats; however, the combination of DST(1 x 2) with JJ316 or JJ319 mAb induced indefinite survival at 100 days following surgery. We found that this protective effect was associated with increased numbers of splenic CD4+ CD45RC- but not CD4+ CD25+ foxp3+ T-cells in long-term survivors. Our data suggest that the combination of suboptimal DST with CD28 mAb induces donor-specific tolerance that correlates with enhanced numbers of regulatory T-cells.     

快速供肝切取与修整的外科技巧

目的总结肝脏移植供肝的快速切取和修整经验。方法分析2004年共186例快速供肝的切取和修整的资料。快速切取技术采用原位腹主动脉、肠系膜上静脉灌注附加下腔静脉引流，快速切取供肝，4℃UW液中保存和修整肝脏。结果供肝热缺血时间为3～10min，平均4．5min；冷缺血时间平均为3-16h，平均7h。供肝的修整时间为26～90min，平均46min。供肝修整时发现肝动脉解剖变异20例。结论快速供肝切取法要求术者技术娴熟、动作迅速和准确，可最大限度地减少供肝热缺血时间。快速切取法能保证供肝的质量和确保供肝切取的成功。     

Kupffer细胞CD14及Toll样受体4介导大鼠肝移植缺血再灌注损伤的机制

目的研究大鼠肝移植缺血再灌注后Kupffer细胞CD14和Toll样受体4(TLR4)的表达及其参与缺血再灌注损伤的机制。方法建立肝移植缺血再灌注模型,并分为正常对照组、缺血再灌注组、抗CD14抗体组,每组均为10只大鼠。分离培养大鼠肝移植缺血再灌注后的Kupffer细胞。检测Kupffer细胞CD14及TLR4的mRNA、蛋白表达、核转录因子κB(NFκB)活性以及培养上清TNFα的分泌量。结果再灌注后Kupffer细胞CD14及TLR4的mRNA和蛋白表达明显高于正常对照组(P<001),再灌注后核转录因子κB活性、培养上清TNFα表达量明显高于对照组(P<001)。用抗CD14抗体后NFκB活性,TNFα表达量明显下降(与再灌注组相比,P<005),但仍然高于对照组(P<001)。结论缺血再灌注后肠道内毒素(脂多糖)能够上调Kupffer细胞CD14及TLR4的表达,激活NFκB,启动细胞因子的转录和分泌,但除CD14和TLR4以外的其他信号途径参与了缺血再灌注损伤。     

FK506 suppression of heart and liver allograft rejection. II: The induction of graft acceptance in rats.

Lewis recipients of orthotopic ACI livers had permanent graft acceptance induced with 3 doses of i.m. FK506 in the early postoperative period. They were studied 100 and 300 days posttransplantation. The recipients rejected ACI as well as Brown Norway (BN) (third-party) skin grafts, and had lymphocytes with substantial reactivity by mixed lymphocyte culture testing against ACI and third-party (BN) alloantigens. Lymphocyte subset redistribution had not occurred in the peripheral blood or spleens of these animals, and there was no evidence of suppressor cell activation by in vitro and in vivo tests. Graft-versus-host reactivity in splenic lymphoid tissues of these recipients was demonstrated with the popliteal lymph node assay. Attempts at adaptive transfer with recipient lymphocytes were unsuccessful. Heart graft acceptance was far more difficult to accomplish than liver graft acceptance, and probably was never permanent. ACI heart graft prolongation in LEW recipients after a brief induction with FK506 lasted for no more than 3 months in most animals. The temporary heart graft acceptance was specific for hearts of the original ACI donor strain but not for ACI skin. Results of studies of lymphocyte subsets and suppressor cell activity were similar to those in the liver recipients. These studies illustrate how poorly graft acceptance is understood and how badly further work is needed to clarify its mechanism.     

The role of antibody isotype in IFN-gamma and IL-2 production during anti-CD3-induced T cell proliferation.

Murine anti-CD3 mAb of the IgG2a isotype are effective in the reversal of graft rejection. However, the first injection of these mAb causes a transient T cell activation in vivo, resulting in the release of cytokines that are held responsible for the sometimes severe febrile and adverse circulatory reactions associated with this therapy. Previously, we and others have reported that there is a polymorphism in the interaction of human Fc gamma R with mouse IgG1 and mouse IgG2b. This polymorphism implies that IgG1 and IgG2b mAb are mitogenic for T cells from respectively 70% and less than 5% of healthy individuals. By contrast, Fc gamma R interact with murine IgG2a and IgG3 mAb in virtually all individuals. We have now investigated the role of the isotype of the anti-CD3 mAb with respect to in vitro T cell proliferation and production of IFN-gamma and IL-2. IFN-gamma and IL-2 production always accompanied IgG2a- and IgG3-induced mitogenesis, whereas with IgG1 mAb the polymorphism in mitogenic effects completely correlated with IFN-gamma and IL-2 production. With IgG2a, IgG3, and IgG1 mAb, proliferation and IFN-gamma production were, at least in part, dependent on IL-2 production. On the other hand, IgG2b-induced proliferation was not accompanied by measurable IFN-gamma or IL-2 production. This suggests a fundamental difference in the way T cells are activated by IgG2b mAb. Given the role of IFN-gamma and IL-2 in the generation of adverse events during in vivo administration of anti-CD3 mAb, the use of IgG1 anti-CD3 or anti-TCR mAb offers a tool to further analyze the role of isotype in this respect. Moreover, the clinical use of IgG2b might result in immunosuppression without side effects.     

Establishment of practical recellularized liver graft for blood perfusion using primary rat hepatocytes and liver sinusoidal endothelial cells

Tissue decellularization produces a three‐dimensional scaffold that can be used to fabricate functional liver grafts following recellularization. Inappropriate cell distribution and clotting during blood perfusion hinder the practical use of recellularized livers. Here we aimed to establish a seeding method for the optimal distribution of parenchymal and endothelial cells, and to evaluate the effect of liver sinusoidal endothelial cells (LSECs) in the decellularized liver. Primary rat hepatocytes and LSECs were seeded into decellularized whole‐liver scaffolds via the biliary duct and portal vein, respectively. Biliary duct seeding provided appropriate hepatocyte distribution into the parenchymal space, and portal vein–seeded LSECs simultaneously lined the portal lumen, thereby maintaining function and morphology. Hepatocytes co‐seeded with LSECs retained their function compared with those seeded alone. Platelet deposition was significantly decreased and hepatocyte viability was maintained in the co‐seeded group after extracorporeal blood perfusion. In conclusion, our seeding method provided optimal cell distribution into the parenchyma and vasculature according to the three‐dimensional structure of the decellularized liver. LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity. This recellularization procedure could help produce practical liver grafts with blood perfusion.     

大鼠肝肾联合移植供体DC细胞迁移诱导的免疫保护作用

目的探讨供体DC细胞迁移对肝肾联合移植免疫保护的诱导作用及机制。方法60只大鼠肝肾联合移植免疫排斥模型(选择DA大鼠为供体,Lewis大鼠为受体),随机分为3组：A组：白细胞介素(IL)-10处理的DC细胞干预组；B组：未经IL-10处理的DC细胞干预组；C组：空白对照组。利用所建立的大鼠肝肾联合移植模型结合成熟前期DC细胞进行研究,首先进行供体大鼠DC细胞的预先分离、培养和IL-10处理,然后输入肝肾联合移植后的受体大鼠体内,分析其对肝、肾移植物的免疫保护以及其对移植物和受体存活时间的影响。结果A组肝肾移植的急性排斥反应受到明显的抑制,平均存活(20．0±2．6)d；B组和C组均表现为典型的重度急性排斥反应,存活时间分别为(6．7±0．8)和(7．5±0．5)d；A组与B、C两组受体的累积生存率差异有统计学意义(P＜0．05)。结论IL-10预处理的DC细胞可以对大鼠肝肾联合移植中的移植物起到免疫保护作用；供体的成熟前期DC细胞迁移到受体组织内,能够抑制肝肾联合移植急性排斥反应,并延长肝、肾移植物和受体大鼠存活时间。     

Delta opioid peptide augments functional effects and intrastriatal graft survival of rat fetal ventral mesencephalic cells

Delta enkephalin analogue [D-Ala(2),D-Leu(5)]enkephalin (DADLE) has been shown to protect dopamine transporters from methamphetamine-induced neurotoxicity. In the present study, we demonstrate that exposure of embryonic ventral mesencephalic cells to DADLE (0.01 g/ml), prior to intrastriatal transplantation, enhanced functional recovery and graft survival in 6-hydroxydopamine-induced hemiparkinsonian rats. At 6 and 8 weeks posttransplantation, animals that received DADLE-treated cell grafts exhibited significantly higher (near normal) spontaneous locomotor behaviors, as well as trends of greater reversal of motor asymmetrical behaviors compared with animals that received nontreated cell grafts. Histological examination revealed that animals transplanted with DADLE-treated cell grafts exhibited about twice the number of surviving tyrosine hydroxylase-immunoreactive grafted neurons compared with those animals that received nontreated cell grafts. These results suggest that DADLE should be considered as an adjunctive agent for neural transplantation therapy in Parkinson's disease.     

Effects of BWH-4 anti-CD4 monoclonal antibody on rat vascularized cardiac allografts before and after engraftment.

We studied the effects and mechanism of action of BWH-4, an IgG2a mouse antirat CD4 monoclonal antibody that recognizes a distinct epitope on the CD4 molecule, in LEW recipients of (LEW x BN)F1 vascularized heterotopic cardiac allografts. Ten animals received daily injections of 700 micrograms BWH-4 for ten days after engraftment. Median actuarial allograft survival was 37 days for the treated animals compared with 7.5 days for controls (n = 6). There was depletion of peripheral blood CD4+ lymphocytes to 4 +/- 1% one week posttransplant (normal = 48 +/- 4%, n = 6). Six additional animals were treated with a lower dose (100 micrograms) of BWH-4 daily for ten days. Median actuarial allograft survival was 10 days and the circulating CD4+ cells decreased to 30 +/- 6% at one week posttransplant. All six low-dose-treated animals mounted an anti-BN alloantibody response by 3 weeks, while only one of the ten high-dose-treated animals had positive alloantibodies two weeks posttransplant. Lymphocyte-mediated cytotoxicity against donor lymphoblasts was completely abolished in the high-dose-treated animals when compared with acutely rejecting controls. We also used low-dose (100 micrograms) BWH-4 to pretreat eleven experimental animals for 7 days prior to engraftment. The circulating CD4+ cells decreased to only 12 +/- 2% on the day of transplantation and 26 +/- 9% one week posttransplant. However, six (55%) pretreated animals survived more than 55 days. There was a 66% decrease in lymphocyte-mediated cytotoxicity against donor lymphoblasts when compared with acutely rejecting controls. We conclude that the anti-CD4 mAb, BWH-4, prevents acute rejection of vascularized heterotopic rat cardiac allografts; this effect is mediated by depletion of the CD4+ T cell subset, suppression of alloantibody production, and inhibition of lymphocyte-mediated cytotoxicity.