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1 Division of Transplant Surgery, 2 Free Radical Research Center, 3 Cardiovascular Research Center, 4 Biophysics Research Institute, 5 Department of Pathology, and 6 Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we examined the
actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft
rejection heterotopically transplanted rat hearts. Chronic
treatment with DETC-Fe inhibited the increase in plasma nitric oxide
(NO) metabolites and nitrosylation of myocardial heme protein as
determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse
injection with DETC-Fe normalized NO metabolites. We verified
intragraft trapping of NO in vivo by pulse injection with DETC-Fe by
the detection within allografts of an anisotropic triplet EPR signal
for DETC-Fe-NO adduct with resonance positions (g tensor
factors for perpendicular and parallel components, respectively
g
= 2.038 and
g
= 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological
rejection scores. DNA binding activity for nuclear factor (NF)-
B and
activator protein-1 was increased in allografts and prevented by
DETC-Fe. Abrogation of the activation of NF-B by DETC-Fe was
associated with increased IB
inhibitory protein. Western blotting
and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO
synthase protein and gene expression. Gene expression for the
proinflammatory cytokine interferon-
was also decreased by DETC-Fe.
Thus DETC-Fe limits NF-B-dependent gene expression and possesses
significant immunosuppressive properties.
nitric oxide synthase; interferon-; electron paramagnetic
resonance spectroscopy; nuclear factor-B; activator protein-1
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACUTE ALLOGRAFT REJECTION is a complex phenomenon that involves various potential inflammatory mediators arising from the interaction of antigen-presenting cells and lymphocytes. The potential mediators include lymphokines, cytokines, and nitric oxide (NO) derived from inducible NO synthase (iNOS). Increased iNOS activity, protein, or mRNA has been documented before rejection in cardiac allografts but not isografts (3, 32, 52, 55). The role of iNOS in acute cardiac allograft rejection is supported by experimental studies showing that treatment with classical immunosuppressive drugs decreased iNOS mRNA or protein (3, 53, 54). Also, iNOS is upregulated in rejecting allografts in humans despite immunosuppressant therapy (18, 46). Supporting a role of iNOS in acute cardiac allograft rejection, several investigators have used treatments in vivo with nonselective and selective iNOS enzyme inhibitors but with variable findings (2, 5, 30, 43, 51). The importance of iNOS was supported as well by studies showing that gene deletion of iNOS decreased histological rejection scores (11).
An alternative approach is to evaluate agents that limit the action of excess NO rather than inhibit enzyme production of NO. This may be accomplished by the design of therapeutic agents that complex NO with a suitable ligand and remove it from the body. Iron complexes of dithiocarbamate derivatives have been used for therapeutic (6, 14) and diagnostic purposes for counteracting or detecting NO (16, 17, 36), respectively, in experimental models of endotoxic shock. Less information is known about the efficacy of dithiocarbamate-iron complexes in acute organ rejection. Previous studies in our laboratory revealed that a hydrophilic dithiocarbamate derivative enhanced acute cardiac allograft survival (33, 37).
Despite this therapeutic efficacy, other studies using electron paramagnetic resonance (EPR) spectroscopy reveal that hydrophilic derivatives such as iron complexes of N-methyl-D-glucamine dithiocarbamate (MGD-Fe) display chemistry that is different from hydrophobic derivatives such as iron complexes of diethyldithiocarbamate (DETC-Fe) (13, 25). Essentially, these studies suggest that DETC-Fe is more resistant to redox changes that might alter the efficiency of NO scavenging in vivo. On the basis of differences in the partitioning characteristics, DETC-Fe would be expected to act intracellularly and in membrane regions, whereas hydrophilic agents like MGD-Fe would likely act primarily in the extracellular space.
To date, no studies have examined the actions of lipophilic derivatives
in rejection in any model of acute organ rejection. The purpose of the
present study was to evaluate the actions of a lipophilic derivative,
DETC-Fe, on scavenging of NO in vivo, nitrosylation of heme protein,
intragraft infiltration of inflammatory cells, activation of key
transcription factors, and gene expression for iNOS and interferon
(IFN)- in acute cardiac transplant rejection.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Transplantation model and protocol.
Rats weighing ~210-230 g were obtained from Harlan
(Indianapolis, IN). The Lewis (Lew: RT11) and Wistar-Furth
(WF: RT1U) rat strains chosen for donor-to-recipient
combinations were Lew 
Lew (for isografts) or WF Lew (for
allografts). In rats anesthetized with an intraperitoneal injection of
50 mg/kg pentobarbital sodium, heterotopic cardiac transplantation was
performed according to techniques established in our laboratory
(33, 37). Graft function was monitored twice daily by
standard external palpation with acute rejection defined as a loss of
palpable contractile activity. Loss of graft function was confirmed on
direct inspection after laparotomy.
Experimental groups and biopsy procedures.
Studies were terminated at either postoperative day 4,
postoperative day 6, or upon the day of rejection. Beginning
the day of surgery until the day of tissue harvest or rejection, a
subset of allograft recipients received twice daily intraperitoneal
injections of 400 mg/kg DETC plus 7.5 mg FeSO4 including
the first dose on the day of tissue harvest or blood samples. This
dosage is equivalent to or lower than that routinely used to detect NO
in a variety of experimental models of endotoxemia or
ischemia-reperfusion injury (47, 50). Grafts were
arrested and flushed with cold University of Wisconsin solution,
minced, and frozen in liquid N2. Tissue was stored at
80°C (for Western blotting and gel shift assays). In a few cases,
the native heart of the recipient was used as an internal control. For
EPR analysis, samples were frozen in 4.0-mm quartz EPR tubes and stored
in liquid N2. Plasma was obtained for determination of NO
by-products, nitrate + nitrite, using a commercial kit (Cayman
Chemical; Ann Arbor, MI).
Histological rejection scoring. Tissue from a portion of grafts was fixed at postoperative day 6 in 4% phosphate-buffered formalin, and paraffin-embedded sections were stained with hematoxylin and eosin. Rejection scoring was performed in a blinded manner based on six-point graded criteria established by the International Society for Heart and Lung Transplantation (ISHLT) as described in our previous studies (34).
EPR spectroscopy. Nitrosylation of myocardial heme protein was detected at liquid N2 temperature using X-band EPR spectroscopy using a liquid N2 finger dewar in a Varian E-109 spectrometer (Palo Alto, CA). Samples from each group were analyzed on the same day under similar instrument settings consisting of a 1,000-G scan range, 4-min scan time, 0.25-s time constant, 2-G 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.
Electrophoretic mobility gel shift assay for the nuclear proteins
NF-B and AP-1.
Nuclear protein from homogenates of cardiac allografts was extracted as
described (37). Doublestranded nuclear factor
(NF)-B or activator protein (AP)-1 oligonucleotides (Promega;
Madison, WI) were end labeled with [-32P]ATP and T4
polynucleotide kinase (Promega) for 10 min at 37°C. After incubation,
the labeled oligonucleotide was desalted and resuspended in Tris-EDTA
buffer. DNA binding reactions were performed at room temperature using
12 µg of nuclear extract, 0.5 ng of labeled oligonucleotide, and 3 µg of poly(dI-dC) (Pharmacia-Upjohn; Kalamazoo, MI) and
electrophoresed using published procedures (37).
Specificity for NF-B or AP-1 binding activity was verified by
competition with 100-fold excess of unlabeled mutant or wild-type oligonucleotides. Antibodies for NF-B supershift assays including p50, p65, c-Rel, and Rel B were obtained from commercial sources (Santa
Cruz Biotechnology; San Diego, CA). Gels were dried on Whatman 3-mm
filter paper and exposed to Kodak XAR film (Eastman Kodak; Rochester,
NY). Band intensity was determined by phosphorimaging (Molecular
Dynamics; Sunnyvale, CA) or by an AlphaImager 2000 image-analysis
system (Alpha Innotech; San Leandro, CA).
Western blotting.
Frozen tissue was homogenized in ice-cold PBS with 1% Triton X-100, 1 mmol/l phenylmethylsulfonyl fluoride, 35 ng/ml pepstatin A, and 10 ng/ml leupeptin. Homogenates were centrifuged at 10,000 g
for 10 min at 4°C. Protein concentration of the supernatant was
determined using the Bio-Rad DC protein assay. Fifty micrograms of each
sample were precipitated with 12.5% trichloroacetic acid containing
0.5 mg/ml deoxycholate. After the pellet was washed in ice-cold
acetone, it was resuspended in SDS-PAGE loading buffer, neutralized
with 1 mol/l Tris base, and electrophoresed on 7.5% SDS-polyacrylamide
gels (for iNOS) or 12% SDS-PAGE gels (for IB) using a Bio-Rad
Mini-Trans-Blot apparatus (Hercules, CA). Proteins were transferred to
Nytran membranes, and blots were probed with a 1:1,000 dilution of
rabbit anti-iNOS (Santa Cruz Biotechnology) or anti-IB (Cell
Signaling Technology; Beverly, MA) and visualized using a 1:5,000
dilution of donkey anti-rabbit IgG horseradish peroxidase conjugate and
enhanced chemiluminescence (Amersham) according to manufacturer's specifications.
Gene expression.
Total RNA was purified from ~60 mg of frozen tissue per sample using
the Promega SV Total RNA Isolation System according to the
manufacturer's directions. RNA concentration was determined spectrophotometrically. cDNA was synthesized from 500 ng of total RNA
and oligo(dT) primer using the Invitrogen Superscript First-Strand Synthesis System for RT-PCR (Carlsbad, CA) according to the
manufacturer's directions. Amplification of iNOS was performed as
previously described (15) with modifications. The 1 µl
of cDNA was mixed with 25 pmol of each primer and Invitrogen PCR
Supermix to a volume of 25 µl, and the reaction was incubated in a
Bio-Rad iCycler under the following conditions: for iNOS, 94°C (60 s), 60°C (60 s), and 72°C (60 s) for 30 cycling times; and for
IFN-, 95°C (30 s), 60°C (30 s), and 72°C (60 s) for 32 cycling
times. One microliter of the PCR product was resolved on a 1.5%
agarose gel in 0.5 × Tris borate-EDTA buffer (pH 8.4, final
concentration 44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA) in the
presence of SyBr green I (Sigma; St. Louis, MO). The PCR product was
visualized by 300-nm ultraviolet transillumination using 
-actin as
the control.
Data analysis. EPR spectra were processed using SUMSPEC and Grapher programs (Golden Software; Golden, CO). Data are presented as means ± SE. Statistics were performed by ANOVA 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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isografts remained functional indefinitely, whereas most
allografts were rejected by postoperative day 7. Chronic
treatment of allograft recipients with DETC-Fe significantly
(P < 0.01) prolonged graft survival (Fig.
1). Histological examination of grafts
harvested at postoperative day 6 revealed pronounced
cellular infiltration in untreated allografts compared with isograft
controls (Fig. 1). ISHLT histological score was decreased by treatment with DETC-Fe at both postoperative day 5 (untreated:
4.25 ± 0.2, n = 8, and DETC-Fe: 3.2 ± 0.5, n = 4; P < 0.03) and postoperative day 6 (Fig. 1).
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On the basis of previous studies in our laboratory, we noted a
pronounced increase in plasma or urine concentrations of nitrate + nitrite and/or iNOS protein in allografts on postoperative day 4 but not on postoperative day 3. The increase in
plasma nitrate + nitrite concentration at postoperative day
4 was decreased in allograft recipients treated chronically with
DETC-Fe (Fig. 2).
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X-band EPR analysis of isografts (Fig. 3)
or native hearts of allograft recipients (data not shown) at
postoperative day 6 revealed background signals for reduced
iron-sulfur (Fe-S) cluster complexes at g = 2.02 and
1.94 and a signal characteristic of semiquinone at g = 2.004. In contrast, EPR analysis of untreated allografts revealed a
strong, broad EPR signal at g = 2.08 and a triplet
signal at g = 2.014 with a hyperfine splitting of 17.5 G. This signal is seen infrequently in any allografts before
postoperative day 4 and is attributed to the nitrosylation
of myocardial heme protein (primarily myoglobin) (2, 33).
Treatment with DETC-Fe decreased the amplitude of the nitrosylheme
protein signal in allograft tissue (Fig. 3).
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To verify in vivo scavenging of NO by DETC-Fe, untreated
allograft recipients received a single-pulse intraperitoneal injection of DETC-Fe on postoperative day 6. Plasma nitrate + nitrite concentration was decreased to near-baseline levels 30 min
later (isografts: 10.24 ± 1.22 µM, n = 9;
allografts: 31.49 ± 2.99 µM, n = 9; DETC-Fe pulse: 13.98 ± 0.54 µM, n = 2). EPR analysis of
DETC-Fe-pulsed isograft controls revealed a four-line EPR signal with
g = 2.025 (Fig. 4). In
these spectra, the background signal at g = 1.94, representing reduced Fe-S cluster protein, is also apparent.
The four-line EPR signal has been described in normal brain tissue after administration of DETC-Fe to represent the g signal
for the copper (II) complex of DETC (44). In contrast to
this four-line EPR signal in isografts, a new signal was clearly
distinguished in DETC-Fe-pulsed allografts that was superimposed
between the composite nitrosylheme signal (i.e., g = 2.08 and the triplet at g = 2.014). The superimposed
signal in allograft recipients was assigned to an anisotropic triplet
signal with resonance positions of g factors of
g = 2.038 and
g = 2.02 representing the mononitrosyl
iron complex of DETC (denoted as DETC-Fe-NO). This signal is similar to
that detected by EPR analysis after the addition of NO solutions
containing DETC-Fe or in cells (27). To document the
source of production of this signal, we observed that the DETC-Fe-NO
adduct was inhibited by a single intravenous injection of 5 mg/kg
L-(1-iminoethyl)lysine, an iNOS inhibitor (4),
30 min before the pulse injection with DETC-Fe (Fig.
5).
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NF-B binding activity was detected by electrophoretic mobility shift
assay (EMSA). Supershift analysis of untreated allograft samples
revealed that dimers were shifted by incubation with antibodies to p50
and p65 but not to Rel B or cRel (Fig.
6). Specificity for NF-B binding in
this assay was verified by the abolition of the band with excess cold
wild-type (Fig. 6, lane 2) but not mutant (Fig. 6,
lane 1) oligonucleotide. NF-B binding activity was
increased in nuclear extracts derived from untreated allografts versus
isograft controls (Fig. 7). NF-B
binding activity was normalized in nuclear extracts isolated from
allograft recipients treated with DETC-Fe (Fig. 7). Western blot
analysis indicates that treatment with DETC-Fe increased IB
protein levels in allografts versus untreated allografts (Fig.
8).
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In addition to activation of NF-B, allotransplantation was
associated with activation of the transcription factor AP-1 (Fig. 9). Specificity for AP-1 binding was
verified by elimination of the band in the presence of excess cold
wild-type (Fig. 9, lane 2) but not NF-B consensus
oligonucleotide (Fig. 9, lane 1). Chronic treatment of
recipients with DETC-Fe inhibited AP-1 activation due to allogeneic
transplantation.
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Western blot analysis of allografts at postoperative day 4 revealed significant upregulation in iNOS protein in untreated allografts compared with isograft controls (Fig.
10). Previous studies have shown that
iNOS protein increases at postoperative day 4 and remains at
this plateau up to postoperative day 6 (unpublished observations). No increase in iNOS protein was found in the
native heart of allograft recipients (data not shown). This
upregulation was nearly blocked or decreased in each recipient treated
with DETC-Fe. RT-PCR revealed increased gene expression of iNOS and the
cytokine IFN- in allografts compared with isografts (Fig. 11). Gene expression for both iNOS and
IFN- was either decreased or absent in allograft recipients treated
with DETC-Fe.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The findings of the present study support the hypothesis that excessive production of NO via the upregulation of iNOS plays a significant role in acute cardiac allograft rejection. Treatment with DETC-Fe complex was shown to inhibit heme protein nitrosylation, decrease peripheral NO by-products, and enhance graft survival. The significance of these findings is that the degree of enhancement in graft survival was similar in magnitude to that observed in our laboratory using low-dose cyclosporine (33, 37). Enhancement of graft survival is roughly equivalent in magnitude to that seen using specific or nonspecific NOS inhibitors (43, 51, 53), although some have reported either no benefit or detrimental effects of these agents on graft survival (2, 30). The enhanced survival by DETC-Fe is consistent with our findings of improved graft survival using either a ruthenium-based NO scavenger (34) or a water-soluble, dithiocarbamate-based iron complex (33, 37), although the potential mechanisms of action of these other water-soluble derivatives have not been examined in detail previously.
DETC-Fe was concluded to cause this benefit, in part, via NO
scavenging. In this context, the rate constant of reaction of NO with
iron-dithiocarbamate compounds in solution has been estimated to be
1.1 × 108
M
1 · s1
(29). Consistent with this property, we showed that
chronic treatment with DETC-Fe decreased plasma nitrate + nitrite
concentration. Our subsequent finding that chronic treatment with
DETC-Fe also decreased iNOS protein levels can partially explain the
decreased plasma NO metabolites. To negate the action on iNOS protein
levels, we performed additional studies to document the in vivo
scavenging properties of DETC-Fe. This was accomplished by
giving a single pulse injection of DETC-Fe in untreated
allograft recipients followed by tissue and plasma harvesting after 30 min. In this strategy, DETC-Fe normalized plasma NO metabolites,
providing indirect evidence of intrinsic activity to scavenge NO in
vivo. In addition, EPR analysis provided direct evidence of the
formation of DETC-Fe-NO adduct after pulse injection with DETC-Fe. The
detection of DETC-Fe-NO adduct in allograft tissue is facilitated by
the superior cell-penetrating property of DETC-Fe compared with more
hydrophilic derivatives, such as MGD-Fe. In the present study, we
provided unequivocal evidence that DETC-Fe scavenges NO under in vivo
conditions. Furthermore, we showed that the NO formation is specific to
allograft tissue. This technique cannot distinguish whether the NO
trapped is derived from cardiac muscle or from trapped infiltrating
cells. The amount contributed from the latter is expected to be limited
because we noted that the amount of iNOS-containing
ED1+-positive macrophage cells peaks at postoperative
day 2 and diminishes to baseline by postoperative day
4 (unpublished findings). On the basis of our knowledge of
the amount of iNOS protein and peripheral NO metabolite levels at the
early time period, detection of DETC-Fe-NO adducts by EPR would be
impractical due to sensitivity considerations. This does not preclude
the possibility that DETC-Fe may act at earlier time periods.
Previously, the detection of iron-nitrosyl complexes ex vivo in
isolated perfused rat hearts after administration of
iron-dithiocarbamates such as MGD-Fe or DETC-Fe to ischemic
myocardium has been attributed to trapping of NO derived from NOS as
the mononitrosyl iron adduct was inhibited by the nonselective NOS
inhibitor N
-nitro-L-arginine
methyl ester (13, 57). In contrast, under certain
conditions, detection of NO by such trapping agents may be independent
of NO derived from NOS due to nonenzymatic conversion of nitrite to NO
(56). We found that the formation of DETC-Fe-NO adduct
within allograft tissue was blocked by prior treatment of recipients
with L-(1-iminoethyl)lysine, a selective iNOS inhibitor. Thus we conclude that detection of DETC-Fe-NO within allograft tissue
arises from NO produced enzymatically from iNOS rather than from
nonenzymatic sources.
We also evaluated the effects of treatment with DETC-Fe on intragraft
infiltration of inflammatory cells. Previous studies using a variety of
NOS inhibitors in acute cardiac allograft rejection have yielded
varying findings. For example,
N-monomethyl-L-arginine, a
nonselective NOS inhibitor that enhanced survival, decreased intragraft
infiltration of inflammatory cells (51). Also, treatment
with the iNOS inhibitor aminoguanidine decreased cell infiltration
(52, 53). In the present study, we observed that treatment
with DETC-Fe inhibited inflammatory cell infiltration. Our findings are
consistent with the findings of decreased cell infiltration with
another water-soluble dithiocarbamate derivative used in experimental
hemorrhagic shock (23). Thus the benefits of the
lipophilic derivative DETC-Fe on acute cardiac allograft rejection may
be related, in part, to inhibiting intragraft infiltration of
inflammatory cells. This anti-inflammatory property may not be unique
to DETC-Fe as our laboratory recently observed a reduction in
histological rejection scores indicative of decreased cell infiltration
by chronic treatment with a ruthenium-based class of NO scavenger
(34). The nature of this effect is incompletely understood
but may relate to actions on alloimmune activation, including cytokine
gene expression, that may be unrelated to NO scavenging (see below). In
this context, diethyldithiocarbamate derivatives have been shown to
possess potent immunosuppressive activity on macrophage or T-lymphocyte
function/activation under in vitro conditions (9, 35) but
have yet to be documented in vivo. Thus it is possible that DETC-Fe had
action in vivo to directly inhibit inflammatory cell activation as a
potential mechanism to limit recruitment and infiltration.
T-cell activation and production of inflammatory mediators is believed
to be mediated, in part, by activation of the transcription factor
NF-B (1). The inactive NF-B transcription factor
exists as a cytosolic protein consisting of a trimeric structure of
NF-B dimers bound to the inhibitory protein IB.
Phosphorylation of IB results in dissociation of the NF-B
dimer subunits usually consisting of homodimers or heterodimers of p50
and p65 subunits. The dissociated dimers translocate to the nuclear
compartment. These dimers bind to sites in the promoter regions of
genes that are important in the development of inflammatory diseases.
NF-B dimers may bind to a plethora of promoters for a variety of
gene products that are believed to be implicated in allograft
rejection, including cell adhesion molecules, major histocompatible
antigen-binding complexes, iNOS, and various inflammatory cytokines.
In the present study, we found that treatment with DETC-Fe normalized
activation of myocardial NF-B. The inhibition of NF-B activation
by DETC-Fe is more pronounced than the modest or partial inhibition
previously shown by our laboratory for water-soluble iron chelators
such as pyrrolidine dithiocarbamate (PDTC) (5) and a
water-soluble dithiocarbamate-based derivative (33). This enhanced activity of DETC-Fe may be related, in part, to more efficient
scavenging of NO by DETC-Fe based on the data of the relative decrease
in NO by-products and inhibition of heme nitrosylation compared with
these other derivatives. It is possible that this is related, in part,
to the higher lipophilicity of DETC-Fe. Indeed, metal complexes of DETC
are six orders of magnitude more lipophilic than similar complexes of
MGD (12). Furthermore, chelation of NO by DETC-Fe in lipid
fractions may be facilitated by the estimated ninefold increased
sequestration of NO in lipid compartments (20). It is also
possible that DETC-Fe might act via other mechanisms independent of NO
scavenging such as an antioxidant mechanism. Indeed, free and
iron-nitrosylated dithiocarbamate derivatives can exhibit antioxidant
potential (19, 21, 48). This action may account for the
inhibition of activation of redox-sensitive transcription factors as
discussed below.
The decrease in NF-B binding activity by treatment with DETC-Fe is
potentially explained, in part, by upregulation of total content of
IB protein levels. Indeed, overexpression of IB protein is
associated with decreased activation of NF-B and decreased NF-B-dependent gene expression (7). Furthermore,
increased myocardial IB protein is associated with an increased
tolerance to endotoxin (40). Reassociation of NF-B
dimers with the increased levels of IB inhibitory protein in
allograft recipients treated with DETC-Fe may serve as a negative
control to limit NF-B activation and downstream effects.
The inhibition of activation of NF-B is likely to counteract
rejection as NF-B is known to participate in T-lymphocyte activation (26). Furthermore, the importance of NF-B was evidenced
in studies showing that NF-B decoy oligodeoxynucleotides limited cell infiltration in renal allografts (49) and enhanced
cardiac allograft survival (8, 45). Our findings that
DETC-Fe prevented NF-B activation, decreased cell infiltration, and
prolonged graft survival are consistent with a link in NF-B
activation and acute cardiac allograft rejection.
We also evaluated the activation of another redox-sensitive transcription factor, AP-1 (39). This transcription factor consists of dimers of Jun and Fos family proteins. DNA binding of these dimers target expression of AP-1-dependent genes. The role of AP-1 activation and its regulation by treatment regimens have not been previously examined in acute cardiac allograft rejection, although AP-1 activation is believed to regulate cell proliferation (39) and AP-1 sites positively regulate transcription of iNOS (22). We found that AP-1 activation occurred in untreated allografts but not in isografts or native hearts of allograft recipients. This indicates that AP-1 activation is specific to alloimmune activation and not related to surgery per se.
The actions of dithiocarbamates on activation of AP-1 in vitro are
complex and incompletely understood as both activation (24,
38) and inhibition (31) have been reported in other experimental models using the iron chelator derivative PDTC. In our in
vivo study, AP-1 activation was prevented by chronic treatment with
DETC-Fe. As there are binding sites for both NF-B and AP-1 in the
iNOS promoter (22), the profound action of DETC-Fe to inhibit activation of both AP-1 and NF-B may provide a concerted dual mechanism to limit inflammatory cell infiltration and inflammatory gene expression.
Accordingly, to examine the potential action of limiting activation of
redox-sensitive transcription factors on the downstream effects on gene
expression, we examined both iNOS protein and mRNA in allograft
recipients treated with DETC-Fe. Previously, the iron chelator PDTC has
been shown to decrease iNOS protein and expression in vitro in
lipopolysaccharide- or cytokine-stimulated macrophage cells (10,
28). Actions of dithiocarbamate-related analogs in vivo on iNOS
have not been examined to date in allograft rejection. Consistent with
a decrease in activation of NF-B and AP-1 by DETC-Fe, we found that
iNOS protein and gene expression were decreased. To our knowledge, this
is the first in vivo study to show that DETC-Fe is able to decrease
iNOS protein in any model of autoimmune or inflammatory disease.
IFN- is released from activated T cells. This cytokine enhances
alloantigen recognition and trafficking of CD4+-T
lymphocytes, and it is a potent stimulant of macrophage-derived NO
production. Thus its expression may indicate an important marker of
early rejection. We also found that treatment with DETC-Fe produced a
marked decrease in IFN- expression. To our knowledge, this is the
first report to show that this class of agents limits expression of
this potent proinflammatory cytokine in acute organ rejection in vivo.
The promoter region for the IFN- gene contains NF-B binding
domains (41). Thus a reduction in iNOS protein by DETC-Fe
may arise secondarily to the reduction in NF-B-dependent IFN-
gene expression. A decrease in IFN- gene expression may indicate
that DETC-Fe also acts on early stages of rejection.
It is important to note that the ability of DETC-Fe to decrease iNOS
protein and mRNA distinguishes it from the known action of NOS
inhibitors that do not alter iNOS protein levels (42). For
example, we noted that an iNOS inhibitor does not alter NF-B binding
activity (unpublished observations). These observations suggest that NO
may not be the molecule that causes activation of NF-B.
Nevertheless, the actions on activation of transcription factors
important for iNOS and IFN- suggest that DETC-Fe has action at early
stages of rejection. These findings raise the hypotheses that agents
such as DETC-Fe might act differently from or are superior to NOS
inhibitors in limiting the upstream and downstream actions of iNOS and
may alter the expression of other unknown mediators of rejection that
might be mediated via NF-B-dependent pathways.
In summary, we conclude that treatment with DETC-Fe prolongs acute
cardiac allograft survival by multiple mechanisms. DETC-Fe appears to
act at upstream and downstream actions of mediators of rejection. These
mechanisms include NO scavenging, decreased intragraft cell
infiltration, inhibited activation of transcription factors (notably
NF-B and AP-1), and limited gene expression for iNOS and IFN-.
Thus DETC-Fe possesses significant immunosuppressive and
anti-inflammatory activities.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health (NIH) Grants HL-64637 (to G. M. Pieper) and AI-41703 (to A. K. Khanna). The authors also acknowledge the support for the EPR Center by NIH Grant RR-01008 to the Biophysics Research Institute at the Medical College of Wisconsin.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. M. Pieper, Div. of Transplant Surgery, Medical College of Wisconsin, 9200 W. Wisconsin Ave., 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.
10.1152/ajpheart.00913.2002
Received 23 October 2002; accepted in final form 2 January 2003.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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P < 0.01 vs.
untreated.
) and semiquinone radical at g = 2.004 (
). Examples show decreased nitrosylation of
myocardial heme protein (g = 2.08 and 2.014) in Allo
recipients treated with DETC-Fe. Each spectra was obtained on the same
day with identical instrument settings.
