Acute cellular xenograft rejection

Abstract: The acute cellular rejection process after xenotransplantation of vascularized organs, i.e. heart, kidney and liver, has so far been difficult to study since the implanted organs are lost in a hyperacute rejection before cellular rejection develops. Primarily non-vascularized xenografts-e.g. pancreatic islets-escape the hyperacute rejection, but succumb to cell-mediated rejection during the first week after transplantation in either mice, rats or cynomolugus monkeys. The present view on the mechanisms orchestrating islet xenograft rejection in these animal models will be summarized and in part generalized to give insight into the process of acute cellular xenograft rejection.     

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     

Thrombin Inhibition in discordant xenograft rejection

Abstract: Microvascular thrombosis and the associated platelet and endothelial cell activation are prominent observations in xenograft rejection. This pathological picture could be related to the excessive generation of thrombin in the context of either inflammation or putative inter-species molecular incompatibilities between activated coagulation factors and their natural anticoagulants. Relatively selective thrombin Inhibition with the serine protease inhibitor SDZ MTH 958 (MTH-958) are independent of heparinoids and anti-thrombin III. MTH-958 has been shown to significantly prolong porcine cardiac function during perfusion with human blood in an ex vivo model. The aim of this study was to validate the role of thrombin generation in a rodent model of discordant xenograft rejection in vivo. The effect of thrombin inhibition with MTH-958 was tested in both hyperacute rejection (HAR) and delayed xenograft rejection (DXR) after decomplementation with cobra venom factor (CVF) in normal Lewis (Lew) rats and Intrinsic C6 deficiency In PVG (C6-/PVG) recipient rats. Recipient rats received heterotopic guinea pig cardiac xenografts and were treated with titrated doses of MTH-958 until the time of graft rejection. Plasma samples at selected time points were examined to confirm effective thrombin inhibition, and rejected grafts were analyzed by immunohistology. MTH-958 significantly improved graft survival in HAR albeit the extent of prolongation was not marked, but the agent failed to prolong survival In CVF-treated Lew rats. In C6-/PVG rats receiving MTH-958, a significantly reduced graft survival time was observed when compared with C6-/PVG controls. The grafts from MTH-958-treated animals showed dense deposits of C3, IgM, and IgG with fibrin levels similar to controls. The thrombin antagonist tested could prolong xenograft survival during HAR but had no benefit in DXR. The relative non-specificity of the serine protease inhibitor MTH-958 with the potential activation of alternative pathway of complement via the inhibition of factor I could account for the failure to prolong xenograft survival in DXR. The pathogenetic significance of thrombin generation in this situation remains to be determined by the use of more selective and pharmacologically acceptable I anti-thrombin agents.     

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.     

Reversibility of cardiac xenograft rejection in primates

More than 500 people died in 1988 because of the shortage of human heart donors. Cardiac xenografts from concordant primates may help to relieve this shortage. Two groups of ABO-matched cynomolgus monkey-to-baboon heterotopic cardiac xenografts were studied. Group 1 (n = 4) control animals had no immunosuppression. Group 2 (n = 6) baboons received cyclosporine, azathioprine, and methylprednisolone acetate. Myocardial biopsy, mixed lymphocyte culture, donor-specific crossmatch, and panel-reactive antibody determinations were performed after transplantation. Biopsy-proven rejection episodes (myocyte necrosis) were treated intravenously with steroids. A follow-up biopsy was performed 7 days after the first biopsy. If rejection persisted, antithymocyte globulin (10 mg/kg/day) was given for 7 days, and another biopsy was performed. Group 1 (control) graft survival was 8, 9, 9, and 10 days (mean, 9 days). Group 2 (immunosuppressed) graft survival was 3, 16, 18, 51, 84, and 392 days (mean, 94 days). Each immunosuppressed baboons' xenograft had myocyte necrosis and variable degrees of edema, cellular infiltrates, and vascular thrombosis consistent with mixed cellular and humoral rejection within 1 week. Only one rejection episode resolved with high-dose steroid therapy alone. Two baboons' rejection episodes resolved with antithymocyte globulin treatments, but rejection recurred in both. Low levels of or delayed progression of panel-reactive antibody was associated with long-term xenograft survival. Severe steroid-resistant rejection with cell-mediated and humoral immune elements developed early in our primate cardiac xenografts despite triple-drug immunosuppression. Use of antithymocyte globulin was associated with temporary resolution of rejection, but progressive increases in lymphocytotoxic antibody led invariably to eventual graft loss despite rare long-term survival.     

The mechanism of discordant xenograft rejection

The mechanism of discordant xenograft rejection using the guinea pig-to-rat heart graft model was studied. In this model, we found that (A) Rejection occurred rapidly, in 17.5 +/- 8.3 min (mean +/- SD) (n = 8). (B) The graft survived longer when the recipient rat was pretreated with cobra venom facter (CVF). (C) Complement hemolytic titers in serum showed significant reduction of C3 in rejection without consumption of C4 and C2, suggesting complement activation through the alternative pathway. (D) No natural antibodies were detected in this combination. Complement-dependent cytotoxicity (CDC) titer, and hemagglutination (HA) titer were lower than x1. (E) Histological examination of the rejected heart xenograft revealed a large area of myocytolysis without interstitial cellular infiltration. (F) In vitro experiments showed that rat complement attacked guinea pig erythrocytes (Egp) via the alternative pathway. These findings indicate that rejection in this discordant xenograft model of guinea pig-to-rat was caused by primary activation of complement via the alternative pathway.     

The effect of immunoglobulin immunadsorptions on delayed xenograft rejection of human CD55 transgenic pig kidneys in baboons

Delayed xenograft rejection (DXR) remains a major obstacle in discordant xenotransplantation. As strategies of complement inhibition and xenogeneic natural antibody (Ab) removal have been shown to give prolonged xenograft survival, we endeavored to determine whether combining these two strategies would lead to an additive effect in terms of graft survival. The study was initiated with two groups, A and B, where group A received normal kidneys and group B received hCD55 transgenic kidneys. Both groups underwent pre-transplant (day-1) total immunoglobulin (Ig) immunoadsorption (IA) and received an immunosuppression of cyclophosphamide, cyclosporine A, mycophenolate mofetil and corticosteroids. Two subsequent groups (C and D) receiving hCD55 transgenic pig kidneys were then performed with an 'optimized' immunosuppression (Cyclophosphamide starting 1 day earlier) but only group D recipients were immunoadsorbed. Biopsies taken during the post-transplantation period were analyzed for Ab deposition, compliment activation and cellular infiltration. No hyperacute rejection was observed. In the initial immunoadsorbed groups A and B, all baboons underwent DXR, which started surprisingly early (day 5 in most cases. In the subsequent two groups, the immunoadsorbed group D baboons also underwent DXR, again as early as day 5. In contrast, group C baboons did not show any signs of DXR on their day 6 biopsy or at their time of death. Analysis of graft biopsies from the kidneys undergoing rejection or with stable function showed strong deposition of anti-Gal IgM in all cases whereas strong complement C5b-9 deposits were only observed in biopsies at rejection. Cellular infiltration consisted mostly of monocytes/macrophages, was more pronounced in biopsies taken at rejection and was associated with a pro-inflammatory environment involving interleukins 1alpha, 6 and 8. Our findings suggest non-specific Ig (anti-Gal and non-Gal Ig of all isotypes) IA or even incomplete IA in immunosuppressed baboon recipients of transgenic pig kidneys is detrimental to graft survival by being associated with an Ab and compliment driven rejection. We speculate that the IA were insufficient in terms of Ig depletion or frequency inducing an Ab rebound or that this total Ig depletion also removed components facilitating graft survival.     

C1-Inhibitor for treatment of acute vascular xenograft rejection in cynomolgus recipients of h-DAF transgenic porcine kidneys

Abstract: At present, the major barrier to successful discordant xenotransplantation of unmodified or complement regulator transgenic porcine xenografts is acute vascular xenograft rejection (AVR). AVR is associated with the intragraft deposition of induced recipient xenoreactive antibodies and subsequent complement activation. In a life-supporting pig to primate kidney xenotransplantation setting using h-DAF transgenic donor organs and postoperative immunosuppression, episodes of AVR were either treated with boluses of cyclophosphamide and steroids or with the same regimen supplemented by a three-day course of C1-Inhibitor, a multifunctional complement regulator. In 8 out of 10 animals stable initial graft function was achieved; in all animals one or more episodes of AVR were observed. When, in 4 animals, C1-Inhibitor was added to the standard anti-rejection treatment regimen, AVR was successfully reversed in 6 out of 7 episodes, while in another group of 4 animals receiving the standard anti-rejection treatment 0 out of 4 episodes of AVR responded to treatment. Response to anti-rejection treatment was associated with a significant increase in recipient survival time. We conclude that AVR of h-DAF transgenic porcine kidneys can be successfully treated by additional short-term fluid phase complement inhibition.     

Discordant xenograft rejection in an antibody-free model.

Newborn pigs prevented from suckling colostrum were shown to have less than 0.05 micrograms/ml total immunoglobulin present in their serum. Rabbit heart xenografts transplanted heterotopically into the neck of such pigs were hyperacutely rejected, with a mean survival time of 92 +/- 14 min (mean +/- SD). Pigs which had been allowed to suckle and whose serum contained 10-15 mg/ml maternal immunoglobulin hyperacutely rejected rabbit heart xenografts in 109 +/- 62 min. Histological studies showed no Ig binding but complement component 3 (C3) binding to rabbit hearts placed in immunoglobulin-negative pigs. Prolongation of rabbit heart xenograft survival was achieved by administering cobra venom factor (1 mg/kg) to the pigs pretransplant. These data show hyperacute xenograft rejection in the absence of antibody and suggest that its cause is activation of complement by the alternative pathway.     

延迟性异种移植排斥反应的病理特征及IgG的作用

目的利用小鼠→大鼠心脏移植模型，探讨延迟性异种移植排斥反应(DXR)的病理特征及IgG的作用。方法将移植大鼠随机分成4组对照组(A组，n＝6)；环孢素A(CsA)组(B组，n＝6)，CsA每2日用药1次，每次20mg/kg，自移植当日(day0)起使用；环磷酰胺(CyP)组(C组，n＝6)，移植前1d给予CyP40mg/kg，自移植后第1天起每2日用药1次，每次20mg/kg；CsA加CyP组(D组，n=5)。血清IgG水平用ELISA法测定；C     

Concordant lung xenograft rejection in a hamster-to-rat model

This study was conducted to investigate the functional and morphological aspects of orthotopic lung xenograft rejection in a concordant hamster-to-rat donor-recipient species combination. By postoperative day (POD) 3, allotransplanted grafts demonstrated good aeration, but infiltrates were seen in all the xenotransplanted lungs. Antihamster lymphocytotoxic antibody titers increased to 5.2±1.1 by POD 3 (P<0.05 vs POD 1) and reached 7.0±0.8 by POD 5 (P<0.05 vs PODs 1 and 3). The CD4+/CD8+ ratio in peripheral blood lymphocytes increased significantly on POD 3 (P<0.05 vs untransplanted), but decreased by POD 5 (P<0.05). Histologically, on POD 3 the xenotransplanted grafts were characterized by perivascular cellular infiltrates and edema. Cytologically, the cells consisted of small round lymphocytes, monocytes, and occasional neutrophils. Immunohistochemical analysis showed heavy IgM and C3 deposits in the vascular endothelium, without any IgG deposits. Allotransplanted grafts showed moderate W 3/25+(28.3±17.3%) and MRC OX8+(38.7±0.7%) cellular infiltrations on POD 3, but ED1+(8.0±3.7%) cells were rare. Conversely, in the xenotransplanted grafts, ED1+(34.2±16.4%) cells were more prevalent than MRC OX8+(18.1±16.5%) cells on POD 3, at P<0.01 and P<0.05 vs allograft, respectively. These results indicate that both antidonor antibodies and macrophages/monocytes play an important role in the concordant lung xenograft rejection.     

异种肾移植超急性排斥反应时血液流变学的变化

采用猪-犬肾移植建立超急性排斥反应模型，以研究此时的血液流变学变化。分别在移植前、移植肾超急性排斥发生时和排斥肾切除后10～15分钟检测外周血的血液流变学参数变化。结果异种肾移植发生超急性排斥时，血小板聚集显著增高，移植肾切除后血中的血小板数和纤维蛋白原明显下降，血小板聚集也恢复原有水平。提示血小板、纤维蛋白原参与了异种移植超急性排斥反应的发生过程，而血小板聚集可能是诊断异种移植超急性排斥反应一个有价值的指标。