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Ann Thorac Surg 1999;67:716-722
© 1999 The Society of Thoracic Surgeons

Original Article

Time course and cellular localization of inducible nitric oxide synthases expression during cardiac allograft rejection

Neil K. Worrall, MDa, Thomas P. Misko, PhDc, Mitchell D. Botney, MDb, Patrick M. Sullivan, MAd, Jia-J Hui, MDa, Gloria M. Suau, MDa, Pamela T. Manning, PhDc, T. Bruce Ferguson, Jr, MDa

a Department of Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
b Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA
c Department of Molecular Pharmacology, Searle Research and Development, Monsanto Company, St. Louis, Missouri, USA
d Department of Molecular and Cellular Biology, Searle Research and Development, Monsanto Company, St. Louis, Missouri, USA

Accepted for publication September 14, 1998.

Address reprint requests to Dr Ferguson, Departments of Surgery and Physiology, Louisiana State University Medical Center, 1542 Tulane Ave, 7th Floor, New Orleans, LA 70112-2822


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. We have demonstrated that inhibition of inducible nitric oxide synthase (NOS) ameliorated acute cardiac allograft rejection. This study determined the time course and cellular localization of inducible NOS expression during the histologic progression of unmodified acute rat cardiac allograft rejection.

Methods. Tissue from syngeneic (ACI to ACI) and allogeneic (Lewis to ACI) transplants were harvested on postoperative days 3 through 10 and analyzed for inducible NOS mRNA expression (ribonuclease protection assay), inducible NOS enzyme activity (conversion of L-[3H]arginine to nitric oxide and L- [3H]citrulline), and nitric oxide production (serum nitrite/ nitrate levels). Inducible NOS mRNA and protein expression were localized using in situ hybridization and immunohistochemistry.

Results. Inducible NOS mRNA and enzyme activity were expressed in allografts during mild, moderate, and severe acute rejection (postoperative days 4 through 10), but were not detected in normals, isografts, or allografts before histologic changes of mild acute rejection (postoperative day 3). Inducible NOS expression resulted in increased serum nitrite/nitrate levels during mild and moderate rejection (postoperative days 4 through 6). Inducible NOS mRNA and protein expression localized to infiltrating mononuclear inflammatory cells in allograft tissue sections during all stages of rejection but were not detected in allograft parenchymal cells or in normals or isografts.

Conclusions. Inducible NOS expression and increased nitric oxide production occurred during the early stages of acute rejection, persisted throughout the unmodified rejection process, and localized to infiltrating inflammatory cells but not allograft parenchymal cells during all stages of acute rejection.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
In human and experimental cardiac transplantation, acute cellular rejection of mild to moderate severity is characterized by perivascular and interstitial inflammatory cell infiltrates, interstitial edema, and focal areas of myocyte injury or necrosis [1]. Acute rejection may become severe and result in irreversible myocyte necrosis and persistent impairment of contractility. Despite many recent advances in the understanding of the immune system, the regulatory and effector mechanisms underlying acute rejection remain incompletely understood.

The free radical nitric oxide (NO) is synthesized from L-arginine by a family of enzymes, the nitric oxide synthases (NOS), and is involved in diverse physiologic and pathophysiologic processes [2]. 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. Constitutive NOS primarily mediates the physiologic action of NO, such as regulation of vascular tone, platelet aggregation, and endothelial function. Conversely, inducible NOS (iNOS) produces much larger amounts of NO for sustained 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 [2, 3].

We recently reported that NO is produced during experimental cardiac allograft rejection by expression of iNOS in the rejecting heart [4–7]. Treatment of cardiac allograft recipients with aminoguanidine, a selective iNOS inhibitor [8], significantly ameliorated acute rejection [4–6]. Inducible NOS protein expression was immunohistochemically localized to infiltrating mononuclear inflammatory cells in allograft tissue sections during late unmodified acute rejection [4]. Others have demonstrated iNOS mRNA or protein expression in (1) infiltrating inflammatory cells and single myocytes isolated from rat cardiac allografts [9] and (2) isolated nontransplanted rat cardiac myocytes and endothelial cells after in vitro stimulation with endotoxin or cytokines [10, 11]. Thus, it is unclear which cell types express iNOS during acute rejection and whether the cell types expressing iNOS change during the histologic progression of acute rejection. Inducible NOS expression has been recently demonstrated in biopsies from human cardiac allografts during acute rejection [12].

The present study used a heterotopic rat cardiac transplant model to further characterize iNOS expression during in vivo allograft rejection by determining (1) when iNOS mRNA and protein were expressed during the histologic progression of unmodified acute rejection (eg, allograft recipients with mild, moderate, and severe rejection), (2) the time course of increased in vivo NO production during acute rejection, and (3) the cell type(s) that expressed iNOS mRNA or protein during the histologic progression of acute rejection.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Heterotopic rat cardiac transplant model
Male 225 to 250 g Lewis (RT-1l) and ACI (RT-1a) rats (Harlan-Sprague-Dawley, Indianapolis, IN) were housed and cared for in compliance with the National Institutes of Health guidelines regarding laboratory animal welfare ("Guide for the Care and Use of Laboratory Animals," NIH publication no. 85-23, revised 1985). Allogeneic (Lewis donor to ACI recipient) and syngeneic (ACI to ACI) heterotopic intraabdominal cardiac transplantation was performed as described [4–7]. After aseptic preparation of the skin of the donor and recipient animals, anesthesia was induced with subcutaneous ketamine (100 mg/kg) and inhaled methoxyflurane. After preparation of the recipient’s infrarenal aorta and inferior vena cava, the donor heart was rapidly excised after hypothermic arrest and ligation of the vena cavae and pulmonary veins. Iced saline was used for topical cooling, and the donor aorta and pulmonary artery were anastomosed to the recipient aorta and inferior vena cava, respectively. Typical cold ischemia times were 20 to 25 minutes and the heart resumed contracting within 1 to 5 minutes after reperfusion.

Inducible nitric oxide synthase ribonuclease protection assay
Hearts were rapidly excised and flash-frozen in liquid nitrogen, and total RNA was extracted using guanidinium thiocyanate as described [5–7]. All reagents were from Sigma Chemical Co (St. Louis, MO) unless otherwise noted. mRNA expression was analyzed by ribonuclease protection assay using an Ambion RPA II kit (Austin, TX) as described [5–7]. Duplicate 5-µg samples of RNA were hybridized to 1 x 105 cpm of 32P-labeled rat iNOS antisense RNA probe. The iNOS probe was generated from lipopolysaccharide-stimulated rat white blood cell RNA by reverse transcriptase-polymerase chain reaction amplification of a 907-base iNOS fragment (bases 509 to 1415 of the rat iNOS coding region) 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 tissue iNOS mRNA leaves a protected fragment of 227 bases in length (bases 1189 to 1415 of the iNOS coding region). Rat glyceraldehyde-3-phosphate dehydrogenase probe 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.

Inducible nitric oxide synthase enzyme activity assay
Inducible NOS enzyme activity was determined by measuring calcium-independent conversion of L-[3H]arginine to NO and L-[3H]citrulline by crude heart homogenates as described [7, 13]. Hearts were rapidly excised, flash-frozen in liquid nitrogen, and homogenized in 1.5 mL of deionized water containing 1 mmol/L dithiothreitol, 1 µmol/L tetrahydrobiopterin, 2 µmol/L flavin adenine dinucleotide, 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. Inducible NOS activity was measured by monitoring conversion of L-[2,3-3H]arginine (DuPont-NEN; Boston, MA) to L-[2,3-3H]citrulline as described [13]. Fifty-microliter samples were run in triplicate with or without the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA, 1 mmol/L). The reaction was started by adding an equal volume of 50 mmol/L Tris (pH 7.6) containing 2 mg/mL bovine serum albumin, 10 mmol/L EGTA (to block calcium-dependent cNOS), 2 mmol/L dithiothreitol, 20 µmol/L flavin adenine dinucleotide, 20 µmol/L tetrahydrobiopterin, 2 mmol/L reduced nicotinamide-adenine dinucleotide phosphate, 40 mmol/L L-valine (to inhibit endogenous arginase), 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 the addition of 300 µL cold stop buffer (10 mmol/L EGTA, 100 mmol/L Hepes (pH 5.5), and 1 mmol/L L-citrulline). L-[3H]citrulline was separated by chromatography on Dowex 50W X-8 cation exchange resin and quantified with a liquid scintillation counter. L-[3H]citrulline production was normalized to the protein content of the homogenate [7]. To most accurately reflect iNOS enzyme activity, total L-[3H]citrulline production is then corrected for any background L-[3H]citrulline produced in the presence of the NOS inhibitor L-NMMA (eg, total L-[3H]citrulline production minus that produced in the presence of L-NMMA).

Serum nitrite/nitrate levels
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 [4–7, 14]. Red blood cells were removed by centrifugation, and the serum was filtered through an Ultrafree-MC microcentrifuge filter (Millipore; Bedford, MA). After conversion of nitrate to nitrite with nitrate reductase, total nitrite was measured by reacting with 2,3-diaminonaphthalene (Aldrich; Milwaukee, WI) under acidic conditions to form 1-(H)-naphthotriazole as described [14]. Formation of 1-(H)-naphthotriazole was quantified using a Pandex fluorescent plate reader (IDEXX Laboratories, Inc, Westbrook, ME) with excitation at 365 nm and emission read at 450 nm.

In situ hybridization
In situ hybridization was performed as described [15]. Briefly, 295-base rat iNOS 35S-labeled sense and antisense RNA probes were transcribed with T7 polymerase using {alpha}-[35S]-UTP (>1,200 Ci/mmole, ICN Biochemicals, Irvine, CA). Paraffin-embedded tissue sections were pretreated with 1 µg/mL nuclease-free proteinase K and washed in 0.1 mol/L triethanolamine/0.25% acetic anhydride buffer. Hybridization solution containing 2.5 x 105 cpm of 35S-labeled probe was added to the processed sections and slides incubated overnight at 55°C. As controls, some sections were treated with 100 µg/mL RNase to remove endogenous RNA. Slides were then washed extensively under stringent conditions, incubated with 20 µg/mL RNase to remove unhybridized probe, and processed for autoradiography and developed after a 2-week exposure.

Immunohistochemistry
Immunohistochemical staining for iNOS was performed using 10-µm frozen sections as described [4]. Tissues were fixed with 1% paraformaldehyde, pH 7.2, for 5 minutes at 22°C and then for 5 minutes in 100% ethanol at 4°C. Nonspecific binding was blocked with 3% normal goat serum in 0.5 mol/L Tris-HCl, pH 7.4, for 1 hour at 22°C. All subsequent incubations were carried out in this buffer. Tissue sections were incubated with a 1:1,000 dilution of either preimmune rabbit sera or an anti-iNOS antisera generated in rabbits to a unique peptide sequence obtained from the carboxyl terminal region of murine iNOS (AVFSYGAKKGSALEEPKATRL) for 16 hours at 4°C. Endogenous peroxidase activity was reduced with periodic acid (Zymed Laboratories, Inc, San Francisco, CA) for 45 seconds at 22°C, followed by sequential incubations with biotinylated anti-rabbit IgG and avidin-biotin-peroxidase complex (Vector Laboratories, Inc, Burlingame, CA) for 2 hours each. The reaction product was visualized using 3,3'-diaminobenzidine intensified with nickel chloride for 6 minutes.

Statistical analysis
Data were logarithmically transformed and then compared by analysis of variance with Tukey’s honest significant difference post hoc correction for multiple comparisons using SYSTAT 5.0 (SYSTAT Inc, Evanston, IL). Data are expressed as mean ± SEM, with a P < 0.05 considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
In the strain cross used in this study (Lewis donor to ACI recipient), acute cellular rejection is first histologically evident in nonimmunosuppressed recipients on postoperative day 3 or 4 (mild perivascular and interstitial infiltrate, mild interstitial edema, and minimal myocyte injury), is of moderate severity on day 6 (increased interstitial infiltrate and edema, focal myocyte damage or necrosis, and distortion of myocardial architecture), is severe on day 8 (heavy infiltrate and diffuse myocyte injury or necrosis), and results in complete cessation of contractile and electrical activity by day 10 or 11.

Inducible nitric oxide synthase expression and in vivo nitric oxide production
Inducible NOS mRNA was very weakly present in 1 of 3 allograft hearts on postoperative day 3 and was strongly present in all allograft hearts examined on days 4, 5, 6, 8, and 10 (Fig 1, n = 3 for each day examined). Inducible NOS mRNA was not detected in normal or day 5 or 8 isograft hearts.



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  		Fig 1. Representative ribonuclease protection assay gel demonstrating inducible nitric oxide synthase (iNOS) mRNA expression in allograft hearts but not isograft or normal hearts. Grafted hearts were harvested on the indicated postoperative day and ribonuclease protection assay performed independently using rat iNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal control) antisense probes. Lanes are as follows: 1, undigested probe; 2, probe hybridized with transfer RNA demonstrating that protection was specific for iNOS mRNA; 3, normal heart; 4 to 9, allograft hearts on postoperative days 3, 4, 5, 6, 8, and 10, respectively; 10 and 11, isograft hearts on days 5 and 8, respectively. The same results were obtained with duplicate samples in these animals and in two additional animals at each time point.

 
The presence of biologically active iNOS enzyme was assessed by the iNOS enzyme activity assay, which is performed under calcium-free conditions to inhibit calcium-dependent cNOS activity in the homogenates and is corrected for any background L-[3H]citrulline production. Allograft hearts harvested on postoperative days 4, 5, 6, 8, and 10 had significantly higher levels of iNOS enzyme activity than normal, isograft, and day 3 allograft hearts (Fig 2). There was no detectable difference in iNOS enzyme activity in allografts on days 4 through 10 (P > 0.9). L-[3H]citrulline production in the homogenates was inhibited more than 80% by inclusion of L-NMMA in the reaction buffer, indicating that there was little background L-[3H]citrulline production, and thus that L-[3H]citrulline production reflected iNOS enzyme activity.



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  		Fig 2. Inducible NOS (iNOS) enzyme activity present in allograft and isograft hearts harvested on the indicated postoperative day. Normal hearts produced 0.11 ± 0.11 pmol L-citrulline · mg protein-1 · min-1 (n = 7, P > 0.9 vs isografts and day 3 allograft). (Mean ± SEM; n = 4 to 8; *P < 0.0005 vs normal, isograft, and day 3 allograft; {dagger}P < 0.0005 vs normal and isograft and P = 0.002 vs day 3 allograft; and {ddagger}P < 0.0005 vs normal, P = 0.002 vs isograft, and P = 0.02 vs day 3 allograft.)

 
Allograft serum nitrite/nitrate levels, which are stable end products of NO metabolism and thus reflect endogenous NO production, were first elevated above isograft and normal levels on postoperative day 4, peaked on days 5 and 6 at threefold to fourfold higher levels than isografts, were significantly decreased by day 8, and returned to baseline isograft levels by day 10 (Fig 3).



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  		Fig 3. Time course of increased in vivo nitric oxide production in allografts. Serum nitrite/nitrate levels were determined in allografts and isografts on the indicated postoperative day. Serum nitrite/nitrate levels in normals were 15 ± 1 µmol/L (n = 13, P > 0.2 vs isografts and day 3 allograft). (Mean ± SEM; n = 7 for isografts and n = 8 to 14 for allografts; *P = 0.0002 vs normal and isograft and P < 0.05 vs day 3 allograft; {dagger}P = 0.0002 vs normal, isograft, day 3 allograft, and P < 0.005 vs day 4 allograft; and {ddagger}P = 0.0002 vs normal, P < 0.002 vs isograft and days 5 and 6 allograft, and P < 0.05 vs day 3 allograft.)

 
Cellular localization of inducible nitric oxide synthase expression
In situ hybridization using the iNOS antisense mRNA probe demonstrated iNOS mRNA expression in the allograft heart on postoperative days 4, 6, and 8 but not in the isograft heart or the day 3 allograft heart (Fig 4). Hybridization to iNOS mRNA occurred in the inflammatory cells in the interstitial spaces but not in the adjacent myocytes or endothelial cells. A brightfield high-power photomicrograph on day 4 further localized iNOS mRNA expression to the infiltrating mononuclear inflammatory cells, which were morphologically suggestive of macrophages (Fig 4B). Similar high-powered views demonstrated iNOS mRNA expression in infiltrating mononuclear inflammatory cells on days 6 and 8 (not shown). There was no hybridization in allografts at any time with the iNOS sense mRNA probe. There was no hybridization with the sense or antisense probes in day 6 isografts.



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  		Fig 4. Representative darkfield and brightfield photomicrographs of in situ hybridization with rat iNOS antisense and sense mRNA probes demonstrating iNOS mRNA expression in infiltrating mononuclear inflammatory cells in allografts (oriented with the left ventricular cavity at top). (A) No iNOS expression in day 3 allograft. (B) iNOS expression in infiltrating mononuclear inflammatory cells in day 4 allograft but not in adjacent myocytes. (C) iNOS expression in interstitial space in day 6 allograft but not in myocytes. (D) iNOS expression in interstitial space in day 8 allograft. (E) No hybridization with the sense probe in the same day 8 allograft section. (F) iNOS mRNA was not detected in day 6 isografts. Similar results were obtained in 3 animals at each time. (Eosin; 100x except 1,000x for [B].)

 
Immunohistochemical staining of the allograft heart on postoperative day 6 with a polyclonal anti-iNOS antibody demonstrated iNOS protein expression in the interstitial spaces surrounding the myocytes (Fig 5). High-power view demonstrated that staining occurred in infiltrating mononuclear inflammatory cells, which were morphologically suggestive of macrophages. There was no staining in allograft myocytes, endothelial cells, or vascular smooth muscle cells. Inducible NOS protein was not detected in isograft or normal hearts, and there was no staining with preimmune sera in any animal.



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  		Fig 5. Representative sections from allograft and isograft hearts stained with iNOS antisera. (A) Interstitial inflammatory cells expressed iNOS and appear to outline the myocytes, which did not stain for iNOS. (B) Perivascular infiltrating cells were positive for iNOS. iNOS was not present in endothelial cells or vascular smooth muscle cells. (C) Higher power demonstrating that iNOS-positive cells are mononuclear inflammatory cells. (D) There was no staining in the isograft heart, the normal heart (not shown), or with preimmune sera in any animal (not shown). Similar results were examined in 3 animals per group. (Mayer’s hematoxylin; 100x except 400x for [C].)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Allograft rejection remains an impediment to the clinical success of cardiac transplantation. Despite recent advances in the understanding of the immune system, the regulatory and effector mechanisms underlying acute rejection remain incompletely understood. We recently demonstrated that NO is an important effector molecule in acute cardiac allograft rejection [4–7]. The present study extends these observations by demonstrating that (1) iNOS mRNA and enzyme activity were first expressed in the nonimmunosuppressed allograft heart during the early stages of mild acute cellular rejection, (2) iNOS mRNA and protein expression persisted throughout the unmodified rejection process (eg, moderate and severe acute rejection), (3) iNOS expression resulted in increased NO production during mild and moderate rejection, and (4) iNOS mRNA or protein was expressed in the infiltrating mononuclear inflammatory cells during mild, moderate, and severe acute rejection, but it was not detected in allograft myocytes, endothelial cells, or smooth muscle cells.

Inducible nitric oxide synthase expression
Inducible NOS mRNA and protein expression first occurred on postoperative day 4, paralleling the development of the first histologic changes of acute cellular rejection in this model. Inducible NOS mRNA and protein expression persisted without detectable change as rejection progressed to moderate, severe, and finally end-stage rejection on days 6, 8, and 10, respectively. Inducible NOS expression resulted in increased in vivo NO production during mild rejection, which subsequently peaked during moderate rejection on days 5 and 6. The serum nitrite/nitrate levels then decreased significantly during severe and end-stage rejection (days 8 and 10).

This reduction in serum nitrite/nitrate levels as rejection progressed in this model is unlikely to reflect reduced iNOS expression or reduced NO synthesis, as iNOS mRNA and protein were strongly present in these animals and we have previously demonstrated a strong electron paramagnetic resonance signal for the NO molecule on postoperative day 8 [4]. The reduced serum nitrite/nitrate levels on days 8 and 10 may reflect destruction of the graft vasculature during severe rejection, leading to reduced perfusion of the graft, and resulting in decreased release of NO or nitrite/nitrate from the graft. Alternatively, reduced perfusion of the graft may lead to reduced NO synthesis because of limited availability of L-arginine, the substrate for iNOS, or of cofactors required for enzyme activity. These observations suggest that systemic serum or urinary nitrite/nitrate levels may not accurately reflect the extent of localized NO production during the latter stages of severe inflammatory conditions.

The observations that iNOS mRNA and enzyme activity were expressed in allografts during acute rejection but were not detected in isografts or in allografts before histologic changes of mild acute rejection suggest that iNOS expression is a specific result of the immune response to allogeneic tissue and that NO may play regulatory or effector roles in in vivo allograft rejection (as stated subsequently). Although the regulation of iNOS expression during allograft rejection remains to be explored, iNOS expression is likely induced by cytokines such as tumor necrosis factor-{alpha}, interleukin-1, and interferon-{gamma}, which are present in the rejecting heart [16] and induce iNOS expression in other model systems [2].

Recent reports demonstrate that iNOS expression and increased systemic serum nitrite/nitrate levels occur in human cardiac transplant recipients during acute rejection [12, 17]. These results, together with our observation that iNOS was expressed and resulted in increased serum nitrite/nitrate levels during mild acute rejection, suggest that increased NO production, detected by the presence of iNOS mRNA, protein, or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection in the clinical setting. The potential diagnostic value of iNOS expression during human allograft rejection requires further investigation.

Inducible NOS mRNA and protein were expressed in the infiltrating mononuclear inflammatory cells but were not detected in allograft myocytes, endothelial cells, or smooth muscle cells. The cell type that expressed iNOS did not appear to vary during the histologic progression of acute rejection. Although the inflammatory cell subtype cannot be precisely identified from these experiments, the cell morphology is suggestive of macrophages or lymphoblasts. Inducible NOS expression in mononuclear inflammatory cells is concordant with demonstration of iNOS expression and NO production in activated macrophages [6]. Our results concur with observations that iNOS mRNA expression is restricted to infiltrating inflammatory cells during cardiac rejection, in contrast to expression in both infiltrating and parenchymal cells during liver allograft rejection [18]. Others have demonstrated iNOS mRNA or protein expression in both the infiltrating cells and in single myocytes isolated from rat cardiac allografts [9]. Inducible NOS expression can be induced in isolated nontransplanted cardiac myocytes and endothelial cells by in vitro stimulation with endotoxin or cytokines [10, 11]. These discordant results may reflect the different strain combinations used in the allograft studies, different methodologies used to detect iNOS, or the detection of iNOS expression in isolated cells or tissue homogenates [9–11] rather than in tissue sections as in the present study. Our present observations suggest that although myocytes and endothelial cells are capable of iNOS expression, the majority of iNOS expression and resultant NO production during acute cardiac allograft rejection in this in vivo model occurs in the infiltrating inflammatory cells rather than in allograft parenchymal cells. Inducible NOS expression in infiltrating inflammatory cells suggests that NO is a nonallospecific effector molecule produced during acute allograft rejection.

Roles of nitric oxide in allograft rejection
Nitric oxide appears to have multiple roles in experimental cardiac allograft rejection. We have demonstrated that systemic treatment of allograft recipients with the iNOS inhibitor aminoguanidine [8] prevented contractile dysfunction during rejection [4]. Similarly, aminoguanidine treatment prevented increased vascular permeation by macromolecules in the allograft heart and systemic vasculature [5]. 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 that is cytotoxic and cytostatic through the nitrosylation and inhibition of cellular enzymes critical to mitochondrial respiration and DNA synthesis [2, 3], (2) NO is cytotoxic to myocytes in vitro [19], (3) NO–protein complexes are formed during acute cardiac allograft rejection [4, 20], and (4) inhibition of iNOS with aminoguanidine prolonged graft survival and ameliorated the histologic changes of acute allograft rejection [4, 6]. Because the regulation and physiologic and pathophysiologic roles of NO vary across species, further investigation is required to delineate whether NO plays similar roles in human cardiac transplantation.

Conclusions
Inducible NOS mRNA and enzyme activity were expressed in the allograft heart during mild, moderate, and severe acute rejection but were not detected in normals, isografts, or allografts before histologic changes of mild acute rejection. Inducible NOS mRNA or protein was expressed in the infiltrating mononuclear inflammatory cells during acute rejection but was not detected in allograft parenchymal cells or in isografts. The cell type that expressed iNOS did not appear to vary during the histologic progression of acute rejection. These findings, together with our previous observations that inhibition of NO production by iNOS ameliorated acute rejection, suggest that iNOS expression and the resultant increased NO production are important mediators of the host immune response to allogeneic tissue. Elucidation of the molecular signals regulating iNOS expression and of the molecular roles of NO in acute rejection may give further insight into the pathogenesis of acute allograft rejection. Furthermore, increased NO production, detected by the presence of iNOS mRNA, protein, or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute cardiac allograft rejection in the clinical setting.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
We thank Richard B. Schuessler, MD, for help with the statistical analysis and Dawn Schuessler for assistance in manuscript preparation. Supported by the NIH (F32HL09021 and HL46387), the Monsanto-Searle/Washington University Biomedical Program, and a grant-in-aid from the American Heart Association.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Billingham M.E. Diagnosis of cardiac rejection by endomyocardial biopsy. Heart Transplant 1981;1:25-30.
  2. Moncada S., Palmer R.M.J., Higgs E.A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109-142.[Medline]
  3. Stuehr D.J., Nathan C.F. Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 1989;169:1543-1555.[Abstract/Free Full Text]
  4. Worrall N.K., Lazenby W.D., Misko T.P., et al. Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med 1995;181:63-70.[Abstract/Free Full Text]
  5. Worrall N.K., Chang K., Suau G.M., et al. Inhibition of inducible nitric oxide synthase prevents myocardial and systemic vascular barrier dysfunction during early cardiac allograft rejection. Circ Res 1996;78:769-779.[Abstract/Free Full Text]
  6. Worrall N.K., Misko T.P., Sullivan P.M., Hui J.-J., Ferguson T.B., Jr Inhibition of inducible nitric oxide synthase attenuates established acute cardiac allograft rejection. Ann Thorac Surg 1996;62:278-285.[Abstract/Free Full Text]
  7. Worrall N.K., Misko T.P., Sullivan P.M., Hui J.-J., Rodi C.P., Ferguson T.B., Jr Corticosteroids inhibit expression of inducible nitric oxide synthase during acute cardiac allograft rejection. Transplantation 1996;61:324-328.[Medline]
  8. Misko T.P., Moore W.M., Kasten T.P., et al. Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur J Pharmacol 1993;233:119-125.[Medline]
  9. Yang X., Chowdhury N., Cai B., et al. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest 1994;94:714-721.
  10. Balligand J.L., Ungureanu-Longrois D., Simmons W.W., et al. Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. J Biol Chem 1994;269:27580-27588.[Abstract/Free Full Text]
  11. Balligand J.L., Ungureanu-Longrois D., Simmons W.W., et al. Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1ß and IFN-{gamma}. Am J Physiol 1995;268:H1293-H1303.[Abstract/Free Full Text]
  12. Lewis N.P., Tsao P.S., Rickenbacher P.R., et al. Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle. Circulation 1996;93:720-729.[Abstract/Free Full Text]
  13. Bredt D.S., Snyder S.H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A 1990;87:682-685.[Abstract/Free Full Text]
  14. Misko T.P., Schilling R.J., Salvemini D., Moore W.M., Currie M.G. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem 1993;214:11-16.[Medline]
  15. Botney M.D., Kaiser L.R., Cooper J.D., Mecham R.P., Roby J., Parks W.C. Extracellular matrix protein gene expression in atherosclerotic pulmonary arteries. Am J Pathol 1991;140:357-364.[Abstract]
  16. Wu C.J., Lovett M., Wong-Lee J., et al. Cytokine gene expression in rejecting cardiac allografts. Transplantation 1992;54:326-332.[Medline]
  17. Benvenuti C., Bories P.N., Loisance D. Increased serum nitrate concentration in cardiac transplant patients: a marker for acute allograft cellular rejection. Transplantation 1996;61:745-749.[Medline]
  18. Kuo P.C., Alfrey E.J., Krieger N.R., et al. Differential localization of allograft nitric oxide synthesis: comparison of liver and heart transplantation in the rat model. Immunology 1996;87:647-653.[Medline]
  19. Pinsky D.J., Cai B., Yang X., Rodriguez C., Sciacca R.R., Cannon P.J. The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor ß. J Clin Invest 1995;95:677-685.
  20. Lancaster J.R., Langrehr J.M., Bergonia H.A., Murase N., Simmons R.L., Hoffman R.A. EPR detection of heme and nonheme iron-containing protein nitrosylation by nitric oxide during rejection of rat heart allograft. J Biol Chem 1992;267:10994-10998.[Abstract/Free Full Text]



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