Vascular rejection in cardiac transplantation. A morphological study of 25 human cardiac allografts.

Between November 1983 and January 1990 103 orthotopic heart transplants were performed at the National Hospital, University of Oslo, Norway. Twenty-two patients died. Acute and/or chronic rejection was the cause of death in nine, disseminated infection in eight, cancer in three, and cerebral haemorrhage in one of the patients. Two patients were successfully retransplanted after graft failure due to AB0 blood group incompatibility because of a communication error. One patient with a positive lymphocytotoxic crossmatch died shortly posttransplant due to acute circulatory collapse. The cumulative one- and five-year survivals were 82% and 68%. Follow-up time was 226.6 graft years, survival range from one to 2,306 days (mean 803 +/- 4.12 SEM). A total of 1,343 endomyocardial biopsies were performed, which revealed 181 acute cellular rejection episodes, and 22 biopsies revealed acute and/or chronic vascular rejection. Autopsy studies showed three general types of vascular damage: acute (necrotizing) vasculitis, atherosclerotic disease in the epicardial arteries and diffuse proliferative arteriopathy in the intramural and smaller branches. In several cases acute vasculitis was concomitant with either of the two chronic types of accelerated graft sclerosis. Tissue immunofluorescence analysis demonstrated vascular deposition of immunoglobulin and complement in acute vasculitis, indicative of humoral immunoreaction. Postoperatively early chronic vascular rejection may develop.     

Extracorporeal photochemotherapy after cardiac transplantation: a new therapeutic approach to allograft rejection

Photopheresis (ECP) is a new immunomodulatory therapy in which recipient lymphocytes are treated extracorporeally with 8-methoxypsoralen and ultraviolet light. The treatment seems to induce an inhibition of both humoral and cellular rejection after transplantation. OBJECTIVE: Since recurrent rejection (RR) continues to be a severe complication after heart transplantation (HTx) and the immunosuppressive regimes used for the treatment are often associated with increased morbidity and mortality, we investigated whether ECP could have a beneficial effect on the number and severity of rejection episodes. METHODS: Eleven HTX recipients (5 M and 6 F, mean age 48.5 yrs) with RR were enrolled in the study. ECP was performed at weekly intervals during the 1st month, at 2 week intervals during the 2nd and 3rd month, and then monthly for another 3 months. RESULTS: The fraction of biopsies (EMB) with a grade 0/1A rejection increased during ECP from 46% to 72% while the EMB showing a 3A/3B rejection decreased from 42% to 18%. It is also noteworthy that out of the 78 EMB performed during ECP only one showed a 3B rejection in comparison with 13 out of 110 EMB in the pre-ECP period. Six rejection relapses were observed in a total follow-up of 60 months, two of them occurring during the tapering of oral steroid. Four relapses were reversed by ECP, one by i.v. steroids and the last by methotrexate after the failure of both i.v. steroids and ECP. The mean doses of immunosuppressive drugs resulted lower after 6 months of ECP: steroids were reduced from 13 to 8.25 mg/day, cyclosporine from 375 to 285 mg/day, azathioprine from 55 to 35 mg/day. CONCLUSIONS: ECP is a well tolerated treatment. Its administration allows better RR control and significant reduction in immunosuppressive therapy.     

Role of RANTES in experimental cardiac allograft rejection

The host response to alloantigen results in upregulation of Class II MHC antigens and associated cytokine production (especially IL-2 and interferon-gamma (IFN-gamma)) as well as lymphocytic infiltration and cellular activation which leads to graft damage/destruction. RANTES (Regulated upon Activation, Normal T-cell Expressed and presumably Secreted) is a member of the beta subfamily (CC) of chemokines and has been shown to function as a lymphocyte chemoattractant. We now describe the requirement for RANTES in cardiac heterotopic allograft (brown Norway into Lewis rats) rejection in rats. By Northern blot analysis, mRNA could be detected in allografts at 6 and 8 days post-transplantation but not in isogenic (Lewis) grafts. RANTES protein could be demonstrated by Western blot analysis in homogenates from allografts at day 8 but not at day 0 and could not be identified in isogenic cardiac transplants. By immunostaining, RANTES protein was present in mononuclear cells of allografts at day 6 but was absent in the isogenic transplants. When rats were treated with anti-RANTES serum, there was a significant delay in rejection time (cessation of beating) of the allografts. These data demonstrate that expression of RANTES in rat cardiac allografts is linked to the rejection phenomenon.     

铁离子沉积与心脏移植急性排斥反应相关性研究

目的：探讨铁离子在心脏移植急性排斥反应的作用。 方法： 建立小鼠颈部异位心脏移植模型，实验分为同系移植组（C57BL/6→C57BL/6）和同种异体移植组（BALB/c→C57BL/6），普鲁士蓝染色观察铁离子在心肌组织的沉积情况，免疫组化法观察HO-1在心肌组织的表达。 结果： 铁离子在浸润的巨噬细胞中沉积，HO-1主要在浸润的炎性细胞中表达，两者随急性排斥反应的加重表达上调。 结论： 铁离子沉积与心脏移植急性排斥反应的病理过程相关，且可作为心脏移植急性排斥反应的监测指标。     

Contribution of Fas ligand to cardiac allograft rejection

Effector mechanisms for allograft injury remain unclear. Inthe present study, we verified the contribution of Fas and Fasligand (FasL) to cardiac allograft rejection by utilizing theFas-deficlent lpr or FasL-deficient gid mice as the donor orrecipient. Cardiac myocytes prepared from normal mice, but notthose from lpr mice, constitutively expressed Fas and were susceptibleto FasL-mediated lysis. Survival of cardiac allografts was substantiallyprolonged when gld or lpr mice were used as the recipient. Incontrast, cardiac allografts from ipr mice were normally rejectedwithout a delay. Histological examination of the grafts in thegld or lpr recipients demonstrated a lesser cellular infiltrationand much milder myocyte damage. Proliferative response and cytotoxicT lymphocyte induction against the donor-type alloantigens werenot impaired in the gld or lpr recipients. These results indicatea substaintial contribution of FasL to cardiac allograft rejection,independent of Fas in the grafts. This raises a possibilitythat FasL may be more generally involved in tissue damage associatedwith various diseases than expected from the expression of Fasin the target organs.     

Treatment with rIFN-gamma has no effect on cardiac allograft rejection.

The effect of recombinant rat interferon-gamma (rRIFN-gamma) on allograft rejection was studied in two rat heart transplantation models. Recipients were treated with rRIFN-gamma by intraperitoneal or intravenous injection, either by bolus or continuous infusion. Treatment was started after transplantation and continued during a period ranging from 4 to 10 days; dosages varied from 2.5 x 10(2) U/kg/day to 3 x 10(6) U/kg/day. Controls were infused with PBS. Treatment with rRIFN-gamma had no effect on allograft survival, irrespective of the route of administration, the dosage used or the duration of treatment. Higher dosages of rRIFN-gamma induced serious morbidity and mortality. In conclusion, systemic treatment of cardiac allograft recipients with rRIFN-gamma has no effect on graft rejection and is associated with serious toxicity and mortality when high dosages are used.     

C6 produced by macrophages contributes to cardiac allograft rejection.

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.     

Role of the endothelial adhesion molecule VCAM in murine cardiac allograft rejection.

Murine heterotopic cardiac isografts (C57B1/6----C57B1/6) undergo transient, non-destructive inflammation that is characterized by the acquisition of microvascular endothelial reactivity with the antibody MECA 32. Cardiac allografts (C57B1/6----DBA/2) undergo destructive inflammation that is characterized by the acquisition of reactivity with the antibody M/K-2, in addition to MECA 32. M/K-2 recognizes the murine endothelial adhesion molecule, VCAM-1. Hence, there appear to be antigen-dependent and antigen-independent forms of graft inflammation. Treatment of cardiac allograft recipients with 200 micrograms/day M/K-2 antibody retarded graft loss by only a few days, and did not interfere significantly with leukocytic infiltration, as detected by limiting dilution analysis of graft-reactive CTL, despite the fact that large amounts of M/K-2 could be detected on graft microvascular endothelia and in the peripheral blood as rejection progressed. These data indicate that VCAM is apparently not essential for the leukocytic infiltration and subsequent rejection of cardiac allografts, and is not involved in leukocytic infiltration of murine cardiac isografts.     

Histologic findings proving the existence of humoral rejection in a cardiac allograft.

Humoral rejection (HR) of the cardiac allograft is neither well characterized nor universally recognized in the transplant/pathology-related literature. One reason for this is that the histologic light-microscopic features are inconsistent, making HR difficult to recognize in the majority of cases. We report an unusual case of cardiac allograft rejection in a patient with a complicated postoperative course who ultimately died within 6 months of transplant. Premortem, HR was not diagnosed in multiple cardiac biopsies. The autopsy revealed unequivocal histologic and immunohistochemical evidence of HR. Vascular distension and endocardial infiltration by numerous macrophages were noted only in the cardiac allograft and were notably absent in the native tissue present within the anastomotic sites. There was capillary deposition of immunoglobulin and complement shown by immunofluorescence staining of frozen tissue, and there was diffuse immunohistochemical staining for C4d within the intramyocardial vessels. This case appears to represent an exaggerated antibody-mediated rejection of the cardiac allograft.     

移植物细胞因子表达与小肠移植排斥反应相关性的实验和临床病例研究

目的结合移植物细胞因子表达的实验和临床病例研究，探索早期诊断小肠移植急性排斥反应的细胞因子相关的敏感指标。方法①两例短肠综合症患者接受活体小肠移植术。定期或病情变化时随时行内镜组织学检查并测定受体大鼠移植物sIL-2R、IL-4、IL-6和IFN-γ表达水平。②BN-LEW大鼠部分小肠移植，A组：SBT（n=20）；B组：SBT＋FK506（2．5mg／kg，n=20），术后第1、4、7、14和30天测定受体大鼠移植物sIL-2R、IL-4、IL-6和IFN-γ水平同时取移植肠黏膜行病理组织学检查。结果首例术后67d发生排斥反应，第2例于术后20d和80d分别发生强烈排斥反应。发生排斥反应相应时相均发生IL-2Rα、IFN-γ表达的显著升高，排斥反应控制后IL-2Rα迅速恢复，但IFN-γ仍在较高水平维持较长时间。A组大鼠术后第1天始即显示IL-2Rα、IFN-γ和IL-6表达的显著升高，于术后7d达到最高，移植后14d仍在高水平。B组仅术后第1天出现IL-2Rα、IFN-γ和IL-6表达的迅速升高，第4天已恢复至基本正常。结论移植物IL-2Rα、IFN-γ表达的升高与小肠移植急性排斥反应密切相关，有望成为早期诊断小肠移植急性排斥反应的敏感指标。     

Graft morphology correlates with fibroblast activity in cardiac allograft rejection

Several extracellular matrix substances, such as hyaluronan and fibronectin, may affect graft viability by their involvement in cell adhesion and in migration. These substances are produced locally in the tissue by fibroblasts. The aim of this study was to investigate the activation state of intragraft fibroblasts under various immunosuppressive treatments and to correlate these with morphological parameters. Syngeneic (n = 5) and allogeneic rat (n = 5-6/group) heterotopic heart transplantations were performed. Allogeneically transplanted animals were immunosuppressed with cyclosporine, mycophenolate mofetil or prednisolone. After 10 days, the transplanted hearts were removed for subsequent isolation of intragraft fibroblasts and for evaluation of graft morphology. The hyaluronan synthesis of graft fibroblasts correlated with the cellular infiltration (p < 0.05) and the interstitial oedema (p < 0.05) of the cardiac grafts. In general, proliferation rate and hyaluronan production were of the same magnitude in fibroblasts from allogeneic hearts under immunosuppression with cyclosporine, mycophenolate mofetil or prednisolone as in fibroblasts from syngeneic grafts. A pool of fibroblasts isolated from cardiac grafts of non-immunosuppressed, allogeneically transplanted rats (n = 4) showed considerably higher levels. We concluded that fibroblast activity correlates to the viability of the tissue rather than to the specific drug used for immunosuppression.     

Granzyme A and perforin as markers for rejection in cardiac transplantation

The use of granzyme A and perforin as markers for rejection after cardiac transplantation has been investigated. Using in situ hybridization we have detected lymphocytes expressing granzyme A and perforin RNA that are infiltrating the donor heart after transplantation. A total of 29 different biopsies from 17 different patients who had undergone cardiac transplantation were examined. Twelve biopsies classified by conventional histological criteria as showing evidence of rejection were found to contain lymphocytes expressing granzyme A and perforin. Seven biopsies classified as showing no histological evidence of rejection infiltrating lymphocytes were found not to be expressing granzyme A or perforin. However, in 10 other biopsies from 5 different patients that had been classified as showing no evidence of rejection by the conventional grading system, lymphocytes expressing granzyme A and perforin were detected. In six of these cases the patient was found to have undergone a subsequent rejection episode. In the other four cases the biopsies were either taken at a very early stage after transplantation and the high doses of immunosuppression used routinely at that stage are likely to have averted any rejection episodes, or it was not possible to follow subsequent rejection episodes. These results, which are statistically significant (p = 0.06), demonstrate that granzyme A- and perforin-expressing lymphocytes can be identified in rejecting biopsies before histological damage is seen. The identification of perforin and granzyme A expression in vivo suggest a possible role for these proteins in the cytolysis that occurs during transplantation rejection. Furthermore, the data presented here suggest that it may be possible to use granzyme A and perforin as early predictive markers of transplantation rejection.     

Immunological monitoring and early prediction of rejection in renal allograft recipients

Graft rejection remains the major problem complicating renal allograft transplantation. A reliable posttransplant predictor of impending rejection will be valuable to help maintain better graft function. We monitored 47 patients with end-stage renal disease treated by renal allograft starting 1 day pretransplantation and continuing for up to 90 days postgrafting. Peripheral blood mononuclear cells (PBMC) from both patients and 71 healthy subjects were compared for: (1) DNA synthesis in T and B lymphocytes in response to mitogens; (2) interleukin-2 (IL-2) production; (3) natural killer (NK) and antibody-dependent cell-mediated cytotoxic (ADCC) activities; (4) induced augmentation of NK and ADCC activities by the biological response modifiers (BRM), lymphoblastoid interferon, recombinant alpha-2-interferon, gamma-interferon and recombinant IL-2. During the 2 weeks preceding rejection we found lower than normal levels of IL-2 production (p less than 0.0005) and DNA synthesis (p less than 0.01) in concanavalin A-stimulated PBMC. IL-2 yield reached its lowest level on the day of rejection and increased sharply the following week after antirejection therapy was started. Mitogen-stimulated DNA synthesis rose in parallel with increasing levels of IL-2 production. Both NK and ADCC activities increased during rejection (p less than 0.05). The ADCC response to BRM activation measured during the first 2 weeks postgrafting was found to correlate with the stability of the graft. Recipients whose graft function remained stable had a minimal ADCC response to BRM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphologic and immunohistochemical findings in antibody-mediated rejection of the cardiac allograft

The recognition and acceptance of the entity of antibody-mediated rejection (AMR) of solid organs has been slow to develop. Greatest acceptance and most information relates to cardiac transplantation. AMR is thought to represent antibody/complement mediated injury to the microvasculature of the graft that can result in allograft dysfunction, allograft loss, accelerated graft vasculopathy, and increased mortality. The morphologic hallmark is microvascular injury with immunoglobulin and complement deposition in capillaries, accumulation of intravascular macrophages, and in more severe cases, microvascular hemorrhage and thrombosis, with inflammation and edema of the affected organ. Understanding of the pathogenesis of AMR, criteria and methods for diagnosis, and treatment strategies are still in evolution, and will be addressed in this review.     

A primate model of hyperacute renal allograft rejection.

Hyperacute renal allograft rejection is initiated by primary immune injury to vascular endothelium and is propagated by secondary vasoconstriction, platelet aggregation and intravascular coagulation. Previous dissociation of these primary and secondary events, with graft survival in one human, suggested that the more usual graft failure was due to secondary injury. As a basis for further modification studies, this primate model most closely resembled its counterpart in man, as the onset and intensity of functional, morphologic and biochemical alterations were similar. Unmodified allografts failed within 5 minutes. The earliest and most abnormal finding was marked reduction in renal blood flow affecting all compartments. By 5 minutes, histologic changes of hyperacute rejection as well as antibody and faint C3 deposits were noted, but biopsies suggested that the initial flow reduction was more likely due to vasoconstriction, which was then followed by vascular obstruction. Glomeruli appeared most damaged, but at the highest antibody titer arterial injury was more prominent. Early red cell sequestration and stasis was marked, followed by progressive platelet clumping and neutrophil infiltration. While the decline in renal venous C3 levels was prompt, as in man, early intrarenal activation of the coagulation, fibrinolytic and kinin-forming systems could not be demonstrated, and fibrin formation was sparse by light and fluorescence microscopy. Qualitatively similar histologic and functional alterations were noted in autograft controls. While the initiating event was unclear and may have been accentuated by the arteriovenous shunts utilized, the final mechanism was probably marked vasoconstriction with renal ischemia. Intrarenal C3 consumption was an important finding and was not associated with tissue deposits of antibody or complement; it may provide a parallel with the progressive complement-mediated injury associated with acute myocardial ischemia noted by others. Endothelial injury was not seen in arteries, and all alterations were delayed in onset and progressed more slowly than in allografts. These findings may elucidate the mechanism of early malfunction of most autografts. Treatment of additional autografts with increasing doses of heparin progressively reversed these changes and even prevented the initial reduction in blood flow. Therefore, many alterations consistent with hyperacute rejection which are probably immediately responsible for graft failure can also be initiated by nonspecific, nonimmunologic events and, where injury is less intense, can be prevented pharmacologically. This model provides a means of dissecting the injurious events and subsequent evaluation of the effectiveness and interaction of various agents on the damaging secondary alterations which occur during hyperacute rejection.     

TLSFJM抑制大鼠心脏移植排斥反应的作用

目的　在同种异体大鼠异位心脏移植模型中 ,探讨TLSFJM对急性移植排斥反应的抑制作用及机制。方法　以F344大鼠作为心脏移植受体 ,以LOU/CN大鼠作为心脏移植供体 ,建立大鼠异位心脏移植模型。受体大鼠分为 3组 ,于移植前后分别施以RPMI16 4 0、CsA或TLSFJM。每天观察移植心脏跳动情况 ,并于停跳当天或之前解剖观察。分别取供体大鼠脾细胞作为刺激细胞 ,取受体大鼠脾细胞作为反应细胞 ,进行单向混合淋巴细胞反应。结果　TLSFJM可明显延长大鼠异位移植心脏的存活时间 :RPMI16 4 0对照组移植心脏的存活期全部为 6d ,TLSFJM治疗组最长均可存活 2 7d ,高剂量 (15mg/kg·d)CsA治疗组存活期超过 2 7d。TLSFJM治疗组大鼠脾细胞增殖的cpm值均低于对照组。结论　TLSFJM具有良好的抗急性移植排斥反应作用。TLSFJM对同种异体抗原诱导T细胞增殖的明显抑制 ,可能是其发挥免疫抑制作用的机制之一