Liver injury caused by ischemia/reperfusion (I/R) insult represents the major problem following orthotopic liver transplantation (OLT). I/R damage has been linked to Th1-like cytokine producers. This study evaluates putative cytoprotective effects/mechanisms of Th2-type IL-13 gene transfer. IL-13 overexpression prevented hepatic insult in a rat model of 24 h cold ischemia followed by OLT, as assessed: (i) profoundly decreased hepatocellular damage (sGOT levels), and ameliorated histological signs of I/R injury (Suzuki criteria), consistent with long-term OLT survival; (ii) prevented hepatic apoptosis (TUNEL stains) and up-regulated expression of antiapoptotic (A20, Bcl-2/Bcl-xl)/antioxidant (HO-1) genes. However, inhibition of HO-1 with tin protoporphyrin reversed cytoprotective/antiapoptotic effects of IL-13. In conclusion, cytoprotection rendered by virally induced IL-13 against hepatic I/R injury in this clinically relevant rat hepatic cold I/R injury model was accomplished via decreased apoptosis and induction of antiapoptotic/antioxidant molecules. HO-1 neutralization studies suggest that HO-1 represents one of putative IL-13 downstream effectors. This study provides the rationale for novel approaches to maximize organ donor pool through the safer use of OLTs despite prolonged periods of cold ischemia.
Ischemia/reperfusion (I/R) injury, the major clinical problem in orthotopic liver transplantation (OLT), results in microcirculatory failure, followed by necrosis and cell death (1–3). Although studies on hepatic I/R insult have provided some etiologic factors, including activation of Kupffer cells, release of pro-inflammatory cytokines, increased expression of vascular cell adhesion molecules, and neutrophil influx (4–7), the exact mechanisms and mediators involved remain to be elucidated.
Apoptosis, or programmed cell death, has been shown to occur in various organs exposed to the damage caused by I/R injury and transplantation (8–10). Indeed, a growing number of reports suggest cell apoptosis as a primary mechanism of I/R injury (11,12). Apoptotic machinery becomes activated during the early phase of reperfusion after liver ischemia and transplantation (13). Furthermore, apoptosis is involved in sinusoidal endothelial cell damage during organ preservation (12). The endothelial cell detachment and rounding associated with prolonged periods of cold ischemia appear to be an early ‘angiogenic response’, and these cells might be primed to undergo apoptosis after oxygenated reperfusion (14). It has been shown that apoptosis of sinusoidal endothelial cells precedes hepatocyte damage in rat liver I/R injury (8,11). Thus, inhibition of apoptosis seems to be a rational strategy to reduce the risk of I/R injury in liver transplants.
IL-13, a type-2 cytokine is known to modulate inflammatory responses by down-regulating production of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, MIP-1α and MIP-2 (15,16). Moreover, in vivo studies in rodents showed that IL-13 prevents LPS-induced lethality and IgG immune complex-induced lung injury (17,18), whereas IL-13 gene therapy reduces inflammation and bony destruction in rheumatoid arthritis (19). Indeed, IL-13 is a potent inhibitor of TNF-mediated activation of NFκB/AP-1 (20), and protection of human synoviocytes from apoptosis could be achieved by overexpressing IL-4 and IL-13 (21). Moreover, IL-13 inhibits pro-inflammatory cytokines and prevents apoptosis in endothelial cells (22).
We have recently shown striking cytoprotective effects of IL-13 overexpression in well-defined and clinically relevant rat model of hepatic cold ischemia followed by OLT (23). Indeed, the survival of liver grafts increased from 50% in controls to 100% after Ad-IL-13 gene therapy. This beneficial effect correlated with improved liver function, preservation of hepatic architecture, and depression of neutrophil infiltration. Ad-IL-13 also diminished activation of macrophage/neutrophil-associated TNF-α/MIP-2 and endothelial-dependent E-selectin. Based on these preliminary findings, we have now focused on apoptotic pathways to assess mechanisms by which IL-13 renders rat OLT resistant to the cold I/R damage.
Materials and Methods
Generation of recombinant Ad-IL-13
We generated recombinant adenovirus encoding IL-13 (Ad-IL-13), as described (24). Briefly, 1282-bp human IL-13 cDNA flanked by Not I and BamHI sites was subcloned into the BamHI site of pACCMVpLpA. The resulting pAC-IL-13 was cotransfected with dL309 viral DNA, which contains 35 kb of the E1-deleted Ad serotype 5 genome in 293 cells. Homologous recombination resulted in a replication-defective Ad-IL-13. The posi- tive viral clone was screened by Southern blot/PCR. The plaque-purified Ad-IL-13 was amplified in 293 cells grown in SMEM medium (Gibco, Grand Island, NY, USA) with 10% newborn calf serum. The titration of the virus was analyzed by plaque assay. Virus stocks of 2.5 × 1010 plaque-forming units (pfu)/mL were stored at − 80 °C. Ad encoding Escherichia coliβ-galactosidase (Ad-β-gal) served as control.
Male Sprague Dawley (SD) rats weighing 250–280 g (Harlan Sprague Dawley, Inc. San Diego, CA, USA). Rats were housed in the UCLA animal facility under specific pathogen-free conditions. All animals received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ and published by National Institute of Health (NIH publication 86–23 revised 1985).
Ad-IL-13 gene transfer in cold ischemia OLT model
Twenty-four to 48 h after i.v. infusion of Ad-IL-13/Ad-β-gal (2.5 × 109 pfu), livers were harvested, flushed with 10 mL of University of Wisconsin (UW) solution, and stored for 24 h at 4 °C prior to being transplanted into syngeneic SD rats. To elucidate the putative role of heme oxygenase-1 (HO-1), we set up a parallel group of rats that were treated with tin protoporphyrin (SnPP; Porphyrin Products, Logan, UT, USA), a competitive HO activity inhibitor. SnPP was administered i.p. (30 μm/kg) to the donor (day −2 and −1) and to the recipient at the time of OLT and every second day thereafter for 7 days (24). Animals were followed for survival at day 14.
Hepatocellular damage assay
Serum glutamic-oxaloacetic transaminase (sGOT) levels were measured serially in blood samples after OLT. Measurements of sGOT were performed using an auto analyzer by ANTECH Diagnostics (Los Angeles, CA, USA).
Serially harvested OLT samples were sliced into small pieces, preserved in 10% neutral-buffered formalin, cut into 5-μm section, and stained with hematoxylin and eosin (H&E). Histological severity of I/R injury in OLTs was graded using Suzuki's classification, in which sinusoidal congestion, hepatocyte necrosis and ballooning degeneration are graded from 0 to 4 (25). No necrosis, congestion/centrilobular ballooning is given a score of 0, while severe congestion/degeneration and >60% lobular necrosis is given a value of 4.
An ELISA assay to detect IL-13 levels was performed, as described (26). Briefly, serum was collected and supernatants were obtained from homogenized OLTs with PBSTDS buffer (50 mm Tris, 150 mm NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% triton X-100, pH 7.2). Flat-bottom 96-well microtiter plates were coated with anti-human IL-13 Ab (Pharmingen, San Diego, CA, USA). Nonspecific binding sites were blocked with Blocking Buffer (10% fetal bovine serum, 10% newborn calf serum or 1% BSA in PBS). Plates were rinsed, and sample supernatants were added, followed by addition of biotinylated mouse anti-human IL-13 Ab (2 μg/mL). After washing, streptavidin-peroxidase conjugate (Pharmingen) was added, and the plates were incubated for color development. Plates were read at 405 nm in ELISA reader. The linear region of IL-13 curves was obtained in a series of eight two-fold dilutions of human IL-13 standard (2000–15 pg/mL).
Detection of apoptosis
A commercial in situ histochemical assay (Klenow-FragEL, Oncogene Research Products, Cambridge, MA, USA) was performed to detect the DNA fragmentation characteristic of apoptosis in formalin-fixed paraffin-embedded OLT sections, as described (24). Biotinylated nucleotides were detected using streptavidin-horse radish peroxidase (HRP) conjugate. Counter-staining with methyl green aids in the morphological evaluation and characterization of normal and apoptotic cells. The results were scored semi-quantitatively by averaging the number of apoptotic cells/microscopic field at 200 × magnification. Six fields were evaluated per tissue sample.
Western blot analysis
Protein was extracted from OLTs with PBSTDS buffer. Proteins (30 μg/sample) in SDS-loading buffer (50 mm Tris, pH 7.6, 10% glycerol, 1% SDS) were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The gel was stained with Coomassie Blue to document protein loading. The membrane was blocked with 3% dry milk + 0.1% Tween 20 (USB, Cleveland, OH, USA). Monoclonal mouse anti-human A20 Ab (Imgenex, San Diego, CA, USA), polyclonal rabbit anti-rat Bcl-2/Bcl-xl, and anti-rat HO-1 Abs (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. The membranes were incubated with Abs, relative quantities of proteins were determined by densitometer, and expressed in absorbance units (AU) (Kodak Digital Science 1D Analysis Software, Rochester, NY, USA).
All data are expressed as mean ± SD. Statistical comparisons between groups were analyzed by Student's t-test. All differences were considered statistically significant at the p-value of <0.05.
Ad-IL-13 prolongs OLT survival, prevents hepatocellular damage and ameliorates histological signs of I/R injury
We have previously shown that in our 24-h ex vivo hepatic cold (4 °C) ischemia model, only 50% untreated or Ad-β-gal pretreated recipients were alive at day 14 post-OLT. In contrast, 100% of OLT recipients transfected with Ad-IL-13 survived at least 14 days (23). We have also shown that by day 1 post-OLT, Ad-IL-13 gene transfer prevented hepatocellular and histological damage, as analyzed by sGOT levels and Suzuki's criteria, respectively.
We have now expanded these studies and, as shown in Figure 1, Ad-IL-13-treated rats revealed significantly lower sGOT levels (IU/L), as compared with Ad-β-gal controls throughout the observation period (day 1: 103 ± 9 vs. 902 ± 30; day 3: 65 ± 18 vs. 487 ± 50, p < 0.02; day 7: 98 ± 18 vs. 754 ± 127, p < 0.01; and day 14: 115 ± 37 vs. 1480 ± 564, p < 0.05).
We performed serial ELISA to assess IL-13 protein levels (pg/mL). As shown in Figure 2 (upper panel), intragraft expression of IL-13 at days 1, 3, 7, and 14 in Ad-IL-13 gene therapy group was significantly higher (p < 0.005), as compared with Ad-β-gal controls (day 1: mean ± SD = 177 ± 18 vs. 16 ± 4; day 3: 167 ± 19 vs. 22 ± 9; day 7: 159 ± 21 vs. 24 ± 10; day 14: 141 ± 16 vs. 17 ± 5). Similarly, as shown in Figure 2 (lower panel), serum IL-13 levels in Ad-IL-13 gene therapy group were significantly higher (p < 0.01), as compared with those in Ad-β-gal controls (day 1: mean ± SD = 179 ± 16 vs. 17 ± 5; day 3: 176 ± 18 vs. 19 ± 7; day 7: 137 ± 15 vs. 21 ± 9; day 14: 118 ± 21 vs. 19 ± 5).
Ad-IL-13 prevents apoptosis and up-regulates expression of cytoprotective molecules in OLT
By day 3, liver grafts in the Ad-β-gal group showed large numbers of apoptotic cells (34.5 ± 15 cells/field; Figure 3A), comparable with untreated controls (28.5 ± 14.6 cells/field; Figure 3B). In contrast, the number of apoptotic cells in OLTs that underwent Ad-IL-13 transfer was markedly decreased (5.5 ± 4.1; p < 0.005 as compared with Ad-β-gal and p < 0.01 as compared with untreated groups) (Figure 3C). We then used Western blots to analyze the expression of antiapoptotic A20, Bcl-2/Bcl-xl, and antioxidant HO-1 gene products. The protein was extracted from OLTs that were transduced with Ad-IL-13 or Ad-β-gal, and harvested at day 3, 7, and 14. The relative expression levels of individual molecules was determined by densitometry, and expressed in absorbance units (AU). As shown in Figure 4, the expression of antiapoptotic A20, Bcl-2/Bcl-xl and antioxidant HO-1 was up-regulated in thr Ad-IL-13 group (1.6–1.9 AU, 2.2–2.3 AU, 1.7–1.9 AU, and 1.7–2.0 AU, respectively), as compared with Ad-β-gal controls (0.05–0.1 AU, 0.05–0.1 AU, 0.01–0.05 AU, and 0.05–0.1 AU, respectively).
HO-1 mediates cytoprotective effects of Ad-IL-13
To elucidate the role of HO-1 in IL-13-mediated cytoprotection, we used SnPP, a competitive HO enzymatic activity inhibitor. SnPP-induced HO-1 depression reversed cytoprotective effect of Ad-IL-13 on OLT survival. Indeed, as shown in Figure 5, only 57.1% of OLTs treated with Ad-IL-13 + SnPP survived 14 days (vs. 100% in the Ad-IL-13 alone group). This was accompanied by increased sGOT levels in the Ad-IL-13 + SnPP, as compared with the Ad-IL-13 only group (Figure 1; day 1: 721 ± 42 vs. 103 ± 9, p < 0.005; day 3: 379 ± 87 vs. 65 ± 18, p < 0.02; day 7: 626 ± 98 vs. 98 ± 18, p < 0.01; day 14: 1158 ± 401 vs. 115 ± 37, p < 0.05). Moreover, day 3 and day 14 OLTs treated with Ad-IL-13 + SnPP revealed moderate to severe hepatocyte necrosis and sinusoidal/vascular congestion (Figure 6C,F; Suzuki score = 3.3 ± 1.2 and 3.2 ± 0.98, respectively) as compared with the Ad-IL-13 alone group (Figure 6B,E; Suzuki score = 0.67 ± 0.8 and 0.83 ± 0.4, respectively; p < 0.005). SnPP treatment was not hepatotoxic on its own, as sGOT levels and liver histology in otherwise naïve rats treated with SnPP alone remained normal (data not shown).
SnPP pretreatment resulted in an increased frequency of TUNEL + cells in OLTs (Figure 3D; 25.5 ± 12.1 cells, vs. 5.5 ± 4.1 cells without SnPP; p < 0.005), and concomitantly suppressed expression of A20, Bcl-2, Bcl-xl, and HO-1 at days 3, 7, and 14 (Figure 4; A20 – lanes 7 A, 8 A and 9 A: 0.4–0.6 AU, vs. 1.6–1.9 AU without SnPP; Bcl-2 – lanes 7B, 8B and 9B: 0.4–0.7 AU vs. 2.2–2.3 AU without SnPP; Bcl-xl: lanes 7C, 8C and 9C: 0.5–0.8 AU, vs. 1.7–1.9 AU without SnPP; and HO-1 – lanes 7D, 8D and 9D: 0.4–0.9 AU, vs. 1.7–2.0 AU without SnPP).
This principal findings of this study on Ad-IL-13 gene therapy in a clinically relevant rat model of cold liver ischemia followed by OLT are as follows: (i) IL-13 overexpression prevented hepatic apoptosis and up-regulated expression of antiapoptotic (A20, Bcl-2/Bcl-xl)/antioxidant (HO-1) genes at the graft site; and (ii) SnPP-mediated HO-1 inhibition reversed the cytoprotective/antiapoptotic effects of IL-13, suggesting HO-1 as one of putative IL-13 down-stream mediators.
Our findings are in agreement with a report in which exogenous recombinant IL-13 inhibited liver injury due to warm I/R in mice (27). However, unlike the latter in situ study, in our previous (23) as well as in the present series, we used a highly selective gene therapy approach to provide evidence that IL-13 overexpression exerts potent cytoprotective function in a rat liver model of cold ischemia followed by transplantation. Indeed, the survival rate increased from c. 50% in Ad-β-Gal treated or untreated rats to 100% in recipients transfected with Ad-IL-13 (23). We have previously shown that HO-1 induction (28), or rPSGL-1 Ig-facilitated blockade of CD62 P selectin (29) resulted in up to 80% OLT survival. Consistent with our OLT survival data, Ad-IL-13 but not Ad-β-gal treatment: (i) prevented hepatocellular damage, as assessed by sGOT levels throughout the 14-day observation period; and (ii) preserved hepatic histological integrity, with less sinusoidal congestion/lobular ballooning at both 1 and 14 post-OLT days, as compared with controls. Collectively, these results are consistent with the ability of virally induced IL-13 to prevent injury induced by cold ischemia and reperfusion in OLTs.
One hundred per cent OLT survival after Ad-IL-13 matches our recently reported effects of gene therapy-induced antiapoptotic Bag-1 in this stringent OLT model (30), consistent with the role of apoptosis in the mechanism of hepatic I/R injury (11,12). IL-13 was initially classed as a Th2-derived cytokine, which inhibits in vitro production of monocyte/macrophage cytokines (15,16). To the best of our knowledge, this study is the first to document a successful use of IL-13 gene transfer to prevent apoptotic development during the course of cold I/R injury. Although decreasing the frequency of OLT infiltrating ‘cytodestructive’ leukocytes by apoptosis could theoretically be beneficial, virally produced IL-13 significantly inhibited apoptosis in OLTs, as assessed by nuclear fragmentation in vivo primarily of hepatocytes. This is consistent with the ability of IL-13 to protect human B cells (31), synoviocytes (22) and endothelial cells (23) from apoptosis in vitro, or to abolish TNF-mediated cytotoxicity/activation of caspase-3 (21). Interestingly, as in our present antigen-independent hepatic I/R model, we have shown a strong synergy between local Ad-IL-13 gene transfer and systemic infusion of regulatory T cells in the infectious tolerance pathway in rat cardiac allograft recipients (24).
Although a variety of mechanisms may be involved, and cell death mediated by TNF and/or FasL may be of importance (32,33), our previous in vitro studies have clearly shown that Ad-IL-13 prevented cytotoxicity and protected HUVEC cultures from TNF-α-mediated apoptosis. This result is consistent with the ability of IL-13 to prevent endothelial cell apoptosis and activation (23). Alternatively, IL-13 may inhibit liver injury induced by I/R via activation of STAT6 transcription pathway (27), consistent with our study in which mice deficient in STAT4 were resistant to hepatic I/R insult (34). Our present Western blot analyses of OLTs have revealed that Ad-IL-13 triggered the expression of antioxidant HO-1 and antiapoptotic A-20, and Bcl-2/Bcl-xl. Consistent with our studies in allo-transplantation models (24,26), these molecules remained up-regulated selectively in ‘cytoprotected’ OLTs. We have previously identified Kupffer cells and macrophages as prime sources of HO-1 in rat livers (35). As HO-1 has been identified as a downstream effector of IL-10 (36) and because of our own interest in HO-1 facilitated cytoprotection (24,28,37), we then asked whether HO-1 may mediate IL-13 effects in this study. Indeed, SnPP-facilitated depression of HO-1 abolished cytoprotection seen otherwise in Ad-IL-13 transfected OLTs, as assessed by animal survival (57% vs. 100%), and increased frequency of TUNEL + cells. Similarly, Ad-IL-13-transfected HUVECs devoid of HO-1 showed an increased number of TUNEL + cells, as compared with cultures without SnPP (24). Collectively, these data provide evidence to support the key role of HO-1 in cytoprotective and antiapoptotic IL-13 function in both antigen-independent and antigen-dependent host pro-inflammatory responses.
In conclusion, we have documented that striking cytoprotection rendered by virally induced IL-13 against hepatic I/R injury in a rat model of cold ischemia followed by OLT is being accomplished via decreasing apoptosis, and by facilitating the induction of antiapoptotic (A20, Bcl-2/Bcl-xl)/antioxidant (HO-1) molecules. Results of HO-1 neutralization studies point to HO-1 as one of the putative IL-13 downstream effectors. These data provide the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.
Supported by NIH Grants RO1 AI23847, AI42223 (JWKW) and The Dumont Research Foundation. B.K. is the recipient of the 2002 American Society of Transplantation (AST) Young Investigator Award.