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Annals of Clinical & Laboratory Science 34:187-194 (2004)
© 2004 Association of Clinical Scientists

Pyrrolidine Dithiocarbamate Added to University of Wisconsin Solution Inhibits Reperfusion Injury after Orthotopic Liver Transplantation in Rats

Xiao-Ping Gu1, Fu-Tao Xu1, Yong Jiang2, Yu-Dong Qiu2 and Yi-Tao Ding2
1 Departments of Anesthesiology and 2 Hepatobiliary Surgery, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, People’s Republic of China

Address correspondence to Yi-Tao Ding M.D., Department of Hepatobiliary Surgery, Drum Tower Hospital, Medical School of Nanjing University, Nanjing 210008, People’s Republic of China; tel 86 25 8330 4616, x10427; fax 86 25 8331 7016; e-mail yys982002{at}yahoo.com.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
This study investigated the effects of pyrrolidine dithiocarbamate (PDTC), a novel NF-{kappa}B inhibitor, on the expression of multiple inflammatory mediators and on neutrophilic inflammation of the graft in rats following liver transplantation. Orthotopic liver transplantation (OLT) was performed after 24 hr of cold storage using University of Wisconsin (UW) solution that contained various concentrations of PDTC. We determined the time course of NF-{kappa}B activation and of the expression of multiple inflammatory signals: tumor necrosis factor-{alpha} (TNF-{alpha}), cytokine-inducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1 (ICAM)-1. Serum alanine aminotransferase (ALT), intrahepatic myeloperoxidase (MPO/ WBC ratio, a measure of neutrophil accumulation), and Mac-1 expression (CD11b/CD18, a measure of circulating neutrophil activity) were also evaluated. The results showed that PDTC decreased OLT-induced NF-{kappa}B activation in a dose-dependent manner (from 20 mmol/L to 60 mmol/L), diminished TNF-{alpha}, CINC, and ICAM-1 protein levels in the graft, and reduced the OLT-induced increase of serum TNF-{alpha} level. Pretreatment with PDTC significantly suppressed OLT-induced neutrophilic inflammation of the graft. The PDTC-exposed livers (PDTC, 40 mmol/L), in comparison with the control livers, had a significant reduction of MPO/WBC ratio (7.04±0.97 vs 14.07±1.31) and Mac-1 expression (181±11.3% vs 281±13.2%) at 6 hr after reperfusion. Furthermore, PDTC inhibited the increase of serum ALT levels after liver transplantation. In conclusion, PDTC inhibited NF-{kappa}B activation and the expression of the inflammatory mediators. These effects were associated with improved graft viability through inhibited intrahepatic neutrophilic inflammation. A therapeutic strategy directed at inhibition of NF-{kappa}B activation within the transplanted liver might be effective in reducing intrahepatic neutrophilic inflammation, and be beneficial to prolonged graft storage.

(received 8 October 2003; accepted 4 January 2004)

Keywords: liver transplantation, pyrrolidine dithiocarbamate, NF-{kappa}B, TNF-{alpha}, , inflammatory mediators


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Liver transplantation has become an accepted therapy for end-stage liver disease, but poor immediate graft function presents a persistent problem and contributes to high mortality rates [1]. Reperfusion after ischemia is responsible for the majority of tissue injury in liver transplantation [2–4]. Recent evidence confirms that liver ischemia/ reperfusion (I/R) injury is a result of activation of inflammatory mediators that occurs early in reperfusion [5].

Treatment of I/R injury is related to the multiplicity of mediators that are involved in its pathogenesis. However, the inhibitory actions of these mediators may be compensated by the release of other mediators. I/R triggers the mediator release by the activation of multiple proinflammatory genes [4–9]. Previous studies showed that a transcription factor, nuclear factor (NF-{kappa}B), plays an essential role in transcriptional regulation of these genes [5,10]. NF-{kappa}B is an appropriate target for the treatment of I/R, because some gene products activated by NF-{kappa}B have major roles in the pathogenesis of I/R injury [11–12]. NF-{kappa}B appears to have both beneficial and harmful effects in liver transplantation. It promotes liver regeneration and prevents apoptosis, but contributes to the inflammatory response of ischemia/reperfusion injury [13].

In the present study, we determined the time course of NF-{kappa}B activation after OLT in relation to the expression of tumor necrosis factor (TNF-{alpha}), intercellular adhesion molecule (ICAM-1), and cytokine-induced neutrophil chemoattractant (CINC). We studied (a) the selectivity of pyrrolidine dithiocarbamate (PDTC) as an in vivo inhibitor of NF-{kappa}B activation; (b) the effects of PDTC on OLT-induced expression of inflammatory gene products and the resultant hepatic neutrophilic inflammation; and (c) the role of NF-{kappa}B activation as a factor that leads to poor immediate graft function after transplantation, showing that inhibition of NF-{kappa}B activation reduces this injury and improves graft function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Reagents.  Pyrrolidine dithiocarbamate (Sigma, St Louis, MO), Nuclear Extract Kit (Active Motif, Carlsbad, CA), NF-{kappa}B p65 Transcription Factor Assay Kits (Active Motif), Pierce BCA protein assay reagent (Jiancheng, Nanjing), MPO assay reagent (Jiancheng, Nanjing), CINC, TNF-{alpha}, and ICAM-1 ELISA assay kit (Amersham, Buckinghamshire, UK), FACS lysing solution (Becton Dickinson, San Jose, CA), FITC-Labeled mouse anti-rat CD11b antibody (Biosource, Camarillo, CA).

Animals.  Male wistar rats (200 to 250 g, body wt) were from the Animal Experimental Unit, Nanjing Medical Center. Prior to use in the study, the rats were fasted for 12 hr and allowed free access to water.

Experimental protocol  Rats were allocated randomly to 4 groups (36 rats per group). In group 1 (control group) the grafts were harvested with injection of University of Wisconsin solution. In group 2, (PDTC20 group) grafts were harvested with the preservation solution altered by addition of PDTC (20 mmol/L). In group 3 (PDTC40 group) grafts were harvested with the preservation solution altered by addition of PDTC (40 mmol/L). In group 4 (PDTC60 group) grafts were harvested with the preservation solution altered by addition of PDTC (60 mmol/L).

Liver harvesting and orthotopic transplantation.  These procedures were performed by the method of Kamada and Calne [14] with minor modifications [15]; the hepatic artery was not reconstructed. The grafted liver was preserved with University of Wisconsin (UW) solution at 4°C for 24 hr. The portal vein of the recipient was clamped for 18 to 20 min; the implantation surgery required <50 min; the entire procedure took about 60 min. There were no significant differences in portal clamping time among the groups. The transplant recipients were killed at 0, 0.5, 1, 2, 4, or 6 hr after reperfusion. At each time point, blood samples were collected from the hepatic vein that drained the left lateral lobe or the liver in order to determine the serum levels of alanine aminotransferase (ALT) and to assay neutrophil activity at the specified time point. The median lobe of the liver was excised at the designated time points and stored at –80°C for later analysis.

NF-{kappa}B activity.  Nuclear extracts were prepared using the nuclear extraction kit; the extracts were analyzed for the p65/relA subunit of NF-{kappa}B using the NF-{kappa}B p65 kit, according to the manufacturer’s protocol. Total nuclear protein content of each specimen was measured by the Pierce BCA protein assay. NF-{kappa}B activity was expressed as µg of p65/ relA subunit per mg of total nuclear protein.

Graft function test.  Hepatocellular function and hepatic injury were assessed by determining the serum alanine aminotransferase activity of 6 rats in each group at specified time points after reperfusion. Serum alanine aminotransferase was measured using the Opera clinical chemistry system (Hitachi, Tokyo, Japan).

Measurements of TNF-{alpha} and CINC.  Serum TNF-{alpha} was measured in duplicate by the immunoassay kit, according to the manufacturer’s protocol. Hepatic levels of TNF-{alpha}, CINC, and ICAM-1 were determined by a modification of the method of Chandrasekar et al [16]. In brief, the medial hepatic lobe was weighed and homogenized at 4°C in 5 ml of 0.1 M phosphate buffer (pH 7.4) containing 0.05% (w/v) sodium azide. The homogenate was centrifuged (2000 x g, 10 min) to remove minute amounts of tissue debris. The supernatant was assayed using a rat ELISA system. The concentration of antigens in liver homogenate was standardized by reference to the total protein content of each specimen, measured by the Pierce BCA protein assay reagent. The results were expressed as pg/g of tissue.

Intrahepatic neutrophil accumulation assessment.  Activity of myeloperoxidase (MPO), an enzyme stored in the azurophilic granules of neutrophils, was used to measure tissue neutrophil sequestration [17]. Frozen lungs were thawed and extracted for MPO according to the homogenization and sonication procedures in the manufacturer’s protocol. One unit of MPO activity is defined as the amount of enzyme that reduces 1 µmol of peroxide/min. MPO activity was expressed as U/g of tissue. To control for intravascular leukocytosis, the ratio of MPO activity in tissue to the WBC count of peripheral blood was calculated and expressed as MPO/WBC.

Circulating neutrophil activity analysis.  Blood samples were prepared for flow cytometric analysis [18]. For CD11b and L-selectin determinations, 50 µl samples were incubated with 10 µl of PE-labeled mouse anti-rat L-selectin antibody and 10 µl of FITC-labeled mouse anti-rat CD11b antibody for 15 min. One ml of FACS lysing solution was added; after 10 min the samples were centrifuged at 1500 x g for 5 min The cells were washed, resuspended in 500 µl of phosphate buffered saline solution, and were analyzed with a flow-cytometer (FACSCalibur, Becton-Dickinson Co, East Rutherford, NJ).

Statistics.  The data were expressed as mean±SD and analyzed by ANOVA using SPSS software (version 11.0) and by the Student-Neuman-Keuls post-hoc test; p values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
PDTC inhibition of NF-{kappa}B activation in grafts of OLT rats.  NF-{kappa}B activity in liver tissue after reperfursion was measured by assaying the p65/relA subunit in nuclear extracts. In the control group, NF-{kappa}B activity in the preserved graft increased markedly at 0.5 hr after reperfusion, reached a peak at 2 hr, and decreased thereafter (Fig. 1Go). Addition of PDTC to the UW solution suppressed the OLT-induced p65/relA subunit increase in a dose-dependent manner, but did not affect the time course for maximal NF-{kappa}B activation after OLT.



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  		Fig. 1. Effect of cold-preservation with PDTC on NF-{kappa}B activation in the graft livers of OLT rats after reperfusion. Hepatic levels of the p65/relA subunit of NF-{kappa}B were significantly inhibited in the PDTC-treated groups, compared to the control group (solid squares). Results are mean±SD, n=6, for each interval and each group. PDTC20 = open squares, PDTC40 = open triangles, PDTC60 = open circles. * p <0.05 vs controls, # p <0.05 vs PDTC40 group.

 
PDTC inhibition of inflammatory mediator expression in grafts of OLT rats.  OLT-induced TNF-{alpha}, CINC, and ICAM-1 expression was determined in the same liver samples that were assayed for NF-{kappa}B p65. The TNF-{alpha}, CINC, and ICAM-1 protein levels in liver homogenates showed different kinetics for the expression of these inflammatory mediators. After reperfusion, the increase in TNF-{alpha} expression was maximal at 1 to 2 hr (Fig. 2AGo), concurrent with NF-{kappa}B activation. The peak expression of CINC (Fig. 2BGo) and ICAM-1 (Fig. 2CGo) was at 4 hr, which followed the peak of NF-{kappa}B activation. Pretreatment with PDTC suppressed the expression of these proteins, although the responses were variable (Fig. 2Go). Maximal inhibition of CINC and ICAM-1 expression was observed at a PDTC concentration of 40 mmol/L, whereas maximal inhibition of TNF-{alpha} expression was observed at a PDTC concentration of 60 mmol/L.





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  		Fig. 2. Effect of cold-preservation with PDTC on inflammatory mediator responses in the graft livers of OLT rats after reperfusion. Hepatic levels of the 3 inflammatory mediators were significantly inhibited in the PDTC-treated groups, compared to the control group (solid squares). Results are mean±SD, n=6, for each interval and each group. PDTC20 = open squares, PDTC40 = open triangles, PDTC60 = open circles. * p <0.05 vs controls, # p <0.05 vs PDTC40 group.

Chart 2A (above, left column) shows the data for hepatic TNF-{alpha} expression, which reached a peak level at 1 hr after reperfusion. TNF-{alpha} expression in the PDTC-treated groups was significantly less than controls at 0.5 hr after reperfusion.

Chart 2B (in the left column) shows that hepatic CINC levels in the 3 PDTC-treated groups were significantly different from controls at 2, 4, and 6 hr after reperfusion. The peak CINC levels in all groups occurred at 4 hr after reperfusion.

Chart 2C (above, right column) shows that hepatic ICAM-1 levels in 2 of the PDTC-treated groups were significantly lower than the controls at 0.5 hr after reperfusion. The peak levels of hepatic ICAM-1 occurred at 4 hr after reperfusion in 3 of the 4 groups.

 
PDTC inhibition of systemic TNF-{alpha} and CINC responses in OLT rats.  The effects of PDTC on serum TNF-{alpha} and CINC are shown in Fig. 3A and Fig. 3BGo, respectively. Pretreatment with PDTC at concentrations of 20, 40, and 60 mmol/L reduced the OLT-induced serum TNF-{alpha} levels to 66%, 51%, and 46%, respectively, at 4 hr after reperfusion. Similarly, the serum CINC levels were reduced to 70%, 48%, and 61%, respectively.




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  		Fig. 3. Effect of cold-preservation with PDTC on the systemic inflammatory responses in graft recipient rats after reperfusion.Results are mean±SD, n=6, for each interval and each group. Control group = solid squares, PDTC20 = open squares, PDTC40 = open triangles, PDTC60 = open circles. * p <0.05 vs controls, # p <0.05 vs PDTC40 group.

Chart 3A (above, left column) shows that serum levels of TNF-{alpha} were significantly inhibited in the 3 PDTC-treated groups, compared to the control group. There was no significant difference between the PDTC40 and PDTC60 groups.

Chart 3B (above, right column) shows that serum levels of CINC were significantly lower in the PDTC-treated groups compared to the control group, at 2, 4, and 6 hr after reperfusion. The serum CINC levels in the 3 PDTC-treated groups were significantly different from each other.

 
PDTC effect on neutrophil sequestration in grafts in OLT rats.  The MPO assay was used to measure tissue neutrophil infiltration over the course of reperfusion. In control transplant livers, the MPO/ WBC value was significantly elevated at 30 min after reperfusion compared to non-reperfused liver tissue and remained significantly elevated throughout the 6 hr after reperfusion (Fig. 4Go). PDTC reduced the reperfusion-induced PMN accumulation; maximal inhibition was at PDTC concentration of 40 mmol/ L, which correlated with the inhibition of OLT-induced intrahepatic CINC and ICAM-1 expression.



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  		Fig. 4. Effect of cold-preservation with PDTC on intrahepatic neutrophil accumulation in recipient OLT rats after reperfusion, as assessed by MPO/WBC. Hepatic neutrophil accumulation was significantly inhibited in the PDTC-treated groups, compared to the control group (solid squares) as early as 0.5 hr after reperfusion. Results are mean±SD, n=6, for each interval and each group. PDTC20 = open squares, PDTC40 = open triangles, PDTC60 = open circles. * p <0.05 vs controls, # p <0.05 vs PDTC40 group.

 
PDTC effect on circulating neutrophil activity in OLT rats.  Increased surface expression of Mac-1 (CD11b/CD18) is important for neutrophil adherence to parenchymal cells. The CD11b content of circulating neutrophils was evaluated by flow cytometry at 0, 0.5, 1, 2, 4, and 6 hr after the onset of reperfusion. In the control group, expression of Mac-1 in controls at the initial time point (0 hr) showed low levels of staining for CD11b (mean fluorescence intensity (MFI) = 76.8±19.2); the CD11b level reached 167% of baseline at 2 hr and 289% at 4 hr; the CD11b level in the control group remained constant at 6 hr (281%) after reperfusion (Fig. 5Go). PDTC inhibited Mac-1 expression in circulating neutrophils in a dose-dependent manner; maximal inhibition was observed at a PDTC concentration of 60 mmol/L, which correlated with the inhibition of the OLT-induced systemic TNF-{alpha} response, but not with the systemic CINC response. This suggests that TNF-{alpha} is a stronger inflammatory mediator in respect to neutrophil activation.



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  		Fig. 5. Effect of cold-preservation with PDTC on circulating neutrophil Mac-1 expression in recipient OLT rats after reperfusion. Mac-1 expression in circulating neutrophils became significantly inhibited in the PDTC-treated groups, compared to the control group (solid squares) at 2 hr after reperfusion.There was no significant difference between the PDTC40 and PDTC60 groups. Results are mean±SD, n=6, for each interval and each group. PDTC20 = open squares, PDTC40 = open triangles, PDTC60 = open circles. * p <0.05 vs controls, # p <0.05 vs PDTC40 group.

 
PDTC effect on postransplantation liver function.  In the controls, the serum ALT level began to increase at 0.5 hr after reperfusion and became progressively elevated throughout 6 hr after reperfusion (Fig. 6Go). PDTC administration caused improvement of liver function after transplantation; the maximal suppression of serum ALT elevation was achieved at a PDTC concentration of 40 mmol/L; PDTC did not affect the time course for serum ALT elevation after OLT (Fig. 6Go).



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  		Fig. 6. Effect of cold-preservation with PDTC on serum ALT activities in OLT rats after reperfusion. Serum ALT activity was significantly inhibited in the PDTC-treated groups, compared to the control group (solid squares) as early as 2 hr after reperfusion. Results are mean±SD, n=6, for each interval and each group. PDTC20 = open squares, PDTC40 = open triangles, PDTC60 = open circles. * p <0.05 vs controls, # p <0.05 vs PDTC40 group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we investigated the mechanisms of neutrophilic graft inflammation following orthotopic liver transplantation (OLT) in rats, assessing the potential contribution of transcription factor NF-{kappa}B. Since transcription factors determine cellular phenotype by specifying protein production [19], it is germane to relate changes in activation of transcription factors, such as NF-{kappa}B, to the expression of specific proteins and the evolution of biologically relevant end-points [20]. We studied the expression of TNF-{alpha}, ICAM-1, and CINC because of their critical roles in the reperfusion-induced inflammatory response, and we observed that reperfusion-induced NF-{kappa}B activation in vivo acompanied the expression of TNF-{alpha} and clearly preceded the expression of CINC and ICAM-1. Thus, in the rat orthotopic liver transplantation, we observed links between NF-{kappa}B activation, systemic and intrahepatic TNF-{alpha}, CINC response, intra-hepatic ICAM-1 expression, neutrophilic inflammation, and graft injury. In addition, we demonstrated that inhibiting NF-{kappa}B activation with PDTC decreases local TNF-{alpha}, CINC, and ICAM-1 expression, ameoliates neutrophilic inflammation, and improves graft function. These observations support the hypothesis that NF-{kappa}B activation is a critical factor for early graft injury in OLT.

Dithiocarbamates, such as PDTC, represent a class of antioxidants that are potent inhibitors of NF-{kappa}B and are capable of suppressing the inflammatory process that is associated with activation of NF-{kappa}B [12,20]. The most effective inhibitor of NF-{kappa}B appears to be PDTC, as a result of its ability to cross cell membranes and its prolonged stability in solution at physiological pH. PDTC, as well as other anti-oxidants, may inhibit NF-{kappa}B by suppressing the intracellular production of reactive oxygen species [21]. Inhibition of NF-{kappa}B is a major mechanism of the anti-inflammatory actions of PDTC [12]. In our study, PDTC probably blocked neutrophilic inflammation in part by diminishing NF-{kappa}B–dependent transcription of CINC and ICAM-1 expression. Blocking NF-{kappa}B activation may also have diminished the transcription of other genes involved in the initiation of inflammation.

At 60 mmol/L, PDTC caused less inhibition of OLT-induced expression of ICAM-1 and CINC than at 40 mmol/L, but the higher PDTC concentration produced greater inhibition of NF-{kappa}B activation. This dissociation of inhibition of NF-{kappa}B activation from the inhibition of ICAM-1 and CINC expression may be explained by the involvement of AP-1 [22]. It is likely that 60 mmol/L PDTC inhibited NF-{kappa}B–mediated stimulation of ICAM-1 and CINC expression and that 60 mmol/L PDTC also augmented AP-1–mediated transcription of ICAM-1. Therefore in respect to ICAM-1 and CINC expression, an inhibitory effect of PDTC that was mediated by NF-{kappa}B suppression may have been offset by an AP-1–mediated stimulatory effect. Further studies are needed to test this supposition.

At 60 mmol/L, PDTC caused less neutrophil accumulation in the graft but more neutrophil activity and less improvement of graft injury than at 40 mmol/L PDTC. This may indicate that local accumulation of activated neutrophils was the definitive factor that influenced local neutrophilic inflammation.

The observation that PDTC administration ameliorated liver injury at 0.5 and 1 hr after reperfusion, as measured by serum ALT levels, was reflected by effects of PDTC on the neutrophil-independent phase of liver injury associated with OLT. In this study, neutrophil sequestration occurred by 0.5 hr after reperfusion, whereas Mac-1 and L-selectin expression were unchanged at the 0.5 and 1 hr time points. According to previous studies, increased expression of Mac-1 [23] and shedding of L-selectin [24] are important to neutrophil-mediated injury. We envision that hepatic injury at the early time points is neutrophil-independent and is mediated by cytosolic xanthine oxidase and mitochondria in damaged hepatocytes [25], and that the relevant oxidant stress occurs initially in the vasculature and is produced by Kupffer cells [26]. This concept is consistent with our data. The protective effect of PDTC in the liver at 0.5 and 1 hr of reperfusion may be mediated by antioxidant modulation of Kupffer cell function. In this study, PDTC inhibited intrahepatic TNF-{alpha} expression in a dose-dependent manner. According to other studies, TNF-{alpha} was probably activated by hepatic Kupffer cells during the 24-hr period of cold-preservation [27]. That is, the presence of primed Kupffer cells during extended cold preservation might result in over-response and excessive production of TNF-{alpha} in the initial phase of reperfusion. The decreased expression of TNF-{alpha} in PDTC treatment groups might partly reflect the Kupffer cell inhibition.

The present study has documented several important observations: (a) the in vivo relationships between NF-{kappa}B activation and the expression of inflammatory genes in the OLT model; (b) the in vivo efficacy of PDTC as an additive to UW solution in inhibiting NF-{kappa}B activation; (c) the effects of inhibiting NF-{kappa}B activation by use of PDTC to reduce systemic and intrahepatic inflammatory responses; and (d) the effects of PDTC-induced alterations of systemic and intrahepatic inflammatory responses on neutrophilic inflammation and liver injury in the graft. The protective effects of PDTC in vivo seem to be pluripotent, comprising its antioxidative properties and its inhibition of NF-{kappa}B. In conclusion, PDTC inhibition of NF-{kappa}B activity provides protection from early graft injury during prolonged cold preservation of liver for orthotopic transplantation.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors are grateful to Dr. Cheng Jun-Bao and Dr Zhang Li-Hua of Drum Tower Hospital for technical assistance. This work received support from the Provincial Educational Foundation of Jiangsu (KJS 02055) and from the Anesthesia Laboratory of Xuzhou Medical Institute.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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