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Ann Thorac Surg 1996;62:378-385
© 1996 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine and Departments of bMolecular Pharmacology and cMolecular and Cellular Biology, Searle Research and Development, Monsanto Company, St. Louis, Missouri
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Abstract |
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 TopFootnotes Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Methods. A rat transplant model was used to determine (1) when inducible nitric oxide synthase was expressed in the allograft heart during unmodified acute rejection and (2) whether pulse therapy with aminoguanidine attenuated the histologic changes of established acute rejection, in comparison with the effects of pulse therapy with corticosteroids.
Results. Inducible nitric oxide synthase messenger RNA and protein were expressed during early and late acute rejection. Pulse therapy with aminoguanidine inhibited nitric oxide production and attenuated the histologic changes of acute rejection, but not as effectively as corticosteroid therapy (rejection scores of 4.1 ± 0.4, 2.5 ± 0.9, and 1.4 ± 0.6 on postoperative day 8, for untreated, aminoguanidine-, and dexamethasone-treated allografts, respectively (scale, 0 to 5; p < 0.05).
Conclusions. (1) Inducible nitric oxide synthase expression first occurs during early acute allograft rejection and persists throughout rejection and (2) nitric oxide is an important effector molecule in acute rejection. Inducible nitric oxide synthase inhibition may offer a therapeutic adjunct in the management of acute rejection.
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Introduction |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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C ardiac transplantation often represents the only effective therapy for patients with end-stage heart disease. However, despite improved immunosuppressive protocols, allograft rejection remains a serious impediment to the clinical success of cardiac transplantation. Acute rejection is mediated by humoral and cellular immune mechanisms, is characterized by accumulation of inflammatory cells within the heart, and can result in progressive destruction of the transplanted heart. Episodes of acute rejection are currently treated with pulses of high-dose corticosteroids or monoclonal or polyclonal antithymocyte or antilymphocyte antibodies, or a combination. Despite many recent advances in the understanding of the immune system, the regulatory and effector mechanisms underlying acute rejection remain incompletely understood.
Nitric oxide (NO) is a free radical that is synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthases (NOS), and is involved in diverse physiologic and pathophysiologic processes, including host immune defense, sepsis, neurotransmission, platelet aggregation, and regulation of vascular tone and endothelial function [1]. Endothelial and neuronal NOS are constitutively expressed (cNOS), are regulated by intracellular calcium concentration, and generate small amounts of NO in response to physical and receptor stimuli. Endothelial cNOS primarily mediates the physiologic actions of NO, such as regulation of vascular tone, platelet aggregation, neutrophil adhesion, and endothelial function. Conversely, the inducible form of NOS (iNOS) produces much larger amounts of NO for sustained time periods, can be expressed in diverse cell types, is principally implicated in the pathophysiologic actions of NO, and in part mediates the cytostatic and cytotoxic effector function of activated macrophages [1, 2].
We recently reported that NO is produced during experimental cardiac allograft rejection by expression of iNOS in the rejecting heart [3]. Maintenance treatment of cardiac allograft recipients with aminoguanidine, a selective iNOS inhibitor [4], significantly attenuated acute rejection. Inhibition of NO production by iNOS resulted in (1) prolonged graft survival [3], (2) reduced myocyte necrosis and mononuclear cellular infiltrate in the latter stages of acute rejection [3], (3) prevention of myocardial contractile and electrophysiologic dysfunction during early rejection [3, 5], and (4) prevention of increased microvascular permeability in the allograft heart and systemic vasculature [6]. Inducible NOS expression has also been demonstrated recently during human cardiac allograft rejection and iNOS expression correlated with impaired ventricular contractility [7].
The present report used a heterotopic rat cardiac transplant model to determine (1) when iNOS was expressed in the allograft heart during unmodified acute rejection and (2) whether pulse therapy with aminoguanidine attenuated or reversed the histologic changes of established acute rejection, in comparison with the effects of pulse therapy with corticosteroids.
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Material and Methods |
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Spectrofluorometric Determination of Serum Nitrite/Nitrate
Systemic serum nitrite/nitrate levels were measured in blood samples taken from the thoracic inferior vena cava at the time of animal sacrifice as described [3, 9]. Red blood cells were removed by centrifugation and the resulting serum filtered through an Ultrafree-MC microcentrifuge filter (Millipore, Bedford, MA) to remove the hemoglobin resulting from cell lysis. After conversion of nitrate to nitrite with nitrate reductase (all reagents from Sigma, St. Louis, MO), unless otherwise noted), total nitrite was measured by reacting with 2,3-diaminonaphthalene (Aldrich, Milwaukee, WI) under acidic conditions to form 1-(H)-naphthotriazole, a fluorescent product, as described [9]. The formation of 1-(H)-naphthotriazole was quantitated using a fluorescent plate reader (Pandex; IDEXX Laboratories, Inc, Westbrook, ME) with excitation at 365 nm and emission read at 450 nm.
Inducible Nitric Oxide Synthase Activity
Inducible NOS enzyme activity was determined by measuring the calcium-independent conversion of L-[3H]-arginine to NO and L-[3H]-citrulline by homogenates of control and transplanted hearts, as described [3, 10, 11]. Hearts were rapidly excised, flash frozen in liquid nitrogen, and homogenized with a Polytron homogenizer (Brinkman Instruments, Inc, Westbury, NY) in 1.5 mL of deionized water containing 1 mmol/L DTT, 1 µmol/L tetrahydrobiopterin , 2 µmol/L FAD, 10 µg/mL pepstatin, 10 µg/mL antipain, 10 µg/mL soybean trypsin inhibitor, 10 µmol/L leupeptin, 10 µmol/L chymostatin, and 0.5 mmol/L phenylmethylsulfonyl fluoride. The iNOS activity was measured by monitoring the conversion of L-[2,3-3H]-arginine (DuPont-NEN, Boston, MA) to L-[2,3-3H]- citrulline [11]. Fifty-microliter samples were run in triplicate. To initiate the reaction, an equal volume of 50 mmol/L Tris (pH 7.6) containing the following components was added: 2 mg/mL bovine serum albumin, 10 mmol/L EGTA (to block calcium dependent cNOS activity), 2 mmol/L DTT, 20 µmol/L FAD, 20 µmol/L tetrahydrobiopterin, 2 mmol/L NADPH, and 60 µmol/L L-arginine containing 0.9 µCi of L-[2,3-3H]-arginine. After incubation at 37°C for 40 minutes, the reaction was terminated by addition of 300 µL of cold stop buffer containing 10 mmol/L EGTA, 100 mmol/L HEPES (pH 5.5), and 1 mmol/L L-citrulline. The L-[3H]-citrulline was separated by chromatography on Dowex 50W X-8 cation exchange resin and radioactivity quantified with a liquid scintillation counter. L-[3H]-citrulline production was normalized to the protein concentration of the homogenate [12].
Inducible Nitric Oxide Synthase Messenger RNA Detection
Tissue was harvested by rapid excision, flash frozen in liquid nitrogen, and total RNA extracted using guanidinium thiocyanate as described [13]. Messenger RNA expression was analyzed by ribonuclease protection assay using an Ambion RPA II kit (Austin, TX) as described [6, 10]. Duplicate 5-µg samples of total RNA were hybridized to 1 x 105 cpm of 32P-labeled antisense rat iNOS RNA probe. The iNOS probe was generated from lipopolysaccharide-stimulated rat white blood cell RNA by reverse transcriptase-PCR amplification of a 907-base iNOS fragment (corresponding to bases 509 to 1,415 of rat iNOS coding region), as described [3], and cloned into the Invitrogen pCRII vector. The 295-base iNOS probe was then generated by linearization with BsaI and transcription with T7 polymerase. RNase digestion after probe hybridization to rat tissue iNOS messenger RNA leaves a protected fragment of 227 bases in length, corresponding to bases 1,189 through 1,415 of the coding region of rat iNOS. Rat glyceraldehyde-3-phosphate dehydrogenase riboprobe was purchased from Ambion and used as an internal control. Fragments were separated by electrophoresis on an 8% polyacrylamide/8 mol/L urea gel and visualized by autoradiography.
Histology
The grafted heart was rapidly excised, fixed in 10% neutral buffered formaldehyde, embedded in paraffin, cross-sectioned, stained with hematoxylin and eosin, and then graded for acute rejection using a modification of Billingham's criteria [14]. In a masked fashion, four separate sections from each specimen were graded for interstitial infiltrate and myocyte necrosis: 0 = no infiltrate or myocyte injury; 1 = mild, scattered mononuclear infiltrate and rare myocyte injury; 2 = moderate infiltrate and patchy myocyte injury; 3 = moderately severe infiltrate and myocyte injury and/or necrosis; 4 = severe infiltrate and myocyte injury, and/or necrosis; 5 = complete rejection.
Statistical Analysis
The iNOS enzyme activity, serum nitrite/nitrate levels, and histologic rejection scores were compared by analysis of variance with Tukey HSD post-hoc test for multiple comparisons using SYSTAT 5.0 (SYSTAT Inc, Evanston, IL). Data are expressed as mean ± standard deviation unless otherwise indicated, with a p value of less than 0.05 considered statistically significant.
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Results |
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). Translation of iNOS messenger RNA into biologically active enzyme was assessed by the iNOS enzyme activity assay (Fig 2). No significant iNOS enzyme activity was detected in controls, POD 4 and 8 isografts, or POD 3 allografts. Allografts had significantly higher iNOS enzyme activity on POD 4 and 8 than isografts, POD 3 allografts, and controls (Fig 2; p < 0.0002). Because iNOS was expressed during early and late acute rejection, the effects of pulse iNOS inhibition or corticosteroid therapy on NO production and the histologic severity of acute rejection were then determined.
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). Serum nitrite/nitrate levels were significantly higher in allografts on POD 6 and 8 than isografts and controls (Fig 3). Pulse therapy with aminoguanidine or dexamethasone on POD 4 through 6 significantly reduced but did not normalize the serum nitrite/nitrate levels in these treated allografts on POD 6. Pulse therapy with aminoguanidine or dexamethasone on POD 6 through 8 normalized the serum nitrite/nitrate levels in these treated allografts on POD 8.
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, Table 1). Allografts treated with aminoguanidine on POD 4 through harvest on POD 6 had significantly reduced inflammatory cell infiltrate, interstitial edema, and myocyte injury compared with untreated POD 6 allografts. Allografts treated with dexamethasone on POD 4 through harvest on POD 6 had significantly reduced histologic changes of acute rejection (eg, myocyte injury, edema, and infiltrate) compared with untreated POD 6 allografts and with aminoguanidine-treated POD 6 allografts.
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and Table 1
)Fig 5. Allografts treated with aminoguanidine on POD 6 through harvest on POD 8 had significantly reduced inflammatory cell infiltrate, interstitial edema, and myocyte injury compared with untreated POD 8 allografts (Fig 5 and Table 1). The histologic severity of rejection in allografts treated with aminoguanidine on POD 6 through 8 was similar to that present in untreated POD 6 allografts. Allografts treated with dexamethasone on POD 6 through harvest on POD 8 had significantly reduced histologic changes of acute rejection compared with untreated POD 8 allografts and with aminoguanidine-treated POD 8 allografts. The rejection score for allografts treated with dexamethasone on POD 6 through POD 8 was significantly reduced compared with untreated POD 6 allografts.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Inducible Nitric Oxide Synthase Expression
The likelihood that iNOS expression during allograft rejection is a specific result of immune-mediated rejection is supported by: (1) increased allograft serum nitrite/nitrate levels and expression of iNOS messenger RNA and protein in allograft hearts but not in controls or isografts, (2) attenuation of acute rejection with pulse iNOS inhibition with aminoguanidine, (3) amelioration of acute rejection with maintenance aminoguanidine treatment [3, 5, 6], and (4) inhibition of iNOS expression in the allograft hearts by pulse or maintenance corticosteroid treatment [10]. The present results of iNOS expression and increased NO production in the allograft heart during mild and severe acute rejection (POD 4 and 8, respectively), together with our previous demonstration of iNOS expression on POD 6 [3], demonstrate that iNOS messenger RNA and protein are induced during the early stages of acute rejection and expression persists throughout the unmodified rejection process.
Previously we used immunohistochemistry to localize iNOS protein expression to the mononuclear inflammatory cells infiltrating the allograft heart on POD 6 [3]. Cells expressing iNOS were morphologically suggestive of macrophages. The iNOS protein was not detected in the endothelial cells or myocytes. Others have demonstrated that iNOS may also be expressed in the allograft myocytes and/or vascular endothelial cells during acute cardiac allograft rejection [15]. The time course of expression of iNOS in these different cell types, the relative contribution of each cell type to the overall production of NO, the specific role of NO produced by each of these cell types, and the signals regulating induction and expression of iNOS messenger RNA and enzyme activity during rejection remain to be elucidated. The iNOS expression is likely induced by cytokines such as tumor necrosis factor-
, interleukin-1, and interferon-
, which are present in the rejecting heart [16] and have been demonstrated to induce iNOS expression in other model systems [1].
Roles of Nitric Oxide in Allograft Rejection
Aminoguanidine is 10- to 100-fold selective for inhibition of iNOS versus cNOS [4]. Aminoguanidine has other effects in addition to inhibiting iNOS, including reducing advanced glycation end product formation [17] and inhibiting diamine oxidase [18] and aldose reductase [19]. However, we are not aware of evidence to suggest that advanced glycation end products or polyol pathway products are elevated in the allografts. Thus, the beneficial effect of aminoguanidine in attenuating allograft rejection in this and previous studies is most likely mediated through inhibition of NO production by iNOS.
Nitric oxide appears to have multiple roles in cardiac allograft rejection. We have demonstrated that aminoguanidine treatment prevented systolic contractile and electrophysiologic dysfunction during early allograft rejection (POD 4), when only mild histologic changes of acute rejection were present in the allografts [3, 5]. Similarly, aminoguanidine treatment prevented increased vascular permeability to macromolecules in the allograft heart during early rejection [6]. Thus, NO appears to have important regulatory roles in myocardial dysfunction during early acute rejection. An effector role for NO in myocyte destruction that occurs as acute rejection progresses is suggested by observations that (1) NO is a primary effector molecule produced by activated macrophages [2], (2) NO is cytotoxic and cytostatic through the nitrosylation and inhibition of cellular enzymes critical to mitochondrial respiration and DNA synthesis [1, 2], (3) NO–protein complexes are formed during acute cardiac allograft rejection [3, 20], and (4) inhibition of iNOS with aminoguanidine prolonged graft survival and ameliorated the histologic changes of acute allograft rejection in this and previous studies [3]. An important effector role for NO in myocyte necrosis during acute rejection is consistent with observations that cytotoxic T-lymphocytes do not fully account for myocyte lysis during acute rejection [21].
Nitric oxide can react with superoxide to form peroxynitrite, which in turn decomposes to various reactive species, including hydroxyl radical and nitrogen dioxide, that are more toxic than NO or superoxide alone [22]. Although peroxynitrite formation during allograft rejection remains to be demonstrated, we have reported evidence suggestive of superoxide formation during allograft rejection [3]. Thus, the cytotoxic effects of NO during allograft rejection may be at least partially mediated by peroxynitrite. Whether peroxynitrite is formed during allograft rejection and whether the combination of iNOS inhibition and oxygen free radical scavengers is more efficacious than aminoguanidine alone remains to be examined.
The present study demonstrated that pulse therapy with aminoguanidine attenuated the histologic progression of established acute rejection, in that the rejection score for POD 8 aminoguanidine-treated allografts was improved versus untreated POD 8 allografts and was similar to untreated POD 6 allografts. These results suggest that iNOS inhibition attenuates further destruction of the allograft, consistent with an important effector function for NO in myocyte destruction. In contrast, dexamethasone appeared to reverse the histologic changes of acute rejection, in that POD 8 dexamethasone-treated allografts had less histologic evidence of rejection than untreated POD 6 allografts. Recently we have demonstrated that corticosteroids inhibit iNOS messenger RNA and enzyme expression during cardiac allograft rejection [10]. Thus, the present finding that dexamethasone-treated allografts had significantly reduced serum nitrite/nitrate levels suggests that the beneficial effects of corticosteroids in the present study were at least partially mediated through inhibition of iNOS expression. The additional efficacy of dexamethasone over aminoguanidine in treating established acute rejection is consistent with the other immunosuppressive actions of corticosteroids, including inhibition of cytokine production, adhesion molecule expression, and T-lymphocyte and macrophage activation, proliferation, and function.
Thus NO appears to be a principal regulatory and effector molecule during early rejection. However, as rejection progresses, other effector molecules (eg, cytokines) and cells (eg, cytotoxic T-lymphocytes, natural killer cells), which are not NO dependent, contribute significantly to destruction of the allograft heart. These results suggest that although aminoguanidine prevents myocardial dysfunction during early rejection [3, 5, 6], it is not an effective single agent for treatment of the histologic changes that develop as rejection progresses. Reduction but not normalization of serum nitrite/nitrate levels in this study, together with our observations that maintenance aminoguanidine treatment incompletely inhibits iNOS activity in the latter stages of rejection (unpublished observations), suggest that newer and more potent iNOS inhibitors may prove to be more efficacious than aminoguanidine. In addition, whether iNOS inhibition modifies the allogeneic response in allograft recipients that are also receiving conventional immunosuppression (eg, cyclosporine) remains to be explored. The potential clinical utility of iNOS inhibition in management of acute rejection requires further investigation and likely depends on (1) development of iNOS inhibitors that are more potent and have a lower side-effect profile than aminoguanidine and (2) demonstration of the efficacy of iNOS inhibition in immunosuppressed recipients.
Nitric oxide is also produced during acute rejection of other transplanted organs. We recently demonstrated iNOS expression and increased NO production during acute lung allograft rejection [23, 24]. Inhibition of iNOS with aminoguanidine attenuated acute rejection [23]. Others have demonstrated increased serum nitrite/nitrate levels, indicative of increased NO production, during (1) experimental skin [25], small bowel [26], and pancreas [27] allograft rejection; (2) experimental and human liver allograft rejection [26, 28]; and (3) experimental and human graft-versus-host disease after bone marrow transplantation [26, 29]. The iNOS expression was not determined in these studies. In contrast to our findings of attenuation of cardiac and lung allograft rejection with selective iNOS inhibition, treatment of allograft recipients with NG-monomethyl-L-arginine, which inhibits both iNOS and cNOS, produced only a small increase in survival of cardiac [30] and skin allografts [25]. However, NG-monomethyl-L-arginine treatment in combination with portal vein pretransplant transfusion of donor spleen cells significantly prolonged skin graft survival more than portal vein transfusion alone, suggesting that NO does contribute to skin allograft rejection [25]. Thus, although the roles of NO in allograft rejection are not completely understood, this report, together with our previous observations [3, 5, 6], demonstrate that selective inhibition of NO production by iNOS attenuates acute cardiac allograft rejection.
Conclusion
Inducible nitric oxide synthase was first expressed during the early stages of acute cardiac allograft rejection and expression persisted throughout the rejection process. Pulse therapy with aminoguanidine inhibited increased NO production and attenuated the progression of established acute rejection, whereas pulse therapy with corticosteroids reversed the histologic changes of acute rejection. Corticosteroids also inhibited increased NO production, suggesting that the beneficial effects of corticosteroids in attenuating established rejection were at least partially mediated through inhibition of iNOS expression. These findings, together with our previous observations, demonstrate that (1) NO is an important regulatory and effector molecule in myocardial dysfunction during early rejection and (2) NO is one of several important effector molecules of the histologic changes that develop as rejection progresses. The potential role of iNOS inhibition in the clinical management of acute rejection requires further investigation.
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Acknowledgments |
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Footnotes |
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Address reprint requests to Dr Worrall, Division of Cardiothoracic Surgery, Washington University School of Medicine, Box 8234-3308 CSRB, 660 S Euclid Ave, St. Louis, MO 63110.
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TopFootnotesAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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p < 0.005 versus controls and isografts; 
p < 0.005 versus controls, isografts, and aminoguanidine- and dexamethasone-treated allografts; p > 0.9 for isografts versus controls; compared by analysis of variance with Tukey HSD post-hoc test.)
