Syngeneic Heart Grafts Research Articles

High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy for the Metabolic Assessment of Acute Rejection After Cardiac Transplantation in Rats

PurposeTo evaluate the potential of high-resolution magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy for metabolite characterization and the differentiation of acute rejection after heart transplantation in rat models. MethodsWe transplanted syngeneic heart grafts from Lewis rats (n = 4) and allogeneic heart grafts from F344 rats (n = 4) heterotopically into Lewis recipients. On day 7 postoperatively, the transplanted hearts were harvested for ex vivo 1H NMR spectroscopy and HR-MAS 1H NMR spectroscopy. 1H NMR spectroscopy and HR-MAS 1H NMR spectroscopy were performed at 4.7 T and 11.7 T, respectively. Metabolomic profiles contributing to the differentiation of allogeneic and syngeneic graft groups were statistically assessed by orthogonal partial least squares discriminant analysis (OPLS/O2PLS-DA). Metabolite concentrations were normalized by total spectral intensities and were compared using Mann-Whitney U tests. ResultsOne allogeneic graft that showed extensive necrotic change suggesting graft failure was excluded from the statistical analysis of the NMR spectroscopy. In the 4.7-T 1H NMR spectroscopy, the creatine peak was decreased in the allogeneic group. The PLS-DA and OPLS/O2PLS-DA score plot demonstrated good discrimination of the allogeneic graft group from syngeneic graft group. The concentrations of creatine, myo-inositol, glucose, niacinamide, hypoxanthine, inosine, and glutamine were significantly decreased in the allogeneic graft group, whereas the concentrations of glycine, phosphoethanolamine, xanthine, sn-glycero-3-phosphocholine, leucine, valine, and tyrosine were significantly increased (P < .05). ConclusionsHR-MAS 1H NMR spectroscopy can metabolically characterize the acute rejection of heart transplantation.

Non-invasive molecular imaging of inflammatory macrophages in allograft rejection.

BackgroundMacrophages represent a critical cell type in host defense, development and homeostasis. The ability to image non-invasively pro-inflammatory macrophage infiltrate into a transplanted organ may provide an additional tool for the monitoring of the immune response of the recipient against the donor graft. We therefore decided to image in vivo sialoadhesin (Sn, Siglec 1 or CD169) using anti-Sn mAb (SER-4) directly radiolabelled with 99mTc pertechnetate.MethodsWe used a heterotopic heart transplantation model where allogeneic or syngeneic heart grafts were transplanted into the abdomen of recipients. In vivo nanosingle-photon emission computed tomography (SPECT/CT) imaging was performed 7 days post transplantation followed by biodistribution and histology.ResultsIn wild-type mice, the majority of 99mTc-SER-4 monoclonal antibody cleared from the blood with a half-life of 167 min and was located predominantly on Sn+ tissues in the spleen, liver and bone marrow. The biodistribution in the transplantation experiments confirmed data derived from the non-invasive SPECT/CT images, with significantly higher levels of 99mTc-SER-4 observed in allogeneic grafts (9.4 (±2.7) %ID/g) compared to syngeneic grafts (4.3 (±10.3) %ID/g) (p = 0.0022) or in mice which received allogeneic grafts injected with 99mTc-IgG isotype control (5.9 (±0.6) %ID/g) (p = 0.0185). The transplanted heart to blood ratio was also significantly higher in recipients with allogeneic grafts receiving 99mTc-SER-4 as compared to recipients with syngeneic grafts (p = 0.000004) or recipients with allogeneic grafts receiving 99mTc-IgG isotype (p = 0.000002).ConclusionsHere, we demonstrate that imaging of Sn+ macrophages in inflammation may provide an important additional and non-invasive tool for the monitoring of the pathophysiology of cellular immunity in a transplant model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0146-7) contains supplementary material, which is available to authorized users.

Origin and Function of Host-Derived Dendritic Cells in Vascularized Organ Transplants.

Donor DC exit organ grafts after transplantation and are replaced by host DC. The origin and function of these tissue-resident, host DC however is unknown. Using flow cytometry and 2-photon intravital microscopy, we observed rapid replacement of donor DC by host DC in both syngeneic and allogeneic heart and kidney grafts within 3 days after transplantation, and demonstrated that the vast majority of host DC were monocyte-derived (Mono-DC). By day 7, allografts harbored >5-fold more DC than syn grafts and >45-fold more than native tissue. Enhanced DC accumulation in allografts was inhibited by treating recipients with cyclosporine A, indicating a crucial role for the host alloimmune response in DC influx. DC accumulation was only partially dependent on fractalkine receptor (CX3CR1) expression on host monocytes. Host DC that infiltrated grafts localized to perivascular areas, extended dendrites into the vessel lumens, mediated the transmigration of cognate effector T cells (Teff) into the graft, and continued to make stable contacts with the Teff after they had transmigrated. Depletion of host Mono-DC by administering DT to CD11b-DTR mice significantly reduced T cell infiltration and IFN-γproduction in the graft. Hence, host Mono-DC amass rapidly in transplanted organs, replacing donor DC, and accelerate rejection by enhancing Teff migration, retention, and activation. Our results provide novel insights into the pathogenesis and treatment of allograft rejection.

Origin and function of host-derived dendritic cells in vascularized organ transplants (TRAN3P.889)

Abstract Donor DC exit organ grafts after transplantation and are replaced by host DC. The origin and function of these tissue-resident, host DC however is unknown. Using flow cytometry and 2-photon intravital microscopy, we observed rapid replacement of donor DC by host DC in both syngeneic and allogeneic heart and kidney grafts within 3 days after transplantation, and demonstrated that the vast majority of host DC were monocyte-derived (Mono-DC). By day 7, allografts harbored &amp;gt;5-fold more DC than syn grafts and &amp;gt;45-fold more than native tissue. Enhanced DC accumulation in allografts was inhibited by treating recipients with cyclosporine A, indicating a crucial role for the host alloimmune response in DC influx. DC accumulation was only partially dependent on fractalkine receptor (CX3CR1) expression on host monocytes. Host DC that infiltrated grafts localized to perivascular areas, extended dendrites into the vessel lumens, mediated the transmigration of cognate effector T cells (Teff) into the graft, and continued to make stable contacts with the Teff after they had transmigrated. Depletion of host Mono-DC by administering DT to CD11b-DTR mice significantly reduced T cell infiltration and IFN-γ production in the graft. Hence, host Mono-DC amass rapidly in transplanted organs, replacing donor DC, and accelerate rejection by enhancing Teff migration, retention, and activation. Our results provide novel insights into the pathogenesis and treatment of allograft rejection.

Effects of Preexisting Autoimmunity on Heart Graft Prolongation After Donor-Specific Transfusion and Anti-CD154

Alloreactive memory T cells prevent costimulatory blockade-induced heart graft survival in mice, but whether and how preexisting autoreactive T cells affect solid-organ transplants under these conditions is unknown. We tested the impact of preexisting cardiac myosin (CM)-specific immunity on murine heart transplant recipients treated with donor-specific transfusion (DST) plus anti-CD154 monoclonal antibody MR1. Preimmunization with CM but not control ovalbumin abrogated the graft prolonging effects of DST/MR1, whether administered 2 weeks or more than 6 weeks before transplantation. Adoptive transfer of spleen cells from CM-immunized mice into naïve recipients had similar effects. CM-specific immunity did not cross-react with donor antigens and preimmunization with CM had no impact on the survival or histology of DST/MR1-treated syngeneic heart grafts, the latter indicating that persistent autoimmunity is insufficient to cause rejection in the context of costimulatory blockade. We observed that the CM preimmunized mice produced higher frequencies of donor-reactive T cells with higher ratios of CD8/CD4Foxp3 cells, suggesting that the autoreactive memory T cells provide help for activation of alloreactive T cells despite the costimulatory blockade. These mechanistic insights linking autoimmunity and alloimmunity in a model of murine heart transplantation have clinical relevance to the known association between autoimmunity and an elevated risk of acute and chronic heart transplant injury in humans.

Analysis of intragraft MicroRNA expression in a mouse-to-rat cardiac xenotransplantation model

Xenograft rejection poses the largest obstacle to successful xenotransplantation. Recent studies have demonstrated that miRNAs play essential roles in embryogenesis, cell proliferation, and pathogenesis of human diseases. However, the role of miRNA in regulating xenograft rejection is relatively unknown. This study was undertaken to analyze the profile of intragraft miRNA expression in a heterotopic mouse-to-rat cardiac xenotransplantation model. Using microarray analysis, a total of 579 miRNAs were detected in the grafts following transplantation. When compared with syngeneic heart grafts, 24 and 25 miRNAs were found to differentially express in xenografts at 24 and 40 hours (endpoint of rejection), respectively, following transplantation. Three major miRNAs were then further analyzed, and it was found that the xenografts showed high expression of miR-146a and miR-155, but low expression of miR-451 when compared with isograft controls. This study suggests that miRNAs detected in this model are potentially involved in the xenogeneic immune response and could play an important role in regulating xenograft rejection.

A novel method of preserving cardiac grafts using a hydrogen-rich water bath

Exogenously administered hydrogen exerts cytoprotective effects through anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms in various disease settings, including organ transplantation. Our objective in this study was to evaluate the efficacy of a novel cold storage device equipped with a hydrogen-rich water bath. The study used an established rat heterotopic transplantation model. Syngeneic heart grafts from elderly donors (60- to 70-week-old Lewis rats) or allografts from adult donors (12-week-old Brown Norway rats) were exposed to prolonged cold preservation. The cardiac grafts were stored in plastic bags containing Celsior, which were immersed in the cold water bath equipped with an electrolyzer to saturate the water with hydrogen. The cardiac grafts then were heterotopically engrafted into Lewis rat recipients. In both experimental settings, serum troponin I and creatine phosphokinase were markedly elevated 3 hours after reperfusion in the control grafts without hydrogen treatment. The grafts exhibited prominent inflammatory responses, including neutrophil infiltration and the upregulation of messenger RNAs for pro-inflammatory cytokines and chemokines. Myocardial injury and inflammatory events were significantly attenuated by organ storage in the hydrogen-rich water bath. The grafts stored using the hydrogen-rich water bath also exhibited less mitochondrial damage and a higher adenosine triphosphate content. Hydrogen delivery to cardiac grafts during cold preservation using a novel hydrogen-supplemented water bath efficiently ameliorated myocardial injury due to cold ischemia and reperfusion. This new device to saturate organs with hydrogen during cold storage merits further investigation for possible therapeutic and preventative use during transplantation.

Effector mechanisms in murine allograft rejection: comparison of skin and heart grafts in fully allogeneic and minor histocompatibility antigen-mismatched strain combinations

Abstract Cytotoxic T lymphocytes (CTLs) and macrophage-mediated delayed-type hypersensitivity (DTH) responses may both mediate allograft rejection. Furthermore, although allograft rejection is classically considered a type [22, 23, 38, 50, 52] 1 cellular immune response, type-2 cytokines can support rejection. This study examines whether the immunogenicity of the transplanted tissue, as determined by type of tissue (skin versus heart) and degree of antigenic mismatch, influences recruitment of these effector mechanisms. Graft survival, histological appearance and intragraft gene expression (IL-2, IFN-γ, IL-12 p40, IL-4, IL-10, perforin, Fas ligand (Fas L), iNOS and TNF-α) were compared for fully allogeneic, minor histocompatibility (mHC) antigen-mismatched and syngeneic skin and heart grafts. We found mRNA characteristic of CTLs and DTH responses in fully allogeneic and mHC antigen-mismatched skin and heart grafts. Concomitant type-1 and type-2 cytokine gene transcription was seen. These findings demonstrate that the tissue grafted and degree of antigenic disparity between donor and recipient do not restrict the repertoire of cellular immune responses involved in graft rejection. This finding has implications in the design of new immuno-suppressive strategies for clinical transplantation.

Early chemokine cascades in murine cardiac grafts regulate T cell recruitment and progression of acute allograft rejection.

The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2(a)) and syngeneic heart grafts in C57BL/6 (H-2(b)) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene alpha (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1beta and 1alpha. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts.

Autoimmune-Mediated Vasculopathy

The use of the immunosuppressive drug cyclosporine A (CsA) in solid organ transplantation can be associated with the development of vasculopathy as part of the complex immune response involved in chronic rejection, including autoimmune recognition. Although CsA can directly affect endothelial cells, this drug alters the T cell repertoire promoting autoimmune recognition. The present studies evaluated the ability of CsA-induced autoreactive T cells to mediate vascular lesions in syngeneic heart grafts. Graft vasculopathy developed in syngeneic heart grafts following either the primary induction of autoimmunity with CsA or the adoptive transfer of CsA-induced autoreactive T cells. Initially, an inflammatory response occurred in the medial wall of the small arterial vessels, accompanied by a perivascular lymphocytic infiltrate (including a lymphocytic infiltrate into the myocardium), followed by progression of vascular disease with endothelial cell proliferation. The development and progression of vascular disease correlated with the cytokine profile of the infiltrating lymphocytes with type 1 cytokines detected early and type 2 cytokines detected as the disease progressed. Initiation of this response correlated with upregulation of the target antigen recognized by the CsA-induced autoreactive T cells, the MHC class II–invariant chain peptide complex. This antigen complex, when upregulated on endothelial cells by interferon, allowed effective targeting by the autoreactive T lymphocytes. Strategies to inhibit the upregulation of MHC class II antigens by treatment of the recipients with chloroquine truncated the disease process. The results of these studies suggest that CsA-induced autoreactive mechanisms can contribute to the development of graft vasculopathy.

Evolution on the immunopathology of Chagas disease.

The pathogenesis of Chagas disease is still con-troversial. The lesions that occur in the acute phaseof infection are initially due to rupture of the heartfibers by the Trypanosoma cruzi. When the para-site antigens sensitize the immune system, the in-flammatory process is due to a sum of the trau-matic mechanism and the immune reaction againstthe parasite.After the acute phase and the development ofthe specific immune response occur an equilibriumbetween the host and the parasite which can lastfor all the life of the host. The T. cruzi as a trueparasite do not kills the host and the immune sys-tem of the host do not kills all the parasites. De-spite it can be detected specific humoral and cellu-lar immune response against the T. cruzi, this para-site evades the immune response mainly penetrat-ing and proliferating in host cells. The equilib-rium phase is called indeterminate phase whenoccur a clearance of parasites from bloodstreamand tissues. Patients present positive serology toT. cruzi antigens but do not present signs and symp-toms of disease.At about 10 to 15% of infected individuals de-velop a chronic heart disease characterized bychronic diffuse and fibrosing myocarditis with ab-sence or very few parasites. Different mechanismshave been proposed to explain that aggressivemyocarditis similar to a rejection of an allogeneicheart transplant. It has been suggested that the le-sions result from gradual destruction of the heartfibers by parasites or by a mechanism of cellularhypersensitivity directed to parasite antigens whichaffect also the heart tissue. Another mechanismproposed is the autoimmune mechanism.Soares and Ribeiro dos Santos presented re-sults of several experiments about immunopathol-ogy of cardiomyopathy in experimental Chagasdisease that indicate autoimmune mechanism as anstrong candidate to explain the pathogenesis ofchagasic myocardiopathy. They show an elegantexperiment where mice chronically infected withT. cruzi reject syngeneic heart grafts. Hearts graftedinto normal or T. cruzi immunized syngeneic re-cipients are not rejected. The study shows that onlyCD4 and not CD8 T cells from chronically infectedmice were able to promote rejection. Mice chroni-cally infected with T. cruzi were treated with anti-CD4 and anti-CD8 monoclonal antibodies beforetransplantation. The in vivo depletion of CD4 cells,but not CD8 cells abrogates rejection.In another experiment it was shown that chroni-cally infected mice treated with anti-CD4 mono-clonal antibodies presented tolerance with abolish-ment of the chronic carditis. The opposite occurredwhen animals were treated with anti-CD8 or anti-Thy1. Tolerant animals presented low mortality andnegative parasitemia. I can not understand whyanimals that had T-CD4 cells destroyed had a nega-tive parasitemia. The expectance was to haveimmunodeficience similar to Aids with a high para-sitemia. The phenotype of cells present in chagasicmyocarditis has been controversial as in mice as inhumans. Some authors refer predominance of CD4cells and others predominance of CD8 cells. In ourexperience with material of necropsy of humanswho died with Chagas disease (Castro-Silva &Sadigursky) we had in 80% of patients predomi-nance of CD4 cells, 10% with predominance ofCD8 cells and 10% with predominance of mac-rophages. Macrophages were seen in variable num-ber in all the cases.Other experiments with lines of autoreactiveCD4 cells from mice chronically infected with T.cruzi reinforce the hypothesis of autoimmunity inchronic Chagas disease.Correa-Oliveira et al. presented “The role ofthe immune response on the development of hu-man severe clinical forms of Chagas disease”. Thesecretion of IFN γ can be correlated with the se-vere cardiac form of Chagas disease. The oppositewas observed for in vitro secretion of IL10. Thestudies indicated that as the cardiac function wors-ens, IFN γ secretion by PBMC increases with aparallel decrease on secretion of IL10. They con-clude that IFN γ secretion can be correlated withthe development of severe cardiac form of the dis-ease. I believe that the high level of IFN γ secre-

A new rat model to study the correlation of cardiac and skeletal muscle allograft rejection.

As a first step to study the correlation of cardiac and skeletal muscle allograft rejection, we describe a new experimental rat model of simultaneous heterotopic heart and cutaneous maximus muscle flap allotransplant. Brown Norway rats were used as donors and Lewis rats as recipients. No immunosuppression was given. The grafts were revascularized with sequential end-to-side anastomosis of each vascular pedicle to the infrarenal aorta and vena cava. Syngeneic heart and cutaneous maximus muscle grafts remained functional and showed no sign of rejection at 7 days after the transplant. In contrast, both allografts developed severe rejection and functional compromise at 7 days after the transplant. Our experimental model is technically feasible and reproducible and may provide important information about the pattern of rejection of cardiac and skeletal muscle allografts.

Effect in supralethally irradiated rats of granulocyte colony-stimulating factor and lisofylline on hematopoietic reconstitution by syngeneic bone marrow or whole organ passenger leukocytes.

We have previously shown the existence of migratory hematopoietic stem cells in adult solid organs. This study demonstrates that granulocyte colony-stimulating factor (G-CSF) and lisofylline, a phosphatidic acid inhibitor that suppresses hematopoiesis-inhibiting cytokines, can enhance the engraftment of organ-based hematopoietic stem cells. When syngeneic heart grafts or liver nonparenchymal cells were transplanted into lethally irradiated (9.5 Gy) Lewis rats, complete hematopoietic reconstitution and animal survival were significantly improved by treating the recipient with G-CSF or, to a lesser extent, with lisofylline. Pretreatment of hepatic nonparenchymal cell donors with G-CSF, but not lisofylline, also resulted in striking improvement of recipient survival which was associated with an augmented subpopulation of donor stem cells. The results suggest that these drugs can be used to enhance the chimerism that we postulate to be the basis of organ allograft acceptance.

An age‐related γδ T cell suppressor activity correlates with the outcome of autoimmunity in experimental Trypanosoma cruzi infection

In this work the suppressive activity of splenic T cells from young and aged BALB/c mice infected with Trypanosoma cruzi were compared and correlated with the development of autoimmune myocarditis. The T cells from young adult BALB/c mice with acute T. cruzi infection exhibit suppressor activity when added to full allogeneic or Mls-disparate mixed lymphocyte cultures. This suppression could not be reverted by exogenous interleukin (IL)-2 and was not directly dependent on the presence of IL-4, IL-10 or transforming growth factor-beta. Further characterization of the T cell lineage responsible for the suppressor activity by in vitro and/or in vivo depletion with monoclonal antibody to alpha beta or gamma delta T cell receptor revealed that splenic gamma delta T cells function as suppressor lymphocytes in young T. cruzi-infected mice. In addition, these young adult BALB/c mice do not develop autoimmune myocarditis and showed a low incidence of syngeneic heart graft rejection in the early chronic phase of the infection. In contrast, T cells from acutely infected aged BALB/c mice lacked demonstrable T suppressor activity. Furthermore, these mice developed a severe autoimmune myocarditis as early as 2 months after the onset of the infection, when the majority of them reject syngeneic heart grafts. These findings suggest that a gamma delta T cell-mediated suppressor mechanism may operate in the avoidance of the breaking of tissue-specific tolerance during the acute infection. Moreover, such a mechanism is likely related to the immune system chronobiology.

EPR detection of heme and nonheme iron-containing protein nitrosylation by nitric oxide during rejection of rat heart allograft.

The paramagnetic molecule nitric oxide (NO), produced from L-arginine by a specific enzyme (NO synthase), has been shown to be involved in a surprising variety of mammalian cellular responses, including the regulation of T cell immunity to alloantigens in vitro. In cytotoxic activated macrophages, NO production results in a characteristic pattern of alteration of iron-containing enzyme function that is mimicked by exposure to NO. Electron paramagnetic resonance (EPR) studies have shown the formation of iron-nitrosyl species during macrophage activation and also during sepsis, indicating that alteration of iron-containing protein function may be the result of the well-documented tendency of NO to bind to metal ions. We have recently shown that the NO synthesis induced during alloantigenic activation of rat splenocytes inhibits lymphocyte proliferation and cytotoxic T-lymphocyte generation. This report demonstrates that iron-nitrosyl EPR signals similar to those observed in macrophages and during sepsis are present in the blood and in the grafted tissue of rats during the rejection of allogeneic (but not syngeneic) heart grafts. These signals are found in the blood and at the site of allograft rejection, but are not found in other tissues (such as spleen and lung), and are obliterated by administration of the immunosuppressant FK506. These results directly demonstrate the formation of iron-nitrosyl complexes during vascularized allograft rejection and suggest that consequent destruction of iron-containing protein function plays an important role in the rejection response.

Anti-CD4 abrogates rejection and reestablishes long-term tolerance to syngeneic newborn hearts grafted in mice chronically infected with Trypanosoma cruzi.

The contribution of autoimmunity in the genesis of chronic Chagas' heart pathology is not clear. In the present study, we show that: (a) BALB/c mice chronically infected with Trypanosoma cruzi reject syngeneic newborn hearts; (b) in vivo treatment with anti-CD4 but not anti-CD8 monoclonal antibodies (mAbs) abrogates rejection; (c) CD4+ T cells from chronically infected mice proliferate in vitro to syngeneic myocardium antigens and induce heart graft destruction when injected in situ; (d) anti-CD4 treatment of chronically infected mice establishes long-term tolerance to syngeneic heart grafts; and (e) the state of tolerance is related to in vitro and in vivo unresponsiveness of the CD4+ T cells. These findings allow us to suggest that autoimmunity is the major mechanism implicated in the rejection of syngeneic heart tissues grafted into the pinna of the ear of mice chronically infected with T. cruzi. The similarity of the lesions to those found in humans suggests that autoimmunity is involved in the pathogenesis of chagasic cardiomyopathy in humans. Moreover, this could imply therapeutic strategies by reestablishing long-term tissue-specific tolerance with anti-CD4 mAb treatment, mediating anergy, or deleting the responder CD4+ T cells to heart tissue antigens.

Dendritic cell-lymphoid cell aggregation and major histocompatibility antigen expression during rat cardiac allograft rejection.

To determine the pattern of cellular expression of donor MHC class I and class II antigens during the course of rat cardiac allograft rejection, ACI cardiac allografts transplanted to BN recipients were examined from day 2 to day 6 using immunohistologic and immunoelectron microscopic methods. We used both monomorphic and donor-specific mouse anti-rat MHC class I and class II mAbs in this study. In normal ACI hearts, MHC class I reactivity was confined to the vascular endothelium and to interstitial cells. Ongoing rejection was characterized by an increased donor MHC class I staining intensity of microvascular endothelium and induction of donor class I surface reactivity on cardiac myofibers. Donor MHC class II reactivity was exclusively confined to interstitial dendritic cells (IDC) in both normal ACI hearts and in rejecting allografts, although rejection was associated with marked fluctuations in class II IDC frequency. An early numerical depression in class II IDC present in both allografts and syngeneic heart grafts was attributed to a direct effect of the transplantation procedure. By days 3-4, allografts showed an absolute overall increase in donor class II IDC frequency, which was associated with the presence of multiple localized high-density IDC-lymphocyte aggregates. The lymphocytes present in the focal areas were predominantly of the class II-reactive Th cell subpopulation. These aggregates may thus represent the in vivo homologue of dendritic cell-lymphocyte clustering, which has been shown to be required for primary class II allosensitization in the rat and mouse in vitro. During the late phase of rejection, there was a marked numerical fall in donor class II IDC, which correlated with extensive overall graft destruction. This study has shown that acute rat cardiac allograft rejection can occur in the absence of donor MHC class II expression by allograft vascular endothelium and cardiac myofibers. The IDC, which are believed to represent the principal class II alloantigen presenting cells in the rat heart, remain the sole class II-expressing cellular constituents of the graft throughout the course of rejection.

Use of indium-111-labeled cells in measurement of cellular dynamics of experimental cardiac allograft rejection.

This study evaluates the kinetics and utility of infused indium-111-labeled cells in detecting rejection in ACI to Lewis rat heart allografts. Syngeneic leukocytes, lymph node lymphocytes, and platelets were isolated and labeled with indium-111 (111In) oxine, respectively, and were infused i.v. into Lewis rats carrying beating ACI or syngeneic hearts from post-transplant days 0 to 6. Recipients were imaged serially at 24 hr after infusion of labeled cells followed by excision of both native and transplanted hearts for direct isotope count. Labeled leukocytes accumulative progressively in the allograft with the scan becoming positive by post-transplant day 4. The ratio of allograft to native heart isotope counts rose from 1.25 on day 1 to 10.07 (P less than 0.0001) on day 7. The Lewis recipients infused with labeled lymphocytes showed a positive scan on days 6 and 7 whereas the allograft to native heart isotope count ratio rose from 0.97 on day 1 to 5.33 (P less than 0.001) on day 7. Recipients infused with 111In-labeled platelets showed a positive scan on days 5 to 7 and the allograft to native heart isotope count ratio rose sharply from 2.56 on day 4 to 16.98 (P less than 0.005) on day 7. Syngeneic heart grafts failed to demonstrate significant accumulation of any of the labeled cell population. These studies confirm the importance of nonlymphocytic cells in cellular rejection, evaluate the kinetics of graft invasion by the various cell types, and suggest that the techniques used afford a method for a safe and an early detection of allograft rejection.