Ann Thorac Surg 1998;65:1604-1609
© 1998 The Society of Thoracic Surgeons
Original articles: cardiovascular
Apoptosis Is Involved in Acute Cardiac Allograft Rejection in Rats
Yoshihiko Kageyama, MDa,
Xiao-Kang Li, MDb,
Seiichi Suzuki, MDb,
Hidetoshi Suzuki, MDc,
Kazuya Suzuki, MDa,
Teruhisa Kazui, MDa,
Yukio Harada, MDa
a First Department of Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan
c Second Department of Anatomy, Hamamatsu University School of Medicine, Shizuoka, Japan
b Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, Tokyo, Japan
Accepted for publication January 11, 1998.
Address reprint requests to Dr Kageyama, First Department of Surgery, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, Shizuoka 431-3192, Japan
e-mail: (kageka{at}hama-med.ac.jp)
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Abstract
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Background. Allograft rejection remains a major obstacle to long-term survival in heart transplantation. Recent studies have demonstrated that apoptotic cell death may occur in acute allograft rejection. The purpose of this study was to determine whether apoptotic cell death is involved in rat cardiac allograft rejection through both the perforin/granzyme pathway and the Fas/Fas ligand (Fas-L) pathway.
Methods. Groups of Lewis (RT11) rats underwent heterotopic heart transplantation from disparate DA (RT1a) or syngeneic Lewis rats. The cardiac grafts were harvested 1, 3, or 5 days after transplantation and analyzed for apoptotic cell death using DNA nick-end labeling, immunocytochemistry, and electron microscopy. In addition, the expression of granzyme B, perforin, Fas, and Fas-L messenger RNA were analyzed by reverse transcriptase-polymerase chain reaction.
Results. Apoptotic cell death of cardiac myocytes was prominent in allografts on day 5 after transplantation. Fas gene transcripts were constitutively expressed in both syngeneic and allogeneic grafts, whereas expression of Fas-L was only upregulated in allografts with ongoing rejection. Granzyme B and perforin gene expression were also upregulated in allografts with ongoing rejection.
Conclusions. These results suggest that myocyte apoptosis through both the perforin-granzyme pathway and the Fas–Fas-L pathway may be involved in cardiac allograft rejection in rats.
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Introduction
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Apoptosis and necrosis can be distinguished by morphologic and biochemical parameters. Apoptosis is characterized by cell shrinkage, chromatin condensation, nuclear collapse, and fragmentation of DNA into multiple nucleosome-sized pieces [1]. Apoptotic cell death occurs when cells are signaled to activate an internally encoded suicide program. Recently it has been reported that several genes appear to be responsible for the positive and negative regulation of apoptosis [2]. Cytotoxic T lymphocytes (CTL) induce apoptosis in target cells by two independent pathways: the perforin-mediated pathway acts through the degranulation of proteases (ie, granzymes) together with the pore-forming protein perforin to induce the rapid death of target cells [3, 4]; and the interaction of Fas ligand (Fas-L), presented on the surface of activated CTL, and Fas receptor, expressed on a variety of target cells, also leads to the death of the targets [5]. Recent studies demonstrated that apoptotic cell death is involved in rejection of the liver, and that the extent of apoptosis was correlated with classic indicators of graft rejection [6].
The purpose of this study was to determine whether apoptotic cell death occurs in cardiac allograft with ongoing rejection through the two pathways mentioned above. Electron microscopy and immunohistochemistry, as well as in situ DNA nick-end labeling method, were used to detect apoptosis. The gene expression of granzyme B, perforin, Fas, and Fas-L was also investigated by reverse transcriptase-polymerase chain reaction (RT-PCR).
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Material and methods
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Animals
Male inbred Lewis rats (RT-11), weighing 200 to 250 g, and male inbred DA rats (RT-1a), weighing 200 to 250 g, were purchased from Nippon SLC (Shizuoka, Japan). All animals had unrestricted access to water and food, and were housed in accordance with institutional animal care policies.
Heterotopic cardiac transplantation
Allogeneic (DA donor to Lewis recipient) and syngeneic (Lewis to Lewis) heterotopic cardiac transplantations in the cervical portion were performed under ether anesthesia, essentially as described previously [7]. Recipient rats were divided into two groups: group 1 (n = 24) consisted of syngeneic transplantations, and group 2 (n = 24) consisted of allogeneic transplantations. Briefly, the right external jugular vein and the right common carotid artery of the recipient were exposed, and then the pulmonary artery of the donor heart was anastomosed to the right external jugular vein of the recipient, and the donor ascending aorta was anastomosed to the recipient right common carotid artery using a cuff technique [8]. The cuff, composed of a polyethylene tube, was shaped into two consecutive parts having a 2.5-mm body and a 1.5-mm extension. The procedures were clean, but not sterile. No antibiotic prophylaxis was used. The grafted hearts were checked daily by palpation, and the day of cardiac arrest was defined as the last day of graft survival. Twelve recipients from each group were observed for the survival study.
Specimens
In addition to the survival study, four recipients from each group were sacrificed on days 1, 3, and 5 after grafting and the cardiac grafts were harvested. Tissue blocks from the grafts approximately 1 cm3 were embedded in OCT compound (Tissue-Tek, Elkhart, IN), and snap-frozen in isopentane, after which four frozen sections were cut on a cryostat for DNA fragmentation analysis and immunohistology. Another part of the graft was fixed in 10% neutral buffered formalin for routine histology. Other animals from group 1 and group 2 were sacrificed on day 3 after grafting for electron microscopy.
In situ dna fragmentation assay
An ApopTag Plus Kit (Oncor, Gaithersburg, MD) was used to detect DNA fragmentation. Cryosections (6 µm) were cut, fixed in 10% neutral buffered formalin in a coplin jar, and quenched in 0.5% to 1% hydrogen peroxide in phosphate-buffered saline for 5 minutes at room temperature. Then each section was incubated with deoxynucleotidyl transferase (TdT) and digoxigenin-conjugated dUTP in 38 µL of reaction buffer at 37°C for 1 hour. The reaction was terminated by incubation with a prewarmed working strength stop/wash buffer for 30 minutes at 37°C. For visualization of incorporated dUTP, sections were washed three times and further incubated with 3,3'-diaminobenzidine (DAB) working solution for 3 to 6 minutes at room temperature. The reaction was terminated by washing with H2O, and the sections were counterstained with hematoxylin and then mounted. Negative controls were prepared by substituting phosphate-buffered saline for TdT in the reaction mixture. An apoptotic index (AI) [9], the ratio of the positively stained area to the whole area, was calculated for each sample in a randomly chosen appropriate field at a magnification of 200x using FUJIX, HC-2000 (Fuji Film, Tokyo, Japan) and Mac SCOPE/PPC software (Mitani, Fukui, Japan).
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total cellular RNA was extracted from frozen heart tissue using ISOGEN (Nippon Gene, Tokyo, Japan) as directed by the manufacturer. The quality of the RNA was confirmed on formaldehyde-agarose gel. One microgram of total RNA was used for first-strand complementary DNA (cDNA) synthesis in 20 µL of 100 mmol/L Tris-HCL, 500 mmol/L KCL, 5 mmol/L MgCl2, 1 mmol/L dNTP, 1 U/µL RNase inhibitor, random 9mer primer, and 0.25 U/µL avian myeloblastosis virus reverse transcriptase. Polymerase chain reaction amplification was done in 100 µL of reaction mixture containing 200 µmol/L of each of the usual dNTPs, 10 pmol of each rat primer (granzyme B: forward—5'CCC AGG CGC AAT GTC AAT3', reverse—5'CCA GGA TAA GAA ACT CGA3'; perforin: forward—5'AAC TAA ATA ATG AGA GAC GCC3', reverse—5'ATG CTC TGT GGA GCT GTT AAA3'; Fas: forward—5'ATG CTG TGG ATC TGG GCT GTC3', reverse—5'TCA CTC CAG ACA TTG TCC TTC A3'; Fas-L: forward—5'GAG AAG GAA ACC CTT TCC TG3', reverse—5'ATA TTC CTG GTG CCC ATG AT3') [10–12], and 2.5 U of Taq DNA polymerase (TaKaRa, Shiga, Japan). We used the TaKaRa Thermal Cycler 480 PCR system and the "hot start" technique to increase specificity. The PCR was done for 40 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension for 90 seconds at 72°C. Polymerase chain reaction products (10 µL) were analyzed on 1.8% agarose gel, and the prominent bands of the correct size were visualized with ethidium bromide staining.
Cardiac histology
Cardiac grafts were excised, fixed in formalin, and embedded in paraffin. Tissue sections were cut and stained with hematoxylin and eosin.
Immunohistology
Cardiac samples harvested on days 1, 3, and 5 after grafting were snap-frozen in liquid nitrogen and stored at -70°C until sectioning on a cryostat. T cells were detected using an anti–
/ß T-cell receptor R73 monoclonal antibody (mAb) (Serotec, Oxford UK). Slides were air-dried and fixed in acetone at -20°C overnight, followed by air drying for 1 hour. R73 was a goat anti–mouse Ig conjugated to horseradish peroxidase (Pharmingen, Sandiego, CA) and was diluted at 1:100 in the abovementioned working solution. Color was developed with a DAB peroxidase substrate Tablet Set (Sigma, St. Louis, MO). Optimum morphologic presentation was obtained when the sections were air-dried for 1 hour before counterstaining with hematoxylin (Sigma).
Transmission electron microscopy
Immediately after removal of the grafts, samples from the left ventricle were fixed for 2 hours at 4°C in phosphate buffer containing 2.5% glutaraldehyde (pH 7.4). The samples were postfixed in 0.1 mol/L phosphate buffer containing 1% osmium tetraoxide (pH 7.4), dehydrated in a graded ethanol series, and embedded in Quetol 812. Thin sections were cut on an Ultracut (Leica, Vienna, Austria) ultramicrotome equipped with a diamond knife. Ultrathin sections were stained with saturated uranyl acetate (5 minutes) and 0.4% lead citrate (3 minutes) before observation with a JEM-100CX (JEOL, Tokyo, Japan) electron microscope at 80 kV.
Statistical analysis
The differences of AI values between the groups were assessed with Student’s unpaired t test. A probability value of less than 0.05 was considered significant.
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Results
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Graft survival
Twelve cardiac grafts in syngeneic recipients survived indefinitely. In contrast, the median survival time of DA cardiac grafts in Lewis recipients was 6 days (range, 5 to 7 days), by which time the allografts showed extensive infiltration with inflammatory cells.
Immunohistochemistry and DNA fragmentation
There was marked infiltration of mononuclear cells in the left ventricular wall of the allografts at 5 days after transplantation (Fig 1). These cells included a large number of R73-positive T cells in all specimens observed (Table 1). A few cardiac myocytes and lymphocytes undergoing apoptosis were observed in the allografts on day 1 after grafting, and the number of apoptotic cells in the grafts reached a peak on day 5 (Fig 2). Few apoptotic cells were seen in syngeneic grafts on day 3, whereas the number of apoptotic cells was significantly larger in allografts on day 3 (AI, 0.065 ± 0.007, p = 0.015) and day 5 (AI, 0.094 ± 0.017, p = 0.023) (Fig 3). The apoptotic cells in the allografts were mainly cardiac myocytes. In the samples collected at different times after transplantation, the presence of apoptosis varied with rejection grade (Table 2). DNA fragmentation, as visualized by immunostaining of nick-end labeled DNA fragments, was clearly detected in myocytes of the allografts undergoing acute rejection. Five days after allogeneic heart transplantation, DNA fragmentation was detected in myocytes with dense nuclear margination.
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Fig 1. Hematoxylin and eosin staining of left ventricular (A) isograft and (B) allograft sections at 5 days after transplantation. Infiltrating cells in the allografts showed an increase in number when compared with the syngeneic grafts.
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Fig 2. Detection of DNA fragmentation in the (A–C) isograft and (D–F) allograft left ventricle at (A, D) 1, 3 (B, E), and 5 (C, F) days after transplantation. The allografts display positively stained myocytes with morphologic features of apoptosis, including dense nuclear margination.
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Fig 3. Histogram of the apoptotic index (AI) values. Apoptotic cells were detected using in situ DNA nick-end labeling. Values were calculated for randomly chosen appropriate fields at a magnification of 200x using FUJIX, HC-2000, and Mac SCOPE/PPC software. The AI of allografts (Allo) was increased in comparison with that of isografts (Iso). (*p = 0.015, **p = 0.023.)
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Gene expression (granzyme B, perforin, Fas, and Fas-L)
Fas transcripts were detected in both syngeneic and allogeneic grafts, and the level of expression was not dramatically altered after either type of grafting. In contrast, Fas-L transcripts were essentially undetectable in syngeneic grafts, but were strongly induced in allografts. Fas-L showed enhanced expression as early as day 1 after transplantation, and was strongly upregulated on days 3 and 5. Expression of granzyme B and perforin mRNA was also upregulated in rejecting allografts (Fig 4).
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Fig 4. Messenger RNA expression for Fas, Fas ligand (Fas-L), granzyme B (Gr. B), and perforin in syngeneic (Syn) and allogeneic (Allo) grafts. Fas transcripts were constitutively expressed in both the syngeneic and allogeneic grafts, and the level of expression was not dramatically altered after grafting. Fas ligand messenger RNA was undetectable in syngeneic grafts, but was upregulated in rejecting allografts. Expression of granzyme B and perforin messenger RNA was also upregulated in rejecting allografts.
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Transmission electron microscopic findings
Morphologic evidence of myocytes and lymphocytes undergoing apoptosis was detected in the allografts on day 3, although the number of apoptotic lymphocytes was smaller (Fig 5). The changes included cell rounding, appearance of cell surface irregularities, intact mitochondria, bleb formation, and cell fragmentation.
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Fig 5. Ultrastructural observation of the left ventricle from an allograft 3 days after transplantation. The characteristic features of apoptosis, such as chromatin condensation (bold arrow), formation of apoptotic bodies (fine arrow), and absence of surface microvilli, can be seen.
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Comment
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T cell-mediated cytotoxicity plays an important role in cellular immunity. Recently, two mechanisms have been shown to account for the cytotoxic activity of in vitro and in vivo generated CTL and T cell lines [13–15]. One mechanism is the granule exocytosis pathway that depends on perforin and granzyme. The other involves the Fas/Fas-L pathway [16, 17]; both pathways lead to apoptotic cell death. The present study was designed to determine whether apoptotic cell death occurs during ongoing rejection of cardiac allografts. We found that apoptotic cell death did occur during cardiac allograft rejection and that it was apparently mediated by both the perforin and Fas pathways. Apoptotic myocytes were detected in hearts undergoing rejection using DNA nick-end labeling and electron microscopy.
The number of infiltrating lymphocytes was not significantly different between syngeneic and allogeneic grafts on 1 day after transplantation. However, there was a marked increase of infiltrating lymphocytes in the allografts by 3 to 5 days after transplantation, but this was not observed in the isografts. Electron microscopy and in situ DNA nick-end labeling demonstrated a large number of apoptotic cardiac myocytes in the grafts with ongoing rejection. Apoptotic cell death was involved in the early phase of allograft rejection.
Cytotoxic T lymphocytes are immune effector cells involved in allograft rejection that destroy their target cells by either a calcium-dependent (perforin/granzyme) or calcium-independent (Fas/Fas-L) pathway. In the present study, RT-PCR analysis showed that Fas transcripts were constitutively expressed in syngeneic and allogeneic grafts, whereas Fas-L transcripts were up-regulated in the rejecting allografts. Fas transcripts were undetectable in normal hearts, and the level of expression was not dramatically altered in either syngeneic or allogeneic grafts after transplantation. However, Fas-L mRNA was essentially undetectable in normal hearts and syngeneic grafts, whereas it was strongly expressed in allogeneic grafts. Thus, expression of Fas-L may play an important role in allograft destruction as well as in immunoregulation, even though Fas/Fas-L interactions are not essential mediators of T cell-induced allograft damage [18].
The expression of perforin and granzyme B genes was also upregulated during rejection. These results support other studies on cardiac, small intestine, and kidney allografts. Perforin and granzyme B have been demonstrated by in situ hybridization in heart transplant patients with acute rejection [19] and in a murine heart model [20]. In a rat small intestine model, McDiarmid and colleagues [21] showed by semiquantitative RT-PCR that prominent upregulation of mRNA for perforin and granzyme B occurred after day 7 in rejecting rat allografts. In kidney allografts, immunostaining with an antiperforin antibody showed a marked increase in the number of positively staining cells within the graft from 1 hour after transplantation to the time of acute rejection [22]. Finally, the in vivo expression of granzyme and perforin has been correlated with activated CTL and allograft rejection [23].
Our results demonstrated that apoptotic cell death may be involved in rat cardiac allograft rejection. Cytotoxic T lymphocytes in rejecting allografts are likely to induce myocyte death by the interaction between Fas and Fas-L or by the release of perforin and granzyme B, and another study has also concluded that apoptosis of myocardial cells occurs during cardiac allograft rejection [24]. On the other hand, there has been a report that apoptosis did not correlate with graft failure or parenchymal cell damage, suggesting that CTL-mediated destruction of graft tissues is rare in cardiac allografts [25]. It remains to be determined whether apoptosis is an indicator of acute rejection. However, the present data suggest that the strong upregulation of Fas-L and granzyme B together with perforin mRNA may be useful markers for the diagnosis of early graft rejection in heart transplantation.
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Acknowledgments
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We thank Naoko Funeshima, Chie Komatsu, and Isao Ohta for their skillful technical assistance, and Dr Ko Bando for his helpful advice.
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Abstract
Introduction
