Acute Cardiac Allograft Rejection in Nitric Oxide Synthase-2−/− and Nitric Oxide Synthase-2+/+ Mice
Effects of Cellular Chimeras on Myocardial Inflammation and Cardiomyocyte Damage and Apoptosis
Background—The contribution of nitric oxide synthase (NOS)-2 to myocardial inflammation and cardiomyocyte necrosis and apoptosis during allograft rejection was investigated through heterotopic cardiac transplantation in mice.
Methods and Results—In the first experiments, hearts from C3H donor mice were transplanted into NOS-2−/− and NOS-2+/+ C57BL/6J.129J recipients. A second series of experiments included NOS-2−/− donor hearts transplanted into NOS-2−/− recipients and wild-type NOS-2+/+ donor hearts transplanted into wild-type NOS-2+/+ recipients. (All donors were C57BL/6J and recipients were C57BL/6J.129J.) In the first series of experiments, no significant differences were observed in allograft survival, rejection score, total number of apoptotic nuclei (TUNEL), total number of apoptotic cardiomyocytes, or graft NOS-2 mRNA and protein. Positive NOS-2 immunostaining occurred in endothelial cells and cardiomyocytes in the allografts; the inflammatory infiltrate was NOS-2 positive only when recipients were NOS-2+/+. In the second series of experiments, cardiac allograft survival was significantly increased in the NOS-2−/− mice (26±13 versus 17±8 days, P<0.05), along with significant reductions in inflammatory infiltrate, rejection score, and total number of apoptotic nuclei (23.5±9.5 versus 56.4±15.3, P<0.01) and of apoptotic cardiomyocytes (2.9±1.6 versus 6.9±2.7, P<0.05). No NOS-2 or nitrotyrosine, a marker of peroxynitrite exposure, was detected in NOS-2−/− allografts transplanted into NOS-2−/− recipients.
Conclusions—The data suggest that NO derived from NOS-2 contributes to the inflammatory response and to cardiomyocyte damage and apoptosis during acute cardiac allograft rejection.
The expression of the inducible isoform of nitric oxide synthase (NOS-2) is upregulated during cardiac allograft rejection in lymphocytes and macrophages of the myocardial inflammatory infiltrate, in endothelial cells, in vascular smooth muscle cells, and in cardiac myocytes.1 2 3 4 5 Increased NOS-2 expression in the setting of the cytokine-rich milieu present during cardiac allograft rejection is consistent with in vitro studies in which NOS-2 expression in macrophages, endothelial and smooth muscle cells, and cardiomyocytes was induced by tumor necrosis factor-α, interferon-γ, and interleukin-1β.6 7 8 Increased NOS-2 expression during acute cardiac allograft rejection has been associated with impaired contractility, with pathological changes, and with the death of cardiac muscle cells by necrosis and apoptosis.1 2 3 4 In the studies by Szabolcs et al3 in a rat heterotopic heart transplantation model, there was a parallelism between the time and extent of NOS-2 expression in the allografts and apoptosis of cardiac muscle cells. The presence of NOS-2 and nitrotyrosine, a marker of exposure to peroxynitrite that is formed by interaction of NO with superoxide, has also been observed in association with apoptosis of cardiomyocytes in acute experimental and human heart transplant rejection.3 5 In contrast, NOS-2 expression in chronic cardiac allograft rejection has been associated with an amelioration of the transplant-associated vasculopathy that occurs in this situation.9 10 The present study was designed to investigate the role of NOS-2 in the pathological changes and cardiomyocyte apoptosis associated with acute cardiac allograft rejection in strains of mice in which the NOS-2 gene was functionally deleted (NOS-2−/−). In the first series of experiments, NOS-2+/+ hearts that differed in major histocompatibility antigens from the recipients were transplanted into recipient animals that were either NOS-2+/+ or NOS-2−/−. In the second series of experiments, cardiac transplantation was performed with NOS-2−/− donors and recipients and NOS-2+/+ donors and recipients. The donor and recipient mice differed in minor histocompatibility antigens. In both series, cardiac allograft survival, the histological degree of rejection, the extent of apoptosis, NOS-2 mRNA, and NOS-2 protein were measured along with immunostaining for NOS-2 and for nitrotyrosine in the different cells within the rejecting allografts.
Heterotopic Heart Transplantation
Hearts from donor mice were transplanted into recipient mice by a modification of the technique of Ono and Lindsay11 and were harvested as previously described1 3 for pathological and biochemical examination.
Mouse Genotyping by Polymerase Chain Reaction
Female C3H/J(H-2 k) mice 6 to 8 weeks old (obtained from Jackson Laboratory, Bar Harbor, Me) were used as heart donors. Male mice deficient in inducible NOS (NOS-2−/−) in a C57BL/6J.129J(H-2b) background were used as organ recipients. Results of the C3H/J heart transplantations into the NOS-2−/− recipients were compared with the results of C3H/J heart transplantations into NOS-2+/+ wild-type C57BL/6J.129J(H-2b) mice. The NOS-2–deficient and the wild-type mice were obtained at the F3 generation from Dr V. Laubach.12 Both the NOS-2−/− and wild-type NOS-2+/+ C57BL/6J.129J(H-2b) mice were back-crossed for >10 generations in our laboratory for the second set of experiments. For these experiments, additional NOS-2−/− mice in a C57BL/6J background (ie, back-crossed for 10 generations) and NOS-2+/+ wild-type C57BL/6J mice were also obtained from Jackson Laboratory. The genotypes of the NOS-2−/− and wild-type mice were confirmed by polymerase chain reaction (PCR) analysis of mouse tail DNA and by measurement of NO3− synthesis by peritoneal macrophages from the mice incubated with lipopolysaccharide and interferon-γ.12
PCR for NOS-2
NOS-2 mRNA in myocardial tissue or cells was measured with a semiquantitative reverse transcription (RT)–PCR system using a recombinant mRNA (rcRNA) internal standard that contained target mRNA primer sequences that were 60 bp shorter than the target gene (NOS-2) sequences.3 Semiquantitative competitive RT-PCR was carried out with the rcRNA as a competitive template. For each sample, a dilution series of the rcRNA internal standard was spiked into each aliquot of sample. RT-PCR products generated from the internal standard were distinguished from the products generated from the target gene mRNA by their size difference and by use of different probes in the fluorescein-antifluorescein–based enzyme-linked oligonucleotide sorbent assay. An RNA sample that was not subjected to RT was also run as a control for contamination by genomic DNA.
Western Analysis for NOS-2
Myocardial homogenates were lysed in a buffer containing 150 mmol/L NaCl, 1.0% SDS, 1 mmol/L EDTA, and 50 mmol/L Tris (pH 7.7), supplemented with protease inhibitors (10 μg/mL of antipain and leupeptin, and 1 mmol/L PMSF), and centrifuged at 14 000 rpm for 20 minutes at 4°C. The cytosolic proteins (60 μg/lane) were electrophoresed on a 75% SDS-polyacrylamide gel, transferred to a nitrocellulose filter, and immunoblotted with rabbit polyclonal antiserum raised against the amino acid 117 to 128 sequence of the (mouse) macrophage-inducible NOS at a 1:2000 dilution. Anti-rabbit horseradish peroxidase–conjugated Ab was used as a secondary Ab. Blots were detected with the enhanced chemiluminescence method. The lysates of cytokine-treated macrophages of NOS-2+/+ and NOS-2−/− mice were used as controls.
Immunohistochemistry and Apoptosis Labeling: Antibodies
Two antibodies (Abs) were used to detect NOS-2. The rabbit polyclonal Ab raised against the amino acid sequence 117 to 128 of the mouse macrophage-inducible NO synthase was used for Western blot (dilution 1:2000) and immunohistochemistry (1:500). A second polyclonal anti–NOS-2 Ab was obtained from Transduction Laboratories and was used for immunohistochemistry (1:50). A polyclonal anti-nitrotyrosine Ab (1:100; Upstate Biotechnology) was used to determine whether cells had been exposed to peroxynitrite. Cardiac myocytes were highlighted with polyclonal anti-desmin Abs, 1 produced in rabbit (1:200; Sigma Chemical Co) for immunoperoxidase staining and 1 produced in goat (1:20, Santa Cruz Biotechnology) for immunofluorescence labeling with FITC. Endothelial cells were labeled with the lectin fluorescein Ricinus communis agglutinin I (1:1000, Vector Laboratories).
NOS-2 immunolabeling using either polyclonal anti–NOS-2 Abs, immunolabeling for peroxynitrite, or double labeling for apoptotic nuclei (terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling, TUNEL) and desmin (which highlights cardiac myocytes) was performed as previously described.5
Immunofluorescence staining for labeling of NOS-2 was performed with Cy3-conjugated donkey anti-rabbit IgG (1:2000, Jackson Immuno Research Laboratories, Inc) yielding a red fluorescence signal. Thereafter, sections were incubated with goat anti-mouse desmin (1:20, Santa Cruz Biotechnology, Inc) followed by fluorescein (FITC)-conjugated rabbit anti-goat IgG (1:100) or by fluorescein-conjugated R communis agglutinin I (1:1000). Sections were mounted in Vectashield mounting medium with DAPI. As negative controls, primary antibodies were replaced with nonspecific immunoglobulins from the respective animals in equal concentrations.
Selected frozen sections fixed in paraformaldehyde were dually labeled with rabbit anti–NOS-2 (Transduction Laboratories, 1:50 for 90 minutes) and TUNEL to correlate the distribution of apoptotic nuclei with NOS-2. All reagents for immunofluorescence labeling were obtained from Vector and from Boehringer-Mannheim Co for TUNEL labeling.
The total number of apoptotic nuclei and the number of apoptotic cardiac myocytes were enumerated for an entire coronal section dually labeled for apoptosis (TUNEL) and desmin and are expressed as the number of apoptotic cells per square millimeter. The degree of rejection was evaluated on hematoxylin-eosin–stained sections and quantified by use of the International Society of Heart and Lung Transplantation (ISHLT) grading system for cardiac allograft rejection,13 with ISHLT grades 0, 1A, 1B, 2, 3A, 3B, and 4 rejection converted to a linear scale from 0 to 6.
Graft survival times for the different treatment groups were analyzed by the nonparametric Kruskal-Wallis test. Analysis of apoptosis and pathology scores used a multifactorial extension of the Kruskal-Wallis test to account for the effect of treatment group and time after transplantation.
Hearts from C3H donor mice were transplanted into NOS-2+/+ and NOS-2−/− C57BL/6J.129J recipients. The survival of the C3H allografts in the NOS-2−/− recipients was not significantly different from that observed in the NOS-2+/+ recipients, 7.1±0.8 days (n=14) versus 6.6±1.1 days (n=11).
Additional C3H cardiac allografts were harvested on days 1, 3, and 5 after transplantation. The C3H allografts in NOS-2−/− and in NOS-2+/+ recipients manifested pathological changes and ISHLT rejection scores that were not significantly different at each time point (Table 1⇓). The total numbers of apoptotic nuclei (TUNEL) and of apoptotic cardiac myocytes were also not significantly different (Table 1⇓).
Densitometric measurements in day 5 C3H cardiac allografts showed only insignificant reductions of NOS-2 mRNA and protein in the allografts of NOS-2−/− recipients compared with those in NOS-2+/+ recipients (Figure 1A⇓ and 1B⇓). Immunohistochemistry, however, revealed differences in the cellular distribution of NOS-2 immunoreactivity. In C3H allografts in NOS-2+/+ recipients, positive NOS-2 immunostaining was apparent by dual immunofluorescence labeling in cardiac myocytes (Figure 2A⇓) and endothelial cells (Figure 2B⇓); there was also strong NOS-2 labeling of infiltrating inflammatory cells (Figure 2A⇓ and 2C⇓). In C3H allografts in NOS-2−/− recipients, positive NOS-2 immunostaining was observed in cardiac myocytes and endothelial cells, but NOS-2 staining was not observed in the inflammatory infiltrate (Figure 2D⇓). In additional control experiments, hearts from NOS-2−/− and from NOS-2+/+ C57BL/6J.129 mice were transplanted into C3H recipients. In these experiments, there were no significant differences in survival, ISHLT rejection score, or apoptosis between the NOS-2−/− and NOS-2+/+ allografts (Table 2⇓). There was no immunoreactivity for NOS-2 in the endothelial cells or cardiomyocytes in the NOS-2−/− allografts, whereas strong NOS-2 immunoreactivity was noted in the myocardial inflammatory infiltrate (Figure 2E⇓). Overall, NOS-2 activity was observed in all allografts of this first series of experiments. In the group in which the donor was NOS-2+/+ and the recipient was NOS-2−/−, the donor myocytes and endothelial cells expressed NOS-2 and the inflammatory infiltrate lacked NOS-2 staining. In the groups with NOS-2−/− donors and NOS-2+/+ recipients, the allografts showed strong NOS-2 reactivity in the inflammatory infiltrate derived from the NOS-2+/+ recipient.
Accordingly, a second series of cardiac transplantations was performed to test the hypothesis that the elimination of NOS-2 in both the donor and recipient would result in less severe rejection than that observed when NOS-2 was expressed in both donor and recipient animals. In these experiments, the differences between the 2 groups of transplantations were restricted to the NOS-2 gene and to differences in minor histocompatibility antigens among mice of the inbred C57BL/6J and C57BL/6J.129J strains. Hearts from NOS-2−/− C57BL/6J donors were transplanted into NOS-2−/− C57BL/6J.129J recipients, and hearts from NOS-2+/+ C57BL/6J donors were transplanted into NOS-2+/+ C57BL/6J.129J recipients. The mean survival of the NOS-2−/− allografts that had been transplanted into NOS-2−/− recipients (26±13 days, n=11) was significantly longer (P<0.05) than the mean survival of the NOS-2+/+ cardiac allografts that had been transplanted into the NOS-2+/+ recipients (17±8 days, n=20).
Additional cardiac transplants were harvested on day 12 after transplantation for biochemical and histological measurements. At day 12, NOS-2 mRNA and protein were lower in the NOS-2−/− cardiac allografts than in the NOS-2+/+ allografts and were comparable to values found in native hearts (Figure 3A⇓ and 3B⇓). The magnitudes of the inflammatory infiltrate, tissue edema, and damage to cardiac muscle cells were also lower in the NOS-2−/− cardiac allografts than in the wild-type NOS-2+/+ allografts (Figure 4A⇓ and 4B⇓). The ISHLT rejection score and the total number of apoptotic cells and of apoptotic cardiac myocytes were significantly lower (P<0.05) in the NOS-2−/− cardiac allografts (Table 3⇓) than in the wild-type NOS-2+/+ allografts (Figure 4C⇓ and 4D⇓, Table 3⇓).
Positive immunostaining for NOS-2 protein was observed in endothelial cells, macrophages, and cardiac myocytes in the NOS-2+/+ allografts. Many apoptotic cells, including cardiac myocytes, were strongly NOS-2 positive (Figure 4E⇑). Allografts in which both the donor and recipient were NOS-2−/− showed no NOS-2 reactivity at all (Figure 4F⇑). Similarly, immunostaining for nitrotyrosine, which reflects exposure to peroxynitrite, was strong in the inflammatory infiltrate and adjacent cardiac myocytes in the NOS-2+/+ allografts (Figure 4G⇑). No labeling for nitrotyrosine was identified in NOS-2−/− allografts transplanted into NOS-2−/− recipients (Figure 4H⇑).
The experimental design of our first set of experiments was similar to that reported by Koglin et al.10 14 These investigators transplanted hearts from NOS-2+/+ CBA/CaJ donor mice into NOS-2−/− C57BL/6J.129SvEV recipients and into NOS-2+/+ wild-type C57BL/6J.129SvEv recipients.10 14 They found increased graft survival and a reduction in the mean ISHLT histopathological rejection score and in 3 markers for apoptosis in the NOS-2+/+ CBA/CaJ cardiac allografts transplanted into NOS-2−/− recipients. In our first set of experiments, hearts from NOS-2+/+ C3H donor mice were transplanted into NOS-2−/− and into NOS-2+/+ C57BL/6J.129J recipient mice. Although we anticipated that our results would confirm the findings of Koglin et al,10 14 the data indicated that cardiac allograft survival was not significantly increased, and there was no significant reduction in the ISHLT score or in the number of apoptotic cardiomyocytes in cardiac allografts transplanted into recipient C57BL/6J.129J mice deficient in NOS-2. The difference between our results and those of Koglin et al was unexpected. One likely explanation is that NOS-2 expression in donor myocytes and endothelial cells was more intense in the C3H rejecting allografts used in our experiments than the CBA/CaJ allografts used by Koglin et al. In our experiments, the mRNA at day 5 in the rejecting allografts from C3H donors transplanted into NOS-2−/− recipients was not significantly different from the mRNA at day 5 in the rejecting allografts from C3H donors transplanted into NOS-2+/+ recipients, whereas Koglin et al found that the NOS-2 mRNA was significantly reduced in cardiac allografts from CBA/CaJ mice transplanted into NOS-2−/− mice compared with the mRNA present in allografts from CBA/CaJ donors transplanted into NOS-2+/+ recipients.10 14 In our first set of experiments, immunostaining revealed that the expression of NOS-2 protein was upregulated in endothelial cells and cardiac myocytes in the acutely rejecting cardiac allografts from C3H donor mice placed into NOS-2−/− recipients. Positive NOS-2 immunostaining was not present in the majority of macrophages infiltrating the myocardium of the rejecting C3H allografts, macrophages that presumably originated from the NOS-2−/− recipients. In the study by Koglin et al of acute cardiac allograft rejection,14 positive immunostaining for NOS-2 was not observed in endothelial cells, vascular smooth muscle cells, or cardiomyocytes in the acutely rejecting CBA/CaJ allografts but was observed in macrophages in the allografts. In other studies analyzing chronic allograft rejection, however, Russell et al9 and Koglin et al10 found positive NOS-2 immunostaining in endothelial cells, vascular smooth muscle cells, and cardiac myocytes at later time points (eg, 50 days after transplantation) in association with chronic vascular rejection. This suggests that the time course of NOS-2 expression and the chimera of cells expressing different NOS isoforms in the graft may vary during allograft rejection among the different strains of mice used.
In our second series of heart transplantations, the degree of antigenic disparity between donor and recipient mice was less than in the first series. NOS-2−/− C57BL/6J donors were transplanted into NOS-2−/− C57BL/6J.129J recipients, and NOS-2+/+ C57BL/6J donors were transplanted into NOS-2+/+ C57BL/6J.129J recipients. We observed in the NOS-2−/− cardiac allografts placed in NOS-2−/− recipients that (1) allograft survival was significantly prolonged, (2) the intensity of rejection (ISHLT score) was less, and (3) the mean total number of apoptotic nuclei and the mean number of apoptotic cardiac myocytes were significantly less than in allografts in which both the donor and recipient mice were NOS-2+/+. These data are consistent with the findings of Koglin et al during acute rejection.10 14 Our finding that apoptosis of cardiac myocytes was significantly reduced in the total absence of NOS-2 is new and is consistent with the hypothesis that NO-mediated apoptosis of cardiac muscle cells occurs during acute cardiac allograft rejection.3 5 14 15
NO exerts a bifunctional effect on apoptosis depending on the cell type and features of the microenvironment in which it is synthesized.16 NO released from NO-donor drugs or synthesized by activated macrophages has been demonstrated to trigger apoptosis of macrophages, lymphocytes, mast cells, chondrocytes, vascular smooth muscle cells, pancreatic islet cells, and both neonatal and adult cardiac myocytes.15 Pinsky et al,8 Ing et al,17 and Arstall et al18 found that cytokine induction of NOS-2 in adult rat or neonatal cardiac myocytes was followed by an increase in NO-mediated apoptosis of the cardiomyocytes. These results are consistent with a report by Kawaguchi et al,19 who used a viral vector to transfect NOS-3 into the myocardium of rats and found that the increased expression of NOS triggered apoptosis-like cell death of the transfected cardiac muscle cells.
In contrast, NO has been shown to inhibit apoptosis of hepatocytes, of cardiac myocytes subjected to stretch, and of vascular endothelial cells.16 The disparate biological effects of NO are determined by various factors. These include (1) the amount of NO synthesized (influenced by the number and types of cells producing NO and the NOS isoforms present in the tissue), (2) diffusion of NO (influenced by distance and the presence or absence of carrier molecules), (3) competing target molecules within the diffusion range and their affinities for NO, and (4) the redox state of the microenvironment. (In the presence of superoxide, NO combines with it to form peroxynitrite, which is a strong oxidant that can cause lipid peroxidation, nitration of cell proteins, and DNA strand breaks that have the potential to initiate apoptosis.)20
The prolonged survival, reduced ISHLT rejection score, and significantly reduced total and cardiomyocyte apoptosis in the NOS-2–deficient cardiac allografts found in our second set of experiments confirm and extend previous work that suggests that the NO produced by NOS-2 contributes to heart muscle damage and impaired contractility during cardiac allograft rejection in humans and rats.1 2 3 4 5 9 10 14 The results are consistent with previous reports that cardiac allograft survival was prolonged, contractility was improved, and the intensity of histological changes during rejection was reduced in cardiac allografts treated with the semiselective NOS-2 inhibitor aminoguanidine. They are also consistent with the modest increase in cardiac allograft survival observed in rats treated with a nonselective inhibitor of NOSs.21 The gradual loss of cardiomyocytes over time by NO-triggered apoptosis may contribute to the decline in ventricular performance and development of heart failure that is observed in some cardiac allografts.4 In addition, the reduced inflammatory response in the rejecting NOS-2−/− cardiac allografts suggests that large amounts of NO, particularly in conjunction with peroxynitrite-induced cell damage, may amplify the inflammatory response in allograft rejection. Such interactions between inflammatory mediators and apoptosis may provide new targets for pharmacological and genetic strategies to ameliorate cardiac allograft rejection.
This work was supported in part by NIH grants HL-54764 and HL-56984.
- Received September 6, 2000.
- Revision received December 31, 2000.
- Accepted January 16, 2001.
- Copyright © 2001 by American Heart Association
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