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Am J Physiol Heart Circ Physiol 286: H525-H534, 2004; doi:10.1152/ajpheart.00356.2003
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Variable efficacy of N6-(1-iminoethyl)-L-lysine in acute cardiac transplant rejection

Galen M. Pieper,1,2 Vani Nilakantan,1 Gail Hilton,1 Xianghua Zhou,1 Ashwani K. Khanna,2,3 Nadine L. N. Halligan,1 Christopher C. Felix,4 Bal Kampalath,5 Owen W. Griffith,6 Mike A. Hayward,6 Allan M. Roza,1 and Mark B. Adams1

1Division of Transplant Surgery, Department of Surgery 2Cardiovascular Center, 3Division of Nephrology, Department of Medicine, 4Biophysics Research Institute, 5Department of Pathology and 6Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Submitted 15 April 2003 ; accepted in final form 1 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the efficacy and mechanism of action of N6-(1-iminoethyl)-L-lysine (L-NIL), a highly selective inhibitor of inducible nitric oxide (NO) synthase (iNOS), on acute cardiac transplant rejection. L-NIL produced a concentration-dependent attenuation of plasma NO by-products and a decrease in nitrosylation of heme protein without altering protein levels of iNOS. At postoperative day 4, L-NIL did not alter the increased binding activities for transcription factors nuclear factor-{kappa}B and activator protein-1. Whereas L-NIL decreased inflammatory cell infiltration, graft survival was only prolonged at the dose of 1.0 µg/ml that incompletely blocked NO production. Higher L-NIL concentrations (30 and 60 µg/ml) ablated the increased NO production but failed to improve graft survival and even potentiated NF-{kappa}B binding activity examined at day 6. Alloimmune activation indicated by increased cytokine gene expression for interferon-{gamma}, interleukin-6, and interleukin-10 was inhibited in grafts only by treatment with 1.0 µg/ml L-NIL. These findings suggest a complex role of NO in acute cardiac allograft rejection. Partial inhibition of iNOS is beneficial to graft survival, whereas total ablation may oppose any benefits to graft survival. These studies have important implications in understanding the dual role of NO in acute rejection and help to reconcile discrepancies in the literature.

inducible nitric oxide synthase; nuclear factor-{kappa}B; activator protein-1; interferon-{gamma}; interleukin-6; interleukin-10; electron paramagnetic resonance spectroscopy

ACUTE REJECTION after solid organ transplantation is complex involving a variety of potential mediators. Lymphokines, cytokines, and nitric oxide (NO) derived from inducible NO synthase (iNOS) arising from the interaction of antigen-presenting cells and lymphocytes are among the leading candidates as potential mediators. iNOS is upregulated in experimental models of acute cardiac transplant rejection (51, 53) and in rejecting human cardiac allografts despite immunosuppressant therapy (14, 43).

To date, controversy also exists regarding the role of excess NO production in acute cardiac allograft rejection based on data derived by pharmacological manipulation using NOS inhibitors. Indeed, NOS inhibitors that are analogs of arginine have shown a modest increase (51), no increase (2), or decrease (25) in graft survival. We hypothesized that some of these findings might be explained by the fact that these inhibitors are not selective for iNOS and might inhibit the beneficial activity of the constitutive NOS isoform.

Aminoguanidine, an inhibitor claimed to have selectivity for the iNOS isoform, prolonged cardiac graft survival (47, 51) and enhanced contractile activity in ex vivo tissue preparations (40, 53). Histological rejection scores, a marker of inflammatory cell infiltrate, were either decreased (51, 52) or unchanged (40, 50, 53) by aminoguanidine. Whereas aminoguanidine has been used as a putative inhibitor of iNOS, it is important to note that aminoguanidine also lacks specificity. Indeed, aminoguanidine inhibited diamine oxidase at nanomolar concentrations while inhibiting iNOS at micromolar concentrations (38).

In contrast to aminoguanidine, a newer agent N6-(1-iminoethyl)-L-lysine (L-NIL) is an iNOS dimerization inhibitor that displays a 30-fold higher selectivity for iNOS (21, 41). L-NIL has increased potency (41) and superior oral bioavailability in vivo compared with aminoguanidine (6). The mechanism of inactivation of iNOS by L-NIL is also distinctively different from that produced by aminoguanidine (4).

Currently, there is insufficient information regarding the efficacy of highly selective iNOS inhibitors in acute rejection of solid organ transplants. Whereas a modest increase in cardiac allograft survival was reported for combination therapy of L-NIL plus an experimental cyclooxygense-2 inhibitor (55), the effect of either agent alone was not addressed. Thus, to our knowledge, the action of L-NIL in acute cardiac transplant rejection has not been evaluated. In the present study, we examined, in detail, the effects and potential mechanisms of action of treatment with a range of dosages of L-NIL on the activation of myocardial nuclear factor (NF)-{kappa}B, iNOS protein levels, cytokine gene expression, myocardial heme nitrosylation, graft survival, and histological rejection score in a rodent model of acute cardiac transplant rejection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and transplantation of grafts. All animal protocols were approved according to the guidelines of the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. Rats weighing ~210–230 g were obtained from Harlan Sprague Dawley (Indianapolis, IN). Lewis (Lew: RT11) and Wistar-Furth (WF: RT1U) rat strains were chosen to represent genetic disparity at both the major and minor histocompatibility loci for donor-to-recipient combinations of Lew -> Lew (for isografts) or WF -> Lew (for allografts). Sterile surgery was performed in rats anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium. Heterotopic cardiac transplantation to the abdominal aorta and vena cava was performed using established microsurgical techniques. Graft function was monitored twice daily for the presence or absence of palpable activity and was confirmed on direct inspection following laparotomy.

Experimental groups and biopsy procedures. Experiments were terminated at either postoperative day 4 (POD4), postoperative day 6 (POD6), or on the day of rejection. A subset of allograft recipients received 1–60 µg/ml L-NIL added to the drinking water beginning the day of surgery until the day of tissue harvesting or rejection. These concentrations were chosen as those within the therapeutic range found previously to have benefits in inflammatory diseases (6). In contrast to typical nonspecific NOS inhibitors, concentrations up to 150 µg/ml L-NIL do not alter water consumption in rodents (41). Furthermore, concentrations of L-NIL up to 1 mg/ml do not alter systemic blood pressure in normal rats (6, 34). This finding confirms that L-NIL under these conditions does not impact significantly on constitutive NOS activity.

For POD4 and POD6, grafts were arrested and flushed with cold University of Wisconsin solution, minced, and frozen in liquid N2. Frozen tissue was stored at –80°C (for Western blots and gel shift assays). For electron paramagnetic resonance (EPR) spectroscopy, tissue samples were frozen in liquid N2. Plasma was obtained for determination of NO by-products nitrate + nitrite by using a commercial kit (Cayman Chemical; Ann Arbor, MI).

Histological rejection scoring. Tissue from a portion of grafts was fixed at POD6 in 4% phosphate-buffered formalin, and paraffin-embedded sections were stained with hematoxylin and eosin. Rejection scoring was performed blinded. Scoring was based on a six-point graded criteria established by the International Society for Heart and Lung Transplantation (ISHLT) and described previously (31).

EPR spectroscopy. X-band EPR spectroscopy was performed on a Varian E-109 spectrometer (Palo Alto, CA). Samples from each group were analyzed at 77°K on the same day under similar instrument settings consisting of 1,000 Gauss scan range, 4 min scan time, 0.25 s time constant, 2 Gauss modulation amplitude, 100 kHz modulation frequency, and 5 mW microwave power. The magnetic field was calibrated with Fremy's salt giving a g value of 2.0055 ± 0.0001.

Western blotting. Frozen tissue was processed as previously described in our laboratory (30). Samples were electrophoresed on 7.5% SDS-polyacrylamide gels and transferred to Nytran membranes. Proteins were stained with Ponceau S and photographed. Blots probed with 1:2,000 dilution of rabbit anti-iNOS and 1:100 dilution of {beta}-actin (Santa Cruz Biotechnology; Santa Cruz, CA) were visualized by using 1:5,000 dilution of donkey anti-rabbit IgG horseradish peroxidase conjugate and enhanced chemiluminescence.

Gene expression. Total RNA was purified from ~60 mg of frozen tissue per sample by using the Promega SV Total RNA Isolation System (Promega; Madison, WI) according to manufacturer's directions. RNA concentration was determined spectrophotometrically. cDNA was synthesized from 1 µg of total RNA and oligo(dT) primers using the Invitrogen Superscript First-Strand Synthesis System for RT-PCR (Invitrogen; Carlsbad, CA) according to manufacturer's directions. One microliter of cDNA was mixed with 25 pmol of specific sense and antisense primers and Invitrogen PCR Supermix to a volume of 25 µl, and the reaction was incubated in a Bio-Rad iCycler (Hercules, CA) under the following conditions: for interferon-{gamma} (IFN{gamma}), 95°C (30 s), 60°C (30 s), and 72°C (60 s) for 35 cycling times; for IL-6 and IL-10, 95°C (30 s), 60°C (30 s), and 72°C (60 s) for 35 cycling times. One microliter of the PCR product was resolved by 1% agarose gel electrophoresis. Ethidium bromide-stained specific bands were visualized under UV light. Densitometric analysis of specific bands were made using Alpha Imager (Alpha Innotech; San Leandro, CA) and expressed as a ratio to {beta}-actin gene controls.

Electrophoretic mobility gel shift assay for nuclear proteins, NF-{kappa}B, or AP-1. Extraction of nuclear protein from homogenates of cardiac allografts was performed as described (30). Double-stranded NF-{kappa}B or activator protein-1 (AP-1) oligonucleotides (Promega) were end labeled with [{gamma}-32P]ATP as described (30). DNA binding reactions were performed at room temperature by using 12 µg of nuclear extract, 0.5 ng of labeled oligonucleotide, and 3 µg of poly(dI-dC) (Pharmacia-Upjohn; Kalamazoo, MI). After incubation for 30 min, the reactions were electrophoresed on 4% nondenaturing polyacrylamide gel in 0.5x Tris-borate EDTA at 10 V/cm. Specificity for NF-{kappa}B or AP-1 binding activity was verified by competition with 100-fold excess of unlabeled mutant or wild-type competitor oligonucleotides. Gels were dried and exposed to autoradiographic film. Intensity of NF-{kappa}B or AP-1 binding activity was determined by the AlphaImager2000 image analysis system (Alpha Innotech).

Data analysis. EPR spectra were processed for presentation by using SUMSPEC and Grapher programs (Golden Software; Golden, CO). Statistics were performed by analysis of variance for multiple group means or by Student's t-test for comparisons between two group means. Statistical significance was set at the level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The concentration of plasma NO metabolites nitrate + nitrite was increased in untreated allografts compared with isograft controls (Fig. 1). Treatment with L-NIL produced a concentration-dependent decrease in nitrate + nitrite relative to the elevated concentration seen in untreated allografts. Partial inhibition was observed at 1 or 10 µg/ml L-NIL, whereas complete inhibition was observed at 30 and 60 µg/ml L-NIL.



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  		Fig. 1. Dose-dependent decrease in concentration of plasma nitrate + nitrite at postoperative day 4 and 6 [POD4 (A) and POD6 (B), respectively] in allograft recipients receiving 1, 10, 30, or 60 µg/ml N6-(1-iminoethyl)-L-lysine (L-NIL, n = 3–8 each). Results are means ± SE. {ddagger}P < 0.01 vs. untreated allografts (Allo; n = 8–9 each); ¶P < 0.001 vs. isograft (Iso, n = 9 each) controls.

 

EPR analysis revealed a signal identified as nitrosylated heme protein was seen in allografts (but not in isografts) by the broad signal at g = 2.08 and the triplet hyperfine component at g = 2.014 (Fig. 2). Chronic treatment with 1 µg/ml L-NIL had no marked effect on myocardial heme nitrosylation. In contrast and consistent with peripheral NO metabolite measures, L-NIL at 30 and 60 µg/ml L-NIL blocked most of the EPR signal for myocardial heme nitrosylation (i.e., 85–90% inhibition).



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  		Fig. 2. Examples of electron paramagnetic resonance (EPR) spectra showing nitrosylation of heme protein in cardiac Allo but not Iso at POD6 and inhibition at various concentrations of L-NIL. Findings were observed in 3 replicates each group.

 

To determine that decreases in plasma NO metabolites were not due to changes in iNOS protein as a consequence to chronic treatment with L-NIL, we performed Western blot analysis of iNOS protein in allografts of treated recipients. There was upregulation of iNOS in allografts but not isografts or native hearts of allograft recipients (Fig. 3). Examples shown indicate no decrease in iNOS protein levels at the lowest and even highest concentration of L-NIL. Examples shown (Fig. 4) of densitometric analysis of iNOS normalized to {beta}-actin confirmed this observation.



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  		Fig. 3. Western blot showing lack of change in inducible nitric oxide synthase (iNOS) protein levels at POD4 in cardiac Allo at 30 µg/ml L-NIL (A) or 60 µg/ml L-NIL (B).

 


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  		Fig. 4. Western blot densitometry of iNOS-{beta}-actin at both POD4 (A) and POD6 (B) in untreated Allo vs. L-NIL at the lowest and highest concentrations (i.e., 1.0 and 60 µg/ml, respectively).

 

Allografts at POD6 were examined for histological grade of rejection. Relative to untreated allografts, there were significant decreases in rejection scores indicative of decreased intragraft infiltration of inflammatory cells with each dose of L-NIL examined (Fig. 5). Graft survival time was prolonged significantly (P < 0.01) at 1.0 µg/ml L-NIL (Fig. 5) but not at higher concentrations of L-NIL.



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  		Fig. 5. International Society for Heart and Lung Transplantation (ISHLT) rejection scores (A) and graft survival time (B) after treatment with 1, 10, 30, and 60 µg/ml L-NIL (n = 5 or more each group). Results are means ± SE. *P < 0.05 or {ddagger}P < 0.01 vs. untreated Allo.

 

Electrophoretic mobility shift assays of NF-{kappa}B binding activity of nuclear extracts were performed at POD4. This time was previously observed to reflect peak binding activity (7, 30). Examples shown (Fig. 6) revealed that L-NIL did not alter the NF-{kappa}B nuclear binding activity at either the lowest or highest doses of L-NIL. Likewise, binding activity of another oxidant-sensitive transcription factor AP-1 was not altered by treatment with L-NIL (Fig. 7). To determine whether extended treatment with high concentrations of L-NIL might potentiate activation of NF-{kappa}B or AP-1, we performed electrophoretic mobility gel shift assay in nuclear extracts derived from allografts at POD6. In agreement with previous findings in the laboratory (7), NF-{kappa}B binding activity in untreated allografts at POD6 was attenuated compared with untreated allografts at POD4. Similarly, AP-1 binding activity in untreated allografts at POD6 was attenuated compared with POD4. NF-{kappa}B binding activity was increased at POD6 in allografts treated with high doses of L-NIL relative to untreated allografts at this same time period (Fig. 8). AP-1 binding activity was increased in samples derived from allografts treated with high doses of L-NIL.



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  		Fig. 6. Nuclear factor (NF)-{kappa}B nuclear binding activity at POD4 in Allo vs. Iso and lack of change by treatment at the lowest (A; 1 µg/ml) to highest (B; 60 µg/ml) doses of L-NIL. Specificity of NF-{kappa}B binding is shown by the elimination by addition of 100x cold wild-type but not mutant oligonucleotide.

 


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  		Fig. 7. Treatment with L-NIL does not alter activator protein-1 (AP-1) nuclear binding activity. Results are shown vs. Allo and Iso controls. Specific activity of AP-1 binding is shown by the elimination by addition of 100x cold wild-type AP-1 oligonucleotide but not by NF-{kappa}B oligonucleotide. A: 1 and 10 µg/ml L-NIL; B: 30 and 60 µg/ml L-NIL.

 


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  		Fig. 8. Electrophoretic mobility gel shift assay (EMSA) showing differential increase in NF-{kappa}B (A) or AP-1 (B) binding activity in untreated Allo at POD4 vs. POD6. Results also show enhanced NF-{kappa}B and AP-1 binding activity at POD6 by treatment of recipients with L-NIL vs. untreated Allo at POD6. Identical nuclear samples were chosen for both assays.

 

We also determined intragraft expression for candidate cytokine genes by using RT-PCR to assess the differential action of varying L-NIL concentrations. Increased gene expression for IFN{gamma}, IL-6, and IL-10 was seen in allografts versus isograft controls. Consistent with enhanced graft survival, we found marked inhibition of gene expression for all three cytokines for recipients receiving 1.0 µg/ml L-NIL (Figs. 9 and Fig. 10A). This inhibition was not seen for the various cytokines at any of the other higher concentrations of L-NIL (Fig. 9, B–D and Fig. 10B).



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  		Fig. 9. Summary of RT-PCR analysis showing intragraft expression of inflammatory cytokine genes in Iso (I), untreated Allo recipients (A) and drug-treated Allo recipients (L) receiving 1.0 µg/ml (A), 10.0 µg/ml (B), 30.0 µg/ml (C), and 60.0 µg/ml L-NIL (D). Genes include interferon-{gamma} (IFN), interleukin-6 (IL-6), interleukin-10 (IL-1), and {beta}-actin controls.

 


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  		Fig. 10. Densitometry of cytokine gene expression normalized to {beta}-actin for Iso (i, n = 2 each), untreated Allo (a, n = 3 each), and L-NIL-treated (l) Allo recipients at low dose (A; 1 µg/ml) vs. high dose (B; 60 µg/ml) (n = 4 each). Open bars: INF-{gamma}; solid bars: IL-6; shaded bars: IL-10. {ddagger}P < 0.01 vs. Iso; *P < 0.05 vs. L-NIL and {ddagger}P < 0.01 vs. L-NIL.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We were able to show enhanced graft survival by using a highly selective inhibitor of iNOS. This action was concentration dependent occurring at a suboptimal dose of L-NIL that incompletely blocked NO by-product formation and inhibited intragraft gene expression of inflammatory cytokines. The dose of L-NIL that prolonged graft survival time was the same dose previously shown to limit interstitial inflammation in an experimental model of lupus (34). Interestingly, we found that L-NIL failed to prolong graft survival time at doses of L-NIL that completely ablated the increase in plasma NO metabolites. Thus it appears from our study that maintaining residual NO bioactivity is necessary to improve graft survival time. Previously, a role of NO in acute cardiac allograft rejection was evaluated in studies using nonselective NOS inhibitors, NO scavengers/neutralizers, or gene deletion of iNOS with variable conclusions. The results from this study reconcile and add a better understanding to the discrepancy in conclusions in the literature regarding the role of NO in acute cardiac rejection for reasons outlined below.

Relationship to pharmacological intervention strategies with nonselective NOS inhibitors. Previously, variable results on graft survival were obtained by using nonselective NOS inhibitors such as NG-monomethyl-L-arginine (L-NMMA) or nitro-L-arginine methyl ester (L-NAME) (2, 25, 49, 52). It is important to note in all of these studies that findings were based on data derived from a single dosage regimen. Furthermore, we have noted that the dosage of inhibitor chosen in these studies caused incomplete inhibition of urinary or plasma NO metabolites. In a study in which urinary nitrate production had been completely blocked by using L-NMMA, graft survival time was unchanged (2). Furthermore, the one study that showed decreased graft survival time was conducted with a dose of L-NAME that produced significant hypertension (25). We reason that the detrimental actions of this inhibitor under such conditions are likely due to inhibition of constitutive NOS activity.

We do not believe that L-NIL at any concentration evaluated in our study impacted on constitutive NOS activity based on previous information available in the literature. First, blood pressure was unaltered after L-NIL at oral 30 mg/kg bid for 2 days in Lewis rats (37). This dose is far higher than the daily dose estimated to be ~6 mg·kg–1·day–1 in our study. Second, L-NIL had no effect on systolic blood pressure despite 19 wk of treatment (34). The concentration used in that study (up to 1 mg/ml in drinking water) is ~16.7-fold higher than the highest concentration (i.e., 60 µg/ml) used in our study. The finding that L-NIL at the higher concentration did not decrease plasma NO metabolites below the isograft values is additional evidence that this did not significantly impact constitutive NO production. Thus there is no rationale to presume that the range of L-NIL used in our study had significant action on constitutive NOS.

Previous studies using the hydrazine-based iNOS inhibitor aminoguanidine have shown prolonged graft survival time and decreased histological rejections scores (47, 51, 52). Other studies showed improved electrophysiological and contractile performance in excised papillary muscles (53) or improved compliance in excised hearts (40). In the latter study, the improved function ex vivo was not associated with any significant change in cell infiltration.

In a recent study, graft survival was prolonged by using the experimental pyrimidylimidazole-based iNOS dimerization inhibitors BBS-1 and BBS-2 (developed by Berlex Biosciences, Richmond, CA), although only BBS-2 significantly decreased histological rejection scores (44). Interestingly, in two studies, improved graft survival/rejection was associated with decreased iNOS protein (44, 47). Thus it cannot be excluded that decreases in NO production or iNOS enzyme activity by this inhibitor might be secondary to decreased iNOS protein per se. In our study, Western blot analysis does not support this as a potential mechanism to improve graft survival by using the amino acid-based iNOS dimerization inhibitor L-NIL because iNOS protein level was unchanged.

It is important to note that most of the studies noted above by other investigators using experimental iNOS inhibitors were conducted using single doses and/or at doses that produced incomplete inhibition of NO production. Collectively, the divergent findings using nonselective NOS inhibitors can now be explained in the context of our studies using a full range of doses of the selective iNOS inhibitor L-NIL, which produced a wide range of effects on NO metabolite levels. Our studies using a full range of concentrations of iNOS inhibitor add important information supporting our hypothesis that graft survival is facilitated at levels that incompletely block NO production (see expanded discussion in Graft survival time versus histological rejection scores).

Graft survival time versus histological rejection scores. Previously, we have shown that cyclosporine or NO scavengers each prolonged graft survival time but also displayed decreased histological rejection scores. Rather than monitoring graft survival function, some investigators have also used histological scoring (indicative of cell infiltration) to evaluate rejection. Studies using NOS inhibitors have yielded variable findings. Aminoguanidine decreased inflammatory cell infiltration (51, 52) or had no effect (40, 53) despite findings that it prolonged graft survival time or improved contractile function. Similarly, L-NMMA produced a small increase in graft survival time, but this was not associated with changes in rejection scores.

Whereas there are no previous studies on the actions of L-NIL in acute graft rejection, L-NIL decreased both NO production and cellular infiltration in a model of carrageenan-induced inflammation (37). In the present study, the dose of L-NIL that prolonged graft survival time was associated with a significant decrease in histological rejection scores, suggesting decreased inflammatory cell infiltration. Interestingly, we found that rejection scores were also decreased in allograft recipients receiving higher doses of L-NIL despite our finding that graft survival time was unaltered. One possibility is that rejection scores (although commonly used in the field) are not always reliable as a predictor of rejection. In this context, a similar conclusion has been made between a lack of good correlation among histology, T-cell activation, and cytokine gene expression during rejection (14). Another possibility is that each of these measures of graft rejection measures a different end point. Finally, it is possible that the observed increase in graft survival time by L-NIL involves mechanisms other than changes in cell infiltration.

Relationship to pharmacological intervention strategies with NO scavengers/neutralizers. We have found that water-soluble, iron-dithiocarbamate-based, and a ruthenium polyaminocarboxylate class of NO scavengers/neutralizers prolonged acute cardiac graft survival time (27, 29–31). In each case, we noted that a significant portion of NO escaped scavenging by these agents. Because the dithiocarbamate derivatives were water soluble with relatively poor cellular penetration, and the ruthenium-based derivative displayed rapid clearance, these limitations made it difficult or impossible to design studies in which both incomplete or complete NO scavenging could be accomplished.

The present study using L-NIL allowed us to evaluate acute rejection over a range of iNOS inhibitor doses that partially or completely inhibited NO production as determined by two criteria (determination of plasma NO metabolite levels and by detection of nitrosylation of intracellular heme protein using EPR spectroscopy). Recently, we have reported that 30 µg/ml L-NIL prevented the formation at POD4 of dinitrosyl-iron complexes of nonheme Fe-S cluster protein and the inactivation of mitochondrial aconitase in cardiac allografts (28). Nitrosylation of nonheme protein occurs earlier than nitrosylation of heme protein that reaches its maximum on POD6. In the present study, 60 µg/ml L-NIL blocked the increase in plasma NO metabolite levels and markedly decreased myocardial heme-protein nitrosylation (estimated 85–90% maximum inhibition). We surmise that the small L-NIL-resistant component of nitrosylation is related to NOS-independent protein nitrosylation rather than incomplete inhibition of intracellular NO production by iNOS. Indeed, under conditions of inflammation and tissue necrosis that would be expected in advanced stages of acute rejection, it is known that NO can be generated by conversion of background nitrite or nitrate by xanthine oxidase (15, 16), and a portion of heme nitrosylation can be resistant to NOS inhibition (9). Our present findings of prolonged graft survival time at doses of L-NIL that incompletely inhibit NO production are consistent with our previous findings above using NO scavengers/neutralizers.

It is interesting to note that the degree of decrease in peripheral NO metabolite levels relative to untreated allografts by 1.0 µg/ml L-NIL (from 38.2 ± 3.06 to 24.8 ± 0.53 µM at POD6) was similar in magnitude as that previously shown by dithiocarbamate analogs such as for diethyldithiocarbamate-Fe (DETC-Fe) (27 ± µM) and NOX-700 (24.4 ± 2.2 µM; Medinox, San Diego, CA) that prolonged graft survival (30, 36). This suggests that regardless of the means to effect changes in NO, there is likely a window of residual NO concentration that is beneficial to counteract acute rejection. This conclusion is consistent with the hypothesis that NO may have both detrimental and beneficial actions in acute cardiac allograft rejection.

In this context, NO produced from activated macrophages can have both cytotoxic action but also immunosuppressive action on T cell activation (1, 13). Because NO can inhibit lymphocyte activation and proliferation, this might explain the benefits of low concentration of L-NIL that partially decreased NO levels. Complete blockade of NO production could counter the immunosuppressive activity of NO. In this context, we performed additional studies to examine alloimmune activation in vivo by RT-PCR of candidate cytokines implicated in cardiac graft rejection. To our knowledge, there has been no previous information on the effects of either generalized NOS inhibitors or iNOS inhibitors on inflammatory cytokine gene expression in cardiac allografts. The importance of this possibility has become apparent from earlier studies in our laboratories in which treatment with two different dithiocarbamate derivatives that partially limit NO also decreased expression of IFN-{gamma} (29, 30). Interestingly, in the present study, we found that gene expression for IFN-{gamma}, IL-6, and IL-10 was inhibited at 1.0 µg/ml L-NIL, the dose that prolonged graft survival, whereas higher concentrations of L-NIL caused either no change or even enhanced cytokine gene expression. The observation of enhanced cytokine gene expression at the higher concentrations of L-NIL is consistent with other studies in renal inflammation (33) and arthritis models (8) in which doses of L-NIL that ablated NO production caused enhanced IL-6 or IL-10 expression. In our study, the enhanced inflammatory cytokine gene expression varied for individual cytokines and at various levels of NOS inhibition by L-NIL. This finding suggests the possiblity that NO may have a differential action on the expression of inflammatory cytokines depending on the individual cytokine examined and the level of residual NO production. Collectively, our findings indicate that there may be a window of optimal NO level that promotes immunosuppression but that ablation of the increase in NO production in rejection may counteract the inhibition of alloimmune activation seen at lower doses of L-NIL by upregulating inflammatory gene expression.

Relationship to iNOS deletion strategies. Gene deletion is a powerful tool that can give valuable information regarding the role of selective genes in a pathological process; however, studies to date have been variable as well. The first report indicated that the absence of iNOS in iNOS–/–recipient mice improved acute cardiac allograft rejection scores, although graft survival time per se had not been examined (11). In contrast, a second study showed no change in either histological rejection scores or graft survival time of BALB/c allografts transplanted into iNOS+/+ and iNOS–/–recipient mice nor from iNOS+/+ and iNOS–/–donors transplanted into BALB/c mice (17). In a third study, no difference in graft survival time was found between iNOS+/+ and iNOS–/–donors or iNOS+/+ and iNOS–/–recipients (42). A possible explanation for the lack of change is that the cell source of iNOS may be derived from donor (i.e., graft) or recipient (i.e., infiltrating cells) cells during antigen presentation. Indeed, the cellular distribution of iNOS was altered, but not eliminated, depending on the combination of donor and recipient relative to wild-type and iNOS null strains (42). Thus, whereas these studies using iNOS gene deletion are valuable, they cannot eliminate the role of iNOS in rejection due to shifting contributions from donor and recipient sources in such a model. Thus our pharmacological approach provides information that can circumvent this limitation by blocking NO produced via iNOS from donor heart and from infiltrating inflammatory cells derived from the recipient.

Typically, gene deletion studies are conducted in homozygous animals. Such models have the potential to develop compensatory pathways of rejection that may or may not be readily apparent. Furthermore, a limitation is that the genetic mutation exists for the lifetime of the animals and is present before any experimental insult. It is important to note that pharmacological interventions designed to assess the actions of NOS can bridge the gap in these limitations. In our present study, we conclude that altering iNOS enzyme activity over a range up to complete ablation of activity by pharmacological means yields different information than could be derived from studies using iNOS gene deletion alone.

Gene deletion may also alter the immunological and inflammatory profile of rejection that may be unrelated to iNOS. For example, one report in another model system showed that INF-{gamma} suppresses lipid peroxidation in iNOS+/+ mice, but a pro-oxidant action is unmasked in studies using iNOS–/–mice (22). Also, gene deletion of iNOS is associated with altered cytokine gene expression particularly for IL-1{beta} (17). In this regard, the presence of elevated NO has the potential to regulate expression of iNOS and other NF-{kappa}B-dependent genes. It is possible that the absence of this signal in iNOS null mice may alter the immunological and inflammatory profile of Th1 versus Th2 cytokines during acute rejection. Thus data in iNOS–/–mice must be interpreted with caution taking into account these caveats.

Role of NF-{kappa}B and AP-1 activation and relationship to iNOS protein. We have found that doses of iron-based NO scavengers/neutralizers that prolonged graft survival time also inhibited activation of NF-{kappa}B (35, 36). NF-{kappa}B and AP-1 play a significant role in the expression of various inflammatory genes that are believed implicated in graft rejection. Thus inhibition in the activation of NF-{kappa}B and AP-1 may account for decreased cell infiltration and iNOS gene expression. It is also possible that this action may impact on other NF-{kappa}B- or AP-1-dependent genes that may play roles in acute graft rejection. In contrast to actions with NOS scavengers, we were unable to find any inhibition of activation of NF-{kappa}B or AP-1 at any dose of L-NIL, including the dose that prolonged graft survival time. Thus we conclude that the beneficial actions of L-NIL on graft survival time or rejection scores are likely unrelated to mechanisms involving inhibition of NF-{kappa}B or AP-1 activation or events distal to NF-{kappa}B or AP-1 activation.

We have observed that prolonged graft survival time using NOS scavengers such as NOX700 (36) or DETC-Fe (30) was associated with decreased iNOS protein and/or mRNA. This is likely consistent with the secondary actions of these agents to inhibit activation of NF-{kappa}B and AP-1. Because L-NIL is an inhibitor of iNOS enzyme activity, the lack of efficacy of L-NIL on altering iNOS protein or NO metabolites is an expected finding. Furthermore, the lack of changes in peak myocardial NF-{kappa}B and AP-1 at POD4 by L-NIL is consistent with the finding of unaltered iNOS levels. Accordingly, it is important to note that our present study provides evidence that the lack of L-NIL to decrease NF-{kappa}B and AP-1 binding activity is distinguished from the actions of NO scavengers. This distinction is interesting for reasons outlined below.

Role of NO on activation of NF-{kappa}B and AP-1. It is generally accepted that NF-{kappa}B and AP-1 activation contribute to increased iNOS gene expression and that activation of NF-{kappa}B and AP-1 is redox sensitive, suggesting a role of reactive oxygen. Cytokine-induced expression of iNOS appears to require activation of AP-1 (18), although a more recent report suggests that AP-1 activation may also suppress expression of iNOS (23). Currently, it is unclear whether NO, another radical, might be a molecular species that could contribute to or amplify activation of either NF-{kappa}B or AP-1 in acute rejection. Furthermore, a contrary theory is that NO can directly inhibit DNA binding of NF-{kappa}B (19, 24, 26) or AP-1 (46) dimers in cell cultures or more directly by the action of NO donors within the reaction mixture of electrophoretic mobility gel shift assay per se. This property of NO has the potential to explain the molecular basis for the beneficial action of NO.

Studies in cell culture using macrophages or lymphocytes indicate an inhibitory or stimulatory effect of NO donor agents on NF-{kappa}B (5, 10, 12) or AP-1 (20, 32, 45, 48) activation. It has not been resolved what determines whether NO elicits an inhibitory versus stimulatory signal (3). Despite these findings in vitro, there is no information of the potential role of NO on NF-{kappa}B activation in the setting of acute rejection in vivo. We found that peak NF-{kappa}B and AP-1 binding activity at POD4 was unchanged using high doses of L-NIL that block NO production. Thus we would conclude that NO derived from iNOS or a reactive oxygen species derived from the iNOS reaction is unlikely to contribute to activation of NF-{kappa}B and AP-1 in acute rejection in vivo. This conclusion is modified based on additional studies (see below).

Previously, we were the first to report that myocardial NF-{kappa}B binding activity is transiently increased to a peak level at POD4 in untreated allografts followed by a decrease before full graft rejection at POD7 (7). Thus we decided to evaluate the possibility that high doses of L-NIL that did not alter the increase in NF-{kappa}B or AP-1 binding activity at POD4 might actually enhance the activation of NF-{kappa}B or AP-1 later on at POD6. Indeed, we found that both 30 and 60 µg/ml L-NIL potentiated both NF-{kappa}B and AP-1 binding activity. These findings compliment reports that 30 µg/ml L-NIL exacerbated inflammation and enhanced NF-{kappa}B binding activity in a model of nephropathy (33). This action illustrates the consequences of complete inhibition of NOS for prolonged periods of time. Thus we surmise that the potentiation of NF-{kappa}B and AP-1 activation might explain the influences of continued treatment with higher doses to counteract any benefits to graft survival. This action could also account for the observation that prolonged treatment with L-NIL increases intimal thickening in a chronic model of aortic transplant-induced arteriosclerosis (39).

In conclusion, we emphasize that our studies support a role of excess NO in acute cardiac allograft rejection. Our studies also support the notion that total ablation of excess NO production does not improve graft survival, whereas partial inhibition does promote graft survival. This is consistent with the notion that NO may have both adverse and beneficial actions. Finally, the design of the experiments of this study uses a full range of inhibitor doses that gives greater insight and helps to reconcile the divergent findings previously reported using single concentrations of nonselective NOS inhibitors, NO scavengers, and iNOS null mice.


   ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Institutes of Health (NIH) Grants HL-64637 (to G. M. Pieper), AI-41703 (to A. K. Khanna), DK-48423 (to O. W. Griffith) and for the NIH EPR Center Grant RR01008 to the Biophysics Research Institute at the Medical College of Wisconsin.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. Pieper, Division of Transplant Surgery, Medical College of Wisconsin, 9200 West Wisconsin Av., Milwaukee, WI 53226 (E-mail: gmpieper{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
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