Interleukin-6 in islet xenograft rejection

Earlier work on primate cardiac xenotransplantation has demonstrated a correlation between interleukin (IL)-6 levels and severity of vascular rejection. IL-6 was originally identified as a lymphokine inducing final maturation of B lymphocytes into antibody-secreting cells. The present study aimed to evaluate the role of IL-6 in fetal porcine islet-like cell cluster (ICC) xenograft rejection. Moreover, other authors have reported that eosinophils dominate the cellular response following discordant islet xenograft transplantation. Here, a technique for specific detection of eosinophils was applied. IL-6-deficient mice and wild-type controls were implanted with fetal porcine ICCs under the kidney capsule and killed 4-, 7-, and 10 days after transplantation. Xenografts were histologically evaluated, and serum samples were analyzed for IgM and IgG antibodies against ICC membrane antigens. IL-6-deficient mice and wild-type controls readily rejected the xenograft. On day 7 after transplantation, abundant numbers of F4/80⁺ and Mac-1⁺ cells were found distributed throughout the collapsing graft accompanied by small amounts of eosinophils and peripherally accumulated CD3⁺ T cells (predominantly CD4⁺). Significantly lower serum levels of IgM and IgG antibodies against ICC membrane antigens were observed in IL-6-deficient mice on day 4 or 7 after transplantation when compared to wild-type controls. No significant differences were seen on day 10 after transplantation. In both experimental groups, specific IgM and IgG antibody levels remained stable over time. In the pig-to-mouse model, IL-6 seems to be of minor importance to fetal porcine ICC xenograft rejection. Macrophages, and not eosinophils, dominate the cellular response associated with this process. Received: 10 August 1999 Revised: 21 August 2000 Accepted: 27 November 2000     

A novel MyD88 inhibitor attenuates allograft rejection after heterotopic tracheal transplantation in mice

BackgroundAfter lung transplantation, the major complication limiting the long-term survival of allografts is obliterative bronchiolitis (OB), characterized by chronic rejection. Innate immune responses contribute to the development of OB. In this study, we used a murine heterotopic tracheal transplantation mouse model to examine the effects of a newtype of innate immune inhibitor, TJ-M2010–5.MethodsSyngeneic tracheal grafts were transplanted heterotopically from C57BL/6 mice to C57BL/6 mice. Allografts from BALB/c mice were transplanted to C57BL/6 mice. The allograft recipients were treated with TJ-M2010–5, and anti-mouse CD154 (MR-1). The grafts were harvested at 7, 14, and 28 days and evaluated by histological and real-time RT-PCR analyses.ResultsIn untreated allografts, almost all epithelial cells fell off at 7 days and tracheal occlusion reached a peak at 28 days. However, the loss of the epithelium and airway obstruction were significantly improved in mice treated with TJ-M2010–5 combined with MR-1. The relative mRNA expression levels of pro-inflammatory cytokines were upregulated in allogeneic tracheal grafts, and treatment with the two drugs reduced the production of pro-inflammatory cytokines and infiltration of inflammatory cells.ConclusionsIn heterotopic tracheal transplantation models, TJ-M2010–5 combined with MR-1 could ameliorate the development of OB.     

FTY720在鼠类异种胰岛移植排斥反应中的作用

目的 在小鼠异种胰岛细胞移植模型上，研究FTY720在控制异种胰岛移植排斥反应中的作用。方法 使用胰管内胶原酶注射和不连续密度梯度纯化法获取大鼠胰岛，建立小鼠肾被膜下移植模型。受体小鼠随机分为3组：对照组，末使用任何免疫抑制药物；实验1组，自移植当日起每日单独喂服FTY720(1.0mg／kg)；实验2组，亦于移植当日起每日联合喂服FTY720(1.0mg／kg)和环孢霉素A(CsA，15mg／kg)，均连续喂饲14d。分别于术后第3，5，7，14天切取移植物，观察并分析排斥反应。结果 对照组和实验1组，植入。肾被膜下的胰岛组织多在1周内完全被排斥，术后第7天胰岛轮廓消失，仅见大量淋巴细胞浸润。实验2组于移植后第7天及第14天肾被膜下仍可见大量完整的胰岛细胞，几乎未见或偶尔可见淋巴细咆浸润。结论 FTY720单独作为免疫抑制剂并不能抑制鼠类异种胰岛移植的排斥反应；联合应用FTY720及CsA则可有效地抑制鼠类异种胰岛移植排斥反应的发生。     

FK506与来氟米特预防异种胰岛移植排斥反应的效果

目的　探讨FK5 0 6、来氟米特 (Lef)应用预防大鼠对小鼠异种胰岛移植排斥反应的效果。方法　将 10 0 0～ 12 0 0个大鼠胰岛移植于化学诱导的糖尿病小鼠肾被膜下 ,实验分为对照组、FK 5 0 6组、Lef组和FK5 0 6+Lef组 ,研究胰岛有功能存活情况 ,并在移植后第 5天行病理组织学观察排斥反应。结果　FK5 0 6( 2和 4mg·kg-1·d-1)、Lef( 5、10、2 0mg·kg-1·d-1)术后用药 10d ,胰岛存活时间分别为 ( 8.9± 2 .1)、( 12 .5± 0 .7)、( 10 .8± 1.9)、( 15 .8± 2 .4)、( 18.8± 2 .2 )d ,较对照组( 5 .9± 1.2 )d明显延长 (P <0 .0 1)。FK5 0 6+Lef组移植物存活时间 ( 2 1.8± 1.2 )d ,较单纯用药组效果更好 (P <0 .0 1)。病理组织学显示Lef组与FK5 0 6+Lef组细胞浸润明显减少 ,有更多的完整胰岛存留。结论　FK5 0 6与Lef对异种胰岛移植有抗排斥作用 ,且两药联合应用有协同作用。     

Chemokine and toll-like receptor signaling in macrophage mediated islet xenograft rejection

BACKGROUND: Adoptive transfer of antigen-primed T-cell-activated macrophages into NOD-SCID mice within 14 days of foetal porcine pancreatic fragment (FPP) or foetal porcine skin (FPS) transplantation had been shown to cause xenograft rejection. In the present study, it was proposed that signaling between the graft and macrophages promoted specific graft recognition and destruction in this setting. METHODS: Exogenous macrophages isolated from rejecting FPP xenografts were transferred to NOD-SCID FPP recipients and tracked by Ly5.1 surface antigen or via CSFE staining. Monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage inflammatory protein-1beta (MIP-1beta), regulated upon activation, normal T-cell expressed and secreted (RANTES), chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), toll-like receptors (TLRs) (1-9) and gene expression in transplanted FPP xenografts was evaluated by real-time polymerase chain reaction. Gene expression of CCR2, CCR5 and TLRs was also analyzed in pooled samples of activated and non-activated macrophages. RESULTS: Exogenous macrophages were shown to track to and reject recently transplanted but not established FPP xenografts. Gene expression for MCP-1, RANTES, MIP-1alpha and MIP-1beta was at least 3-fold greater in recently transplanted compared with established xenografts (P < 0.05), and CCR2 and CCR5 gene expression was 10-fold greater in activated compared non-activated macrophages, suggesting that graft-mediated pro-inflammatory signals were important for macrophage recruitment. Specific graft recognition by macrophages may involve TLR signaling as macrophages exposed to porcine islets had higher levels of TLR gene expression compared with those exposed to allografts regardless of the level of activation. CONCLUSION: Xenografts provide additional activation signals to macrophages that are not seen following allotransplantation. This study identifies chemokines and TLR as important signals in macrophage-mediated recognition and rejection of islet xenografts.     

过继性回输Treg对小鼠同种胰岛移植物免疫排斥反应的影响

目的探讨过继性回输调节性T细胞(Treg)对同种胰岛移植术后胰岛功能恢复及移植物存活时间的影响。方法以C57BL/6小鼠为受体建立糖尿病模型,以Balb/c小鼠为供体进行肾包膜下同种小鼠胰岛移植。根据处理方法不同将受体小鼠分为3组:Treg实验组(Treg组,术前1 d经尾静脉注射1×10~6个Treg细胞)、阳性对照组[西罗莫司(SRL)组,术前1 d开始给予300μg/(kg·d)SRL灌胃]和空白对照组(对照组,术前1 d开始每日给予等体积生理盐水灌胃),每组3只。采用酶联免疫吸附试验(ELISA)检测小鼠术后14 d内血糖、C肽变化情况;通过活体成像技术动态监测小鼠移植物术后14 d内存活情况。结果与移植术前比较,术后3 d各组小鼠血糖水平均显著降低(均为P0.001)。术后1 d各组小鼠C肽水平均出现不同程度的爆发性回升,术后3 d各组小鼠C肽水平较术前明显升高(均为P0.001)。在术后14 d观察期结束时,与对照组比较,SRL组与Treg组C肽水平仍相对较高[(427±50)、(833±57)pmol/L];而两组的血糖水平[(14.5±0.5)、(12.1±0.6)mmol/L]均显著低于对照组(均为P0.001)。与SRL组比较,Treg组C肽爆发性释放量更低,下降趋势明显更加平缓,观察期结束时C肽水平较高(均为P0.001)。术后14 d对照组的移植物几乎完全凋亡,SRL组有50%的移植物存活,Treg组有80%的移植物存活。结论过继性回输Treg能有效保护胰岛移植物,延长移植物存活时间,维持移植小鼠血糖以及C肽的正常水平。     

Therapeutic strategies for xenograft rejection

The increasing demand for transplantable organs over the past several decades has stimulated the idea of using animal organs in lieu of cadaveric organs in clinical transplantation. Pigs are now considered to be the most suitable source of organs for transplantation because of their abundant availability, their appropriate size, their relatively short gestation period, and the recent development in the technology to genetically manipulate them. In the past few years, some of the seemingly complex immunologic responses in pig-to-primate transplantation have been elucidated. This progress has allowed us to focus our efforts on devising specific therapeutic strategies to overcome or prevent some of the responses that contribute to rejection of the xenograft. In this article, we review the various approaches that might allow clinical xenotransplantation to come to fruition.     

Tacrolimus inhibits discordant islet xenograft rejection: a study in the pig-to-rat model

Abstract: The aim of the present study was to evaluate the immunosuppressive effect of tacrolimus (TAC) in discordant islet xenotransplantation. Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in normoglycemic rats treated with TAC monotherapy, TAC plus other immunosuppressive drugs or cyclosporin A (CsA) monotherapy. Twelve or 24 days after transplantation, the extent of a cellular infiltration in the xenografts was evaluated using immunohistochemistry. In some animals, the grafts were examined for antibody and complement deposition and the levels of xenoreactive antibodies in serum were determined. In untreated rats, the xenografts were completely rejected after 12 days and no intact ICCs remained. TAC monotherapy (at 0.5 and 1.0 mg/kg b.w.) almost completely inhibited rejection for up to 12 days. In animals treated with TAC monotherapy (at 0.5 mg/kg b.w.), rejection was markedly inhibited for up to 24 days. However, the effect after 24 days was not consistent and in some grafts there were signs of rejection. The protective effect of TAC observed in this study is in contrast to the findings in rats given CsA monotherapy in which no or only a marginal effect on islet xenograft rejection was observed. Only when CsA was given at 20 mg/kg b.w., an inhibitory effect could be observed. Immunosuppression with TAC at a suboptimal dose (0.3 mg/kg b.w.) plus 15-deoxyspergualin or brequinar also had an inhibitory effect on the rejection. In animals given TAC plus mycophenolate mofetil, a protective effect was observed as well; however, this effect was not consistent.     

Pig islet xenograft rejection in a mouse model with an established human immune system

Abstract: Background: Xenotransplantation from pigs provides a potential solution to the severe shortage of human pancreata, but strong immunological rejection prevents its clinical application. A better understanding of the human immune response to pig islets would help develop effective strategies for preventing graft rejection. Methods: We assessed pig islet rejection by human immune cells in humanized mice with a functional human immune system. Humanized mice were prepared by transplantation of human fetal thymus/liver tissues and CD34⁺ fetal liver cells into immunodeficient mice. Islet xenograft survival/rejection was determined by histological analysis of the grafts and measurement of porcine C‐peptide in the sera of the recipients. Results: In untreated humanized mice, adult pig islets were completely rejected by 4 weeks. These mice showed no detectable porcine C‐peptide in the sera, and severe intra‐graft infiltration by human T cells, macrophages, and B cells, as well as deposition of human antibodies. Pig islet rejection was prevented by human T‐cell depletion prior to islet xenotransplantation. Islet xenografts harvested from T‐cell‐depleted humanized mice were functional, and showed no human cell infiltration or antibody deposition. Conclusions: Pig islet rejection in humanized mice is largely T‐cell‐dependent, which is consistent with previous observations in non‐human primates. These humanized mice provide a useful model for the study of human xenoimmune responses in vivo.     

Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ(-/-) mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.     

Comparative immunohistologic studies in an adoptive transfer model of acute rat cardiac allograft rejection

It has been shown that fulminant acute rejection of rat cardiac allografts across a full haplotype disparity may occur as a direct result of adoptive transfer of sensitized W3/25+ MRC OX8- SIg- T helper/DTH syngeneic spleen cells to sublethally irradiated recipients. In order to establish the immunohistologic parameters of this form of rejection, allografts and recipient lymphoid tissue were analyzed using a panel of monoclonal antibodies of known cellular distribution. These data were compared with those obtained following reconstitution of irradiated allograft recipients with unseparated sensitized spleen cells, with unreconstituted irradiated donor recipient pairs, with unmodified first-set rejection, and with induced myocardial infarction of syngeneic heart grafts transplanted to normal and to sublethally irradiated recipients. Rejecting cardiac allografts transplanted to all reconstituted irradiated recipients were characterized by extensive infiltration with MRC OX8+ (T cytotoxic-suppressor, natural killer) cells even when this subset was virtually excluded from the reconstituting inocula. A similar proportional accumulation of MRC OX8+ cells observed at the infarct margins of syngeneic heart grafts transplanted to irradiated unreconstituted recipients greatly exceeded that present in normal nonirradiated controls. These data provide evidence that under conditions of heavy recipient irradiation, MRC OX8+ cells may be sequestered within heart grafts in response to nonspecific injury unrelated to the rejection process. Although there was no significant degree of MRC OX8+ cellular repopulation within organized secondary lymphoid tissues of irradiated animals over the study period, the density of ileal mucosal MRC OX8+ lymphocytes approximated normal at 7 days post-irradiation, raising the possibility that these cells could share a common origin with those sequestered within the heart grafts. Carbon+ MRC OX6+ macrophages were a significant component of the infiltrate in all rejecting cardiac allografts, as well as in all infarcted syngeneic heart grafts--providing further evidence that macrophage "activation" with expression of class II determinants may occur in response to nonspecific injury. In unmodified first-set rejection there was an intense B cell reaction in recipient spleens and lymph nodes. In the adoptive transfer model, marked B cell expansion was exclusively confined to the parathymic lymph nodes of irradiated allograft recipients reconstituted with the sensitized W3/25+, MRC OX8-, SIg- T helper/DTH donor cell inocula.(ABSTRACT TRUNCATED AT 400 WORDS)     

白细胞介素-2及其受体与胰岛移植排斥反应发生的相关性及其意义

目的：探讨检测血清中白细胞介素－2（IL－2）及其受体（SIL－2R）的含量对异种胰岛移植排斥反应中的意义。方法：昆明小鼠为供体，Wistar大鼠为受体。分4组：A组为糖尿病对照组，B组为单纯胰岛移植组，C组为胰岛和胸腺联合移植组，D组为胸腺细胞胸腺内预注射后，胰岛和胸腺联合移植组。移植后用酶联免疫吸附试验（ELISA法）检测IL－2、SIL－2R含量。结果：血清中IL－2在胰岛排斥前4d开始有显著升高（P＜0．001），而SIL－2R在排斥前3d开始显著升高（P＜0．001）。两者含量一直升高至排斥当日。结论：检测IL－2、SIL－2R含量在异种胰岛移植是否发生排斥反应具有重要意义。     

Detecting rejection after mouse Islet transplantation utilizing islet protein-stimulated ELISPOT

Improved posttransplant monitoring and on-time detection of rejection could improve islet transplantation outcome. The present study explored the possibility of detecting harmful events after mouse islet transplantation measuring the immune responsiveness against islet extracts. Mouse islet transplantations were performed using various donor/recipient combinations, exploring autoimmune (NOD/SCID to NOD, n = 6) and alloimmune events (C57BL/6 to BALB/c, n = 20), a combination of both (C57BL/6 to NOD, n = 8), the absence of both (BALB/c to BALB/c, n = 21), or naive, nontransplanted control mice (n = 14). The immune reactivity was measured by ELISPOT, looking at the ex vivo release of IFN-γ from splenocytes stimulated by islet donor extracts (sonicated islets). The immune reactivity was not altered in the syngeneic and autoimmune models, demonstrating similar levels as nontransplanted controls (p = 0.46 and p = 0.6). Conversely, the occurrence of an allogeneic rejection alone or in combination to autoimmunity was associated to an increase in the level of immune reactivity (p = 0.023 and p = 0.003 vs. respective controls). The observed increase was transient and lost in the postrejection period or after treatment with CTLA4-Ig. Overall, allogeneic rejection was associated to a transient increase in the reactivity of splenocytes against islet proteins. Such a strategy has the potential to improve islet graft monitoring in human and should be further explored.     

Reduced NO production improves early canine islet xenograft function: a role for nitric oxide in islet xenograft primary nonfunction

Isolated canine islets transplanted to hyperglycemic rats fail to restore euglycemia in almost all cases, although the grafted islet tissue appears to be morphologically intact for up to 48 h following transplantation. Cytokines typically produced in the xenograft environment (e.g., IL-1 and TNF) inhibit insulin biosynthesis and secretion from isolated pancreatic islets, and are associated with the production of nitric oxide (NO). To further define the relationship between NO production and islet xenotransplantation, the inhibition of NO in a splenocyte/islet coculture system, and the in vivo effect of this inhibition on canine islet xenotransplantation, was investigated. Splenocytes (SPLC) from Lewis rats were cocultured with canine islets (freshly isolated or cultured 7 days), supernatant removed, and NO concentration (NO2) determined by optical density (Griess reaction, 550 nm, expressed as nmol nitrite/10(6) cells/18 h). Lipopolysaccharide (LPS) was used as a positive control of SPLC production of NO. Stimulation by LPS resulted in maximal NO production (2.20 +/- 0.16 nmol/10(6) cells/18 h, p < 0.001 compared to baseline values of 0.73 +/- 0.04 nmol/10(6) cells/18 h). In the presence of NO inhibitors (NMA, polymyxin B, hydrocortisone, aminoguanidine, DMSO), nitrite levels did not significantly rise above unstimulated values. Freshly isolated canine islets did stimulate NO production (1.26 +/- 0.12 nmol/10(6) cells/18 h, p < 0.001). In contrast, cultured canine islets did not stimulate NO production (0.84 +/- 0.09 nmol/10(6) cells/18 h). Transplantation of freshly isolated canine islets to STZ-diabetic recipient Lewis rats resulted in amelioration of hyperglycemia in only 50% (n = 6) of recipients 12 h posttransplant, with a return to hyperglycemia at all subsequent time points. Transplantation of 7-day cultured canine islets resulted in amelioration of hyperglycemia in 88% of recipients 12 h posttransplant and 63% of recipients 24 h posttransplant [p = 0.028, mean survival time (MST) = 1.0 days, n = 8]. Transplantation of canine islet xenografts with aminoguanidine therapy (BID, n = 11) resulted in amelioration of hyperglycemia in 100% of recipients at 12 h posttransplant, decreasing to 82% by 24 h following transplantation (p = 0.002, MST = 0.9 days). These results demonstrate that freshly isolated canine islets are potent stimulators of NO production by rat SPLC in vitro, and that culture of canine islets, or addition of NO inhibitors, abrogates stimulated NO production. These results also demonstrate a statistically significant improvement (p < 0.001) in early function of canine islet xenografts following 7 days of islet culture prior to transplant, and following recipient treatment with aminoguanidine. These studies suggest that the production of NO in the microenvironment of the graft site may adversely affect engraftment and function of canine islets, and suggest that the abrogation of islet-stimulated NO production may improve engraftment following islet xenotransplantation.