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Inhibition of inducible nitric oxide synthase attenuates established acute cardiac allograft rejection
Inhibition of Inducible Nitric Oxide Synthase Attenuates Established Acute Cardiac Allograft Rejection Nell K. Worrall, MD, Thomas P. Misko, PhD, Patrick M. Sullivan, MA, Jia-J. Hui, MD, and T. Bruce Ferguson, Jr, MD Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine and Departments of Molecular Pharmacology and Molecular and Cellular Biology, Searle Research and Development, Monsanto Company, St. Louis, Missouri
Background. We previously demonstrated that continuous treatment with aminoguanidine, a selective inhibitor of nitric oxide production by inducible nitric oxide synthase, attenuated acute cardiac allograft rejection. 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 in-
hibited 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.
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 m a n y 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 i m m u n e 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, 21. 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 h u m a n cardiac
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Presented at the Thirty-secondAnnual Meeting of The Society of Thoracic Surgeons, Orlando, FL, Jan 29-31, 1996. 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. © 1996 by The Society of Thoracic Surgeons Published by Elsevier Science lnc
(Ann Thorac Surg 1996;62:378-85)
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Ann Thorac Surg 1996;62:378-85
WORRALL ET AL iNOS INHIBITION ATTENUATES ACUTE REJECTION
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.
Material and Methods
Heterotopic Rat Cardiac Transplant Model Male 225 to 250 g Lewis (RT-11 major histocompatability antigen haplotype) and ACI (RT-1 a) rats were purchased from Harlan-Sprague-Dawley (Indianapolis, IN). Animals were housed and cared for in compliance with guidelines set forth by the National Institutes of Health regarding laboratory animal welfare ("Guide for the Care and Use of Laboratory Animals," NIH publication 85-23, revised 1985). Allogeneic (Lewis donor to ACI recipient) and syngeneic (ACI to ACI) heterotopic intraabdominal cardiac transplantation was performed as described [8]. Aminoguanidine-treated allografts received continuous intravenous infusion of aminoguanidine hemisulfate (400 rag. kg -1. d -1 in phosphate-buffered saline solution) through a silicone right external jugular vein cannula connected to an ALZET osmotic infusion pump (model 2ML1; ALZA Corp, Palo Alto, CA) implanted in the intrascapular subcutaneous space. Corticosteroidtreated allografts received dexamethasone (1 mg• kg 1. d -a in phosphate-buffered saline solution) subcutaneously through an implanted ALZET osmotic infusion pump. Aminoguanidine and dexamethasone were administered according to one of two treatment protocols: (1) pulse therapy from the time of p u m p implantation on postoperative day 4 (POD 4) through animal harvest on POD 6, or (2) pulse therapy from the time of pump implantation on POD 6 through animal harvest on POD 8. Untreated allograft and isograft rats received phosphate-buffered saline solution in a similar fashion.
Spectrofluorometric Determination Nitrate
of Serum Nitrite/
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.
379
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-I3H]-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 ~tmol/L tetrahydrobiopterin, 2 txmol/L FAD, 10 txg/mL pepstatin, 10 ~g/mL antipain, 10/zg/mL soybean trypsin inhibitor, 10 /zmol/L leupeptin, 10 /xmol/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-I2,3-3H] - 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 ~mol/L FAD, 20 ~mol/L tetrahydrobiopterin, 2 mmol/L NADPH, and 60/xmol/L L-arginine containing 0.9 /xCi of L-[2,3-3H]-arginine. After incubation at 37°C for 40 minutes, the reaction was terminated by addition of 300 ~L 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, r-[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-/xg samples of total RNA were hybridized to 1 × 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.
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WORRALL ET AL iNOS INHIBITION ATTENUATES ACUTE REJECTION
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Fig 1. Inducible nitric oxide synthase fiNOS) messenger RNA was expressed in postoperative day 4 and 8 allografl hearts, but was not detected in controls or isografts. Ribonuclease protection assay was performed independently with iNOS and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) antisense RNA probes (G3PDH is internal control to facilitate comparison between animals). Lane 1, undigested probe; lane 2, probe hybridized with transfer RNA demonstrating that protection was specific for inducible nitric oxide synthase messenger RNA; lane 3, control; lanes 4 and 5, postoperative day 3 allografts; lanes 6 and 7, postoperative day 4 allografls; lanes 8 and 9, postoperative day 8 allografis; lanes 10 and 11, postoperative day 4 and 8 isografis, respectively.
Histology
The grafted heart was rapidly excised, fixed in 10% neutral buffered formaldehyde, e m b e d d e d 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 A n a l y s i s 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.
POD 8, and results in complete cessation of contractile and electrical activity by POD 10 or 11. Inducible NOS messenger RNA was detected in allograft hearts on POD 4 and 8, but was not present in control hearts, POD 4 or 8 isograft hearts, or POD 3 allograft hearts (Fig 1). 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. Effect o f Inducible N i t r i c O x i d e S y n t h a s e I n h i b i t i o n or Corticosteroids on Nitric O x i d e Production Serum nitrite/nitrate levels, which reflect endogenous NO production, were not different in POD 6 or 8 isografts versus controls (Fig 3). 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 dexarnethasone 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. Effect o f Inducible N i t r i c O x i d e S y n t h a s e I n h i b i t i o n or Corticosteroids on A c u t e Rejection Untreated allografts demonstrated moderately severe histologic changes of acute rejection on POD 6 (Fig 4,
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In the strain cross used in this study (Lewis donor to ACI recipient), mild acute rejection develops by POD 4 in n o n i m m u n o s u p p r e s s e d animals, becomes severe by
Fig 2. Allografls had significantly higher inducible nitric oxide synthase (iNOS) enzyme activity than isografis on postoperative day 6 and 8. Inducible nitric oxide synthase enzyme activity was 0.21 ± 0.14 pmole I_-f~H]-citrulline • m g protein- ~ • rain ~ in controls (n = 4; mean ± standard error qf the mean). (*p < 0.0002 versus controls, isografls, and postoperative day 3 allografls by analysis of variance with Tukey HSD post-hoc test.)
Ann Thorac Surg 1996;62:378-85
WORRALL ET AL iNOS INHIBITION ATTENUATES ACUTE REJECTION
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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. Untreated allografts demonstrated severe histologic changes of acute rejection on POD 8 (Fig 5 and Table 1). 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 aUografts (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 allografls. The rejection score for allografts treated with dexamethasone on POD 6 through POD 8 was significantly reduced compared with untreated POD 6 allografts. Comment The regulatory and effector mechanisms underlying the immune-mediated acute rejection process remain incom-
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Postoperative Day Fig 3. Serum nitrite/nitrate levels were higher in allografls (ALLO, n = 10) than isografts (ISO, n = 6) and were reduced with aminoguanidine (AG, n = 12) or dexamethasone (DEX, n 7) treatment. Nitrite/nitrate levels were 16 +_ 3 in controls (n = 1@ mean +- standard deviation). (*p = 0.0002 versus controls, isografls, and aminoguanidine- and dexamethasone-treated allografts; ~'p < 0.005 versus controls and isografts; ~:p < 0.005 versus controls, isografts, and aminoguanidine- and dexamethasone-treated allografis; p > 0.g for isografts versus controls; compared by analysis of variance with Tukey HSD post-hoc test.)
Fig 4. Representative photomicrographs from (A) untreated postoperative day 6 allograft, (B) allografi treated with aminoguanidine on postoperative day 4 through harvest on postoperative day 6, and (C) allograft treated with dexamethasone on postopen~Hve day 4 through harvest on postoperative day 6. (Hematoxylin and eosin, ×200 be.fore 47% reduction.)
pletely understood. Recently we demonstrated that NO, produced by expression of iNOS in the rejecting heart, is an important effector molecule in acute cardiac allograft rejection [3, 5, 6]. The present study demonstrated that (1) iNOS was first expressed in the nonimmunosuppressed
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W O R R A L L ET A L iNOS INHIBITION ATFENUATES ACUTE REJECTION
Ann Thorac Surg 1996;62:378-85
allograft heart during the early stages of acute rejection, (2) iNOS expression persisted throughout the rejection process, (3) pulse therapy with aminoguanidine or dexamethasone inhibited increased allograft NO production, and (4) pulse therapy with aminoguanidine attenuated established acute rejection, but not as effectively as pulse therapy with corticosteroids. Inducible Nitric O x i d e S y n t h a s e Expression The likelihood that iNOS expression during allograft rejection is a specific result of i m m u n e - m e d i a t e d 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 [15l. 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
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Histologic Score (o-5) Treatment Groups
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POD 8
Untreated allograft (n -- 14, 14) 2.6 + 0.5 4.1 -+ 0.4 Allograft + aminoguanidine (n = 4, 14) 1.6 -+ 0.5 b 2.5 -+ 0.9 d Allograft + dexamethasone (n = 6, 5) 0.8 _+0.4~ 1.4 _+0.6~ a Rejection scores for u n t r e a t e d p o s t o p e r a t i v e d a y (POD) 6 a n d 8 allografts a n d allografts t r e a t e d w i t h a m i n o g u a n i d i n e or d e x a m e t h a s o n e on P O D 4 t h r o u g h 6 or P O D 6 t h r o u g h 8. M e a n +- s t a n d a r d deviation; n = for P O D 6 a n d P O D 8, respectively. b p = 0.004 v e r s u s P O D 6 allografts; Cp = 0.0001 v e r s u s P O D 6 aUografts a n d p = 0.04 v e r s u s a m i n o g u a n i d i n e - t r e a t e d P O D 6 allografts; a p = 0.0002 v e r s u s P O D 8 allografts a n d p = 0.99 v e r s u s P O D 6 allografts; ~ p = 0.0002 v e r s u s P O D 8 allografts a n d p = 0.005 v e r s u s P O D 6 allografts a n d a m i n o g u a n i d i n e - t r e a t e d P O D 8 allografts; c o m p a r e d b y analysis of v a r i a n c e w i t h T u k e y H S D p o s t - h o c test.
c Fig 5. Representative photomicrographs from (A) untreated postoperative day 8 allograft, (B) allograft treated with aminoguanidine on postoperative day 6 through harvest on postoperative day 8, and (C) allograft treated with dexarnethasone on postoperative day 6 through harvest on postoperative day 8. (Hematoxylin and eosin, ×200 before 47% reduction.)
during rejection remain to be elucidated. The iNOS expression is likely induced by cytokines such as tumor necrosis factor-c~, 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].
Ann Thorac Surg 1996;62:378-85
Roles of Nitric Oxide in Allografl 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, 2l, (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 destruc-
WORRALL ET AL iNOS INHIBITION ATTENUATES ACUTE REJECTION
383
tion 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 Tlymphocyte 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 ceils}, 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 sideeffect 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
384
WORRALLET AL iNOS [NHIB|T1ON ATTENUATESACUTE REJECTION
findings of attenuation of cardiac and lung allograft rejection with selective iNOS inhibition, t r e a t m e n t of allograft r e c i p i e n t s with N C - m o n o m e t h y l - L - a r g i n i n e , which inhibits both iNOS and cNOS, p r o d u c e d only a small increase in survival of cardiac [30] a n d skin allografts [25]. However, NC-monomethyl-L-arginine treatm e n t in combination with portal vein p r e t r a n s p l a n t transfusion of donor spleen cells significantly p r o l o n g e d skin graft survival m o r e than portal vein transfusion alone, s u g g e s t i n g t h a t N O d o e s c o n t r i b u t e to skin allograft rejection [25]. Thus, a l t h o u g h the roles of N O in allograft rejection are not c o m p l e t e l y u n d e r s t o o d , this report, t o g e t h e r with our p r e v i o u s o b s e r v a t i o n s [3, 5, 6], d e m o n s t r a t e that selective i n h i b i t i o n of N O p r o d u c t i o n b y i N O S a t t e n u a t e s acute cardiac allograft rejection.
Ann Thorac Surg 1996;62:378-85
6.
7.
8. 9. 10.
11.
Conclusion Inducible nitric oxide synthase was first expressed during the early stages of acute cardiac allograft rejection a n d expression persisted t h r o u g h o u t the rejection process. Pulse t h e r a p y with a m i n o g u a n i d i n e inhibited increased N O production a n d a t t e n u a t e d the progression of established acute rejection, w h e r e a s pulse t h e r a p y with corticosteroids r e v e r s e d the histologic changes of acute rejection. C o r t i c o s t e r o i d s also i n h i b i t e d i n c r e a s e d N O production, suggesting that the beneficial effects of corticosteroids in attenuating established rejection were at least partially m e d i a t e d t h r o u g h inhibition of iNOS expression. These findings, together with our previous observations, d e m o n s t r a t e that (1) NO is an i m p o r t a n t regulatory a n d effector molecule in myocardial dysfunction during early rejection a n d (2) NO is one of several i m p o r t a n t effector molecules of the histologic changes that develop as rejection progresses. The potential role of iNOS inhibition in the clinical m a n a g e m e n t of acute rejection requires further investigation.
12.
13. 14. 15. 16. 17. 18.
19. 20.
This work was supported by the National Institutes of Health (F32 HL09021 [N.K.W.] and R29 HL46387 [T.B.F.]) and by the Monsanto-Searle/Washington University Biomedical Program (T.B.F.). We thank Dr Richard B. Schuessler for help with the statistical analysis, Donna Marquart for technical assistance, and Dawn Schuessler and Gall Agnew for secretarial assistance.
21.
22.
References 1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43:109-42. 2. Stuehr DJ, Nathan CF. Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 1989;169:1543-55. 3. Worrall NK, Lazenby WD, Misko TP, et al. Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med 1995;181:63-70. 4. Misko TP, Moore WM, Kasten TP, et al. Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur J Pharmacol 1993;233:119-25. 5. Worrall NK, Pyo RT, Alexander DG, Misko TP, Lazenby WD, Ferguson TB Jr. Nitric oxide mediates myocardial contractile
23. 24.
25.
26.
and electrophysiological dysfunction during early cardiac allograft rejection. Surg Forum 1995;46:239-41. Worrall NK, Chang K, Suau GM, et al. Inhibition of inducible nitric oxide synthase prevents myocardial and systemic vascular barrier dysfunction during early cardiac allograft rejection. Circ Res 1996;78:769-79. Lewis NP, Tsao PS, Rickenbacher PR, et al. Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle. Circulation 1996;93:720-9. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969;57:225-9. Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem 1993;214:11-6. Worrall NK, Misko TP, Sullivan PM, Hui J-J, Rodi CP, Ferguson TB Jr. Corticosteroids inhibit expression of inducible nitric oxide synthase during acute cardiac allograft rejection. Transplantation 1996;61:324-8. Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990;87:682-5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248 -54. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. Billingham ME. Diagnosis of cardiac rejection by endomyocardial biopsy. Heart Transplant 1981;1:25-30. Yang X, Chowdhury N, Cai B, et al. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest 1994;94:714-21. Wu CJ, Lovett M, Wong-Lee J, et al. Cytokine gene expression in rejecting cardiac allografts. Transplantation 1992;54: 326 -32. Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A. Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science 1986;232:1629-32. Hui JY, Taylor SL. Inhibition of in vivo histamine metabolism in rats by foodborne and pharmacologic inhibitors of diamine oxidase, histamine N-methyltransferase, and monoamine oxidase. Toxicol Appl Pharmacol 1985;81:241-9. Kumari K, Umar S, Bansal V, Sahib MK. Inhibibition of diabetes-associated complications by nucleophilic compounds. Diabetes 1991;40:1079-84. Lancaster JR, Langrehr JM, Bergonia HA, Murase N, Simmons RL, Hoffman RA. EPR detection of heine and nonheme iron-containing protein nitrosylation by nitric oxide during rejection of rat heart allograft. J Biol Chem 1992;267:10994-8. Wagoner LE, Zhao L, Bishop DK, Chan S, Xu S, Barry WH. Lysis of adult ventricular myocytes by cells infiltrating rejecting murine cardiac allografts. Circulation 1996;93: 111-9. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 1990;87:1620-4. Shiraishi T, DeMeester SR, Worrall NK, et al. Inhibition of inducible nitric oxide synthase ameliorates rat lung allograft rejection. J Thorac Cardiovasc Surg 1995:110:1449-60. Worrall NK, Boasquevisque CH, Misko TP, Sullivan PM, Patterson GA, Ferguson TB Jr. Expression of inducible nitric oxide synthase during acute lung allograft rejection [Abstract]. J Heart Lung Transplant 1996;15:$62. Gorczynski RM, Rossi-Bergman B, Sullivan B, Chert Z. Role of reactive nitrogen intermediates in the regulation of allogeneic skin graft survival in mice after portal vein pretransplant transfusion. Transplantation 1995;60:707-13. Langrehr JM, Murase N, Markus PM, et al. Nitric oxide production in host-versus- graft and graft-versus-host reactions in the rat. J Clin Invest 1992;90:679-83.
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27. Tanaka S, Kamiike W, Ito T, et al. Generation of nitric oxide as a rejection marker in rat pancreas transplantation. Transplantation 1995;60:713-7. 28. Devlin J, Palmer RMJ, Gonde CE, et al. Nitric oxide generation: a predictive parameter of acute allograft rejection. Transplantation 1994;58:592-5. 29. Weiss G, Schwaighofer H, Herold M, et al. Nitric oxide
WORRALL ET AL iNOS |NHIBITION ATTENUATES ACUTE REJECTION
385
formation as a predictive parameter for acute graft-versushost disease after h u m a n allogeneic bone marrow transplantation. Transplantation 1995;60:1239-44. 30. Winlaw DS, Schyvens CG, Smythe GA, et al. Selective inhibition of nitric oxide production during cardiac allograft rejection causes a small increase in graft survival. Transplantation 1995;60:77-82.
DISCUSSION DR MEHMET C. OZ (New York, NY): I have spent a fair amount of my research career studying nitric oxide and am still confused by its effects. Even in this meeting's program there are several great things that nitric oxide does and several terrible things. You are always trying to figure out, even when you work in the field, which side of the cutting edge you are on. Do you have any evidence that inducing nitric oxide actually accelerates rejection? For example, did you give L-arginine or any other kind of nitric oxide inducer? DR WORRALL: Many of the actions of nitric oxide depend on the conditions u n d e r which it is produced, including the local redox potential and the concentration of nitric oxide produced. Most of the pathologic effects of nitric oxide, particularly in tissue injury, result from high levels of nitric oxide, as are produced during acute cardiac allograft rejection. We have not specifically examined whether we can accelerate acute rejection through increasing the concentration of r-arginine, which is the substrate for nitric oxide synthase, in the rat's diet or through transfection of the inducible nitric oxide synthase gene into
isografts, for example, although that would be interesting to examine. DR JAMES K. KIRKLIN (Birmingham, AL): If you had a systemic blocker of inducible nitric oxide synthase, what sort of systemic or whole organism effects would you anticipate? DR WORRALL: Systemic administration of nonselective nitric oxide synthase inhibitors such as L-NMA or L-NAME to animals at doses high enough to inhibit high levels of pathologic nitric oxide produced by inducible nitric oxide synthase expression in the allograft heart, as would be required during allograft rejection, would also cause peripheral vasoconstriction through inhibition of constitutive nitric oxide synthase. This would result in reduced blood flow to the allograft, which would probably end up being deleterious. Aminoguanidine and other newer inducible nitric oxide synthase inhibitors that are currently u n d e r development are at least 10- to 100-fold selective for the inducible nitric oxide synthase versus the constitutive nitric oxide synthase. So one would expect minimal side effects.
Background. We previously demonstrated that continuous treatment with aminoguanidine, a selective inhibitor of nitric oxide production by inducible nitric oxide synthase, attenuated acute cardiac allograft rejection. 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 in-
hibited 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.
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 m a n y 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 i m m u n e 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, 21. 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 h u m a n cardiac
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Presented at the Thirty-secondAnnual Meeting of The Society of Thoracic Surgeons, Orlando, FL, Jan 29-31, 1996. 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. © 1996 by The Society of Thoracic Surgeons Published by Elsevier Science lnc
(Ann Thorac Surg 1996;62:378-85)
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Ann Thorac Surg 1996;62:378-85
WORRALL ET AL iNOS INHIBITION ATTENUATES ACUTE REJECTION
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.
Material and Methods
Heterotopic Rat Cardiac Transplant Model Male 225 to 250 g Lewis (RT-11 major histocompatability antigen haplotype) and ACI (RT-1 a) rats were purchased from Harlan-Sprague-Dawley (Indianapolis, IN). Animals were housed and cared for in compliance with guidelines set forth by the National Institutes of Health regarding laboratory animal welfare ("Guide for the Care and Use of Laboratory Animals," NIH publication 85-23, revised 1985). Allogeneic (Lewis donor to ACI recipient) and syngeneic (ACI to ACI) heterotopic intraabdominal cardiac transplantation was performed as described [8]. Aminoguanidine-treated allografts received continuous intravenous infusion of aminoguanidine hemisulfate (400 rag. kg -1. d -1 in phosphate-buffered saline solution) through a silicone right external jugular vein cannula connected to an ALZET osmotic infusion pump (model 2ML1; ALZA Corp, Palo Alto, CA) implanted in the intrascapular subcutaneous space. Corticosteroidtreated allografts received dexamethasone (1 mg• kg 1. d -a in phosphate-buffered saline solution) subcutaneously through an implanted ALZET osmotic infusion pump. Aminoguanidine and dexamethasone were administered according to one of two treatment protocols: (1) pulse therapy from the time of p u m p implantation on postoperative day 4 (POD 4) through animal harvest on POD 6, or (2) pulse therapy from the time of pump implantation on POD 6 through animal harvest on POD 8. Untreated allograft and isograft rats received phosphate-buffered saline solution in a similar fashion.
Spectrofluorometric Determination Nitrate
of Serum Nitrite/
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.
379
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-I3H]-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 ~tmol/L tetrahydrobiopterin, 2 txmol/L FAD, 10 txg/mL pepstatin, 10 ~g/mL antipain, 10/zg/mL soybean trypsin inhibitor, 10 /zmol/L leupeptin, 10 /xmol/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-I2,3-3H] - 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 ~mol/L FAD, 20 ~mol/L tetrahydrobiopterin, 2 mmol/L NADPH, and 60/xmol/L L-arginine containing 0.9 /xCi of L-[2,3-3H]-arginine. After incubation at 37°C for 40 minutes, the reaction was terminated by addition of 300 ~L 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, r-[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-/xg samples of total RNA were hybridized to 1 × 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.
380
WORRALL ET AL iNOS INHIBITION ATTENUATES ACUTE REJECTION
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Fig 1. Inducible nitric oxide synthase fiNOS) messenger RNA was expressed in postoperative day 4 and 8 allografl hearts, but was not detected in controls or isografts. Ribonuclease protection assay was performed independently with iNOS and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) antisense RNA probes (G3PDH is internal control to facilitate comparison between animals). Lane 1, undigested probe; lane 2, probe hybridized with transfer RNA demonstrating that protection was specific for inducible nitric oxide synthase messenger RNA; lane 3, control; lanes 4 and 5, postoperative day 3 allografts; lanes 6 and 7, postoperative day 4 allografls; lanes 8 and 9, postoperative day 8 allografis; lanes 10 and 11, postoperative day 4 and 8 isografis, respectively.
Histology
The grafted heart was rapidly excised, fixed in 10% neutral buffered formaldehyde, e m b e d d e d 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 A n a l y s i s 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.
POD 8, and results in complete cessation of contractile and electrical activity by POD 10 or 11. Inducible NOS messenger RNA was detected in allograft hearts on POD 4 and 8, but was not present in control hearts, POD 4 or 8 isograft hearts, or POD 3 allograft hearts (Fig 1). 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. Effect o f Inducible N i t r i c O x i d e S y n t h a s e I n h i b i t i o n or Corticosteroids on Nitric O x i d e Production Serum nitrite/nitrate levels, which reflect endogenous NO production, were not different in POD 6 or 8 isografts versus controls (Fig 3). 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 dexarnethasone 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. Effect o f Inducible N i t r i c O x i d e S y n t h a s e I n h i b i t i o n or Corticosteroids on A c u t e Rejection Untreated allografts demonstrated moderately severe histologic changes of acute rejection on POD 6 (Fig 4,
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In the strain cross used in this study (Lewis donor to ACI recipient), mild acute rejection develops by POD 4 in n o n i m m u n o s u p p r e s s e d animals, becomes severe by
Fig 2. Allografls had significantly higher inducible nitric oxide synthase (iNOS) enzyme activity than isografis on postoperative day 6 and 8. Inducible nitric oxide synthase enzyme activity was 0.21 ± 0.14 pmole I_-f~H]-citrulline • m g protein- ~ • rain ~ in controls (n = 4; mean ± standard error qf the mean). (*p < 0.0002 versus controls, isografls, and postoperative day 3 allografls by analysis of variance with Tukey HSD post-hoc test.)
Ann Thorac Surg 1996;62:378-85
WORRALL ET AL iNOS INHIBITION ATTENUATES ACUTE REJECTION
38l
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. Untreated allografts demonstrated severe histologic changes of acute rejection on POD 8 (Fig 5 and Table 1). 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 aUografts (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 allografls. The rejection score for allografts treated with dexamethasone on POD 6 through POD 8 was significantly reduced compared with untreated POD 6 allografts. Comment The regulatory and effector mechanisms underlying the immune-mediated acute rejection process remain incom-
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Postoperative Day Fig 3. Serum nitrite/nitrate levels were higher in allografls (ALLO, n = 10) than isografts (ISO, n = 6) and were reduced with aminoguanidine (AG, n = 12) or dexamethasone (DEX, n 7) treatment. Nitrite/nitrate levels were 16 +_ 3 in controls (n = 1@ mean +- standard deviation). (*p = 0.0002 versus controls, isografls, and aminoguanidine- and dexamethasone-treated allografts; ~'p < 0.005 versus controls and isografts; ~:p < 0.005 versus controls, isografts, and aminoguanidine- and dexamethasone-treated allografis; p > 0.g for isografts versus controls; compared by analysis of variance with Tukey HSD post-hoc test.)
Fig 4. Representative photomicrographs from (A) untreated postoperative day 6 allograft, (B) allografi treated with aminoguanidine on postoperative day 4 through harvest on postoperative day 6, and (C) allograft treated with dexamethasone on postopen~Hve day 4 through harvest on postoperative day 6. (Hematoxylin and eosin, ×200 be.fore 47% reduction.)
pletely understood. Recently we demonstrated that NO, produced by expression of iNOS in the rejecting heart, is an important effector molecule in acute cardiac allograft rejection [3, 5, 6]. The present study demonstrated that (1) iNOS was first expressed in the nonimmunosuppressed
382
W O R R A L L ET A L iNOS INHIBITION ATFENUATES ACUTE REJECTION
Ann Thorac Surg 1996;62:378-85
allograft heart during the early stages of acute rejection, (2) iNOS expression persisted throughout the rejection process, (3) pulse therapy with aminoguanidine or dexamethasone inhibited increased allograft NO production, and (4) pulse therapy with aminoguanidine attenuated established acute rejection, but not as effectively as pulse therapy with corticosteroids. Inducible Nitric O x i d e S y n t h a s e Expression The likelihood that iNOS expression during allograft rejection is a specific result of i m m u n e - m e d i a t e d 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 [15l. 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
A
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Histologic Score (o-5) Treatment Groups
POD 6
POD 8
Untreated allograft (n -- 14, 14) 2.6 + 0.5 4.1 -+ 0.4 Allograft + aminoguanidine (n = 4, 14) 1.6 -+ 0.5 b 2.5 -+ 0.9 d Allograft + dexamethasone (n = 6, 5) 0.8 _+0.4~ 1.4 _+0.6~ a Rejection scores for u n t r e a t e d p o s t o p e r a t i v e d a y (POD) 6 a n d 8 allografts a n d allografts t r e a t e d w i t h a m i n o g u a n i d i n e or d e x a m e t h a s o n e on P O D 4 t h r o u g h 6 or P O D 6 t h r o u g h 8. M e a n +- s t a n d a r d deviation; n = for P O D 6 a n d P O D 8, respectively. b p = 0.004 v e r s u s P O D 6 allografts; Cp = 0.0001 v e r s u s P O D 6 aUografts a n d p = 0.04 v e r s u s a m i n o g u a n i d i n e - t r e a t e d P O D 6 allografts; a p = 0.0002 v e r s u s P O D 8 allografts a n d p = 0.99 v e r s u s P O D 6 allografts; ~ p = 0.0002 v e r s u s P O D 8 allografts a n d p = 0.005 v e r s u s P O D 6 allografts a n d a m i n o g u a n i d i n e - t r e a t e d P O D 8 allografts; c o m p a r e d b y analysis of v a r i a n c e w i t h T u k e y H S D p o s t - h o c test.
c Fig 5. Representative photomicrographs from (A) untreated postoperative day 8 allograft, (B) allograft treated with aminoguanidine on postoperative day 6 through harvest on postoperative day 8, and (C) allograft treated with dexarnethasone on postoperative day 6 through harvest on postoperative day 8. (Hematoxylin and eosin, ×200 before 47% reduction.)
during rejection remain to be elucidated. The iNOS expression is likely induced by cytokines such as tumor necrosis factor-c~, 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].
Ann Thorac Surg 1996;62:378-85
Roles of Nitric Oxide in Allografl 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, 2l, (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 destruc-
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tion 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 Tlymphocyte 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 ceils}, 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 sideeffect 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
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findings of attenuation of cardiac and lung allograft rejection with selective iNOS inhibition, t r e a t m e n t of allograft r e c i p i e n t s with N C - m o n o m e t h y l - L - a r g i n i n e , which inhibits both iNOS and cNOS, p r o d u c e d only a small increase in survival of cardiac [30] a n d skin allografts [25]. However, NC-monomethyl-L-arginine treatm e n t in combination with portal vein p r e t r a n s p l a n t transfusion of donor spleen cells significantly p r o l o n g e d skin graft survival m o r e than portal vein transfusion alone, s u g g e s t i n g t h a t N O d o e s c o n t r i b u t e to skin allograft rejection [25]. Thus, a l t h o u g h the roles of N O in allograft rejection are not c o m p l e t e l y u n d e r s t o o d , this report, t o g e t h e r with our p r e v i o u s o b s e r v a t i o n s [3, 5, 6], d e m o n s t r a t e that selective i n h i b i t i o n of N O p r o d u c t i o n b y i N O S a t t e n u a t e s acute cardiac allograft rejection.
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6.
7.
8. 9. 10.
11.
Conclusion Inducible nitric oxide synthase was first expressed during the early stages of acute cardiac allograft rejection a n d expression persisted t h r o u g h o u t the rejection process. Pulse t h e r a p y with a m i n o g u a n i d i n e inhibited increased N O production a n d a t t e n u a t e d the progression of established acute rejection, w h e r e a s pulse t h e r a p y with corticosteroids r e v e r s e d the histologic changes of acute rejection. C o r t i c o s t e r o i d s also i n h i b i t e d i n c r e a s e d N O production, suggesting that the beneficial effects of corticosteroids in attenuating established rejection were at least partially m e d i a t e d t h r o u g h inhibition of iNOS expression. These findings, together with our previous observations, d e m o n s t r a t e that (1) NO is an i m p o r t a n t regulatory a n d effector molecule in myocardial dysfunction during early rejection a n d (2) NO is one of several i m p o r t a n t effector molecules of the histologic changes that develop as rejection progresses. The potential role of iNOS inhibition in the clinical m a n a g e m e n t of acute rejection requires further investigation.
12.
13. 14. 15. 16. 17. 18.
19. 20.
This work was supported by the National Institutes of Health (F32 HL09021 [N.K.W.] and R29 HL46387 [T.B.F.]) and by the Monsanto-Searle/Washington University Biomedical Program (T.B.F.). We thank Dr Richard B. Schuessler for help with the statistical analysis, Donna Marquart for technical assistance, and Dawn Schuessler and Gall Agnew for secretarial assistance.
21.
22.
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DISCUSSION DR MEHMET C. OZ (New York, NY): I have spent a fair amount of my research career studying nitric oxide and am still confused by its effects. Even in this meeting's program there are several great things that nitric oxide does and several terrible things. You are always trying to figure out, even when you work in the field, which side of the cutting edge you are on. Do you have any evidence that inducing nitric oxide actually accelerates rejection? For example, did you give L-arginine or any other kind of nitric oxide inducer? DR WORRALL: Many of the actions of nitric oxide depend on the conditions u n d e r which it is produced, including the local redox potential and the concentration of nitric oxide produced. Most of the pathologic effects of nitric oxide, particularly in tissue injury, result from high levels of nitric oxide, as are produced during acute cardiac allograft rejection. We have not specifically examined whether we can accelerate acute rejection through increasing the concentration of r-arginine, which is the substrate for nitric oxide synthase, in the rat's diet or through transfection of the inducible nitric oxide synthase gene into
isografts, for example, although that would be interesting to examine. DR JAMES K. KIRKLIN (Birmingham, AL): If you had a systemic blocker of inducible nitric oxide synthase, what sort of systemic or whole organism effects would you anticipate? DR WORRALL: Systemic administration of nonselective nitric oxide synthase inhibitors such as L-NMA or L-NAME to animals at doses high enough to inhibit high levels of pathologic nitric oxide produced by inducible nitric oxide synthase expression in the allograft heart, as would be required during allograft rejection, would also cause peripheral vasoconstriction through inhibition of constitutive nitric oxide synthase. This would result in reduced blood flow to the allograft, which would probably end up being deleterious. Aminoguanidine and other newer inducible nitric oxide synthase inhibitors that are currently u n d e r development are at least 10- to 100-fold selective for the inducible nitric oxide synthase versus the constitutive nitric oxide synthase. So one would expect minimal side effects.