Ischemia/reperfusion (I/R) injury, an antigen (Ag)-independent event surrounding removal, storage, and engraftment of solid organs primarily from cadaver sources, remains an important clinical problem. In the case of liver transplantation, despite advances in organ preservation and immunosuppression, primary and delayed hepatic nonfunction due to I/R injury continue to affect both early and long term liver allograft outcomes (1).
The injurious events elicited during hepatic I/R injury are thought to occur in a biphasic pattern. The acute onset of reperfusion is characterized by oxidative injury (2), Kupffer cell activation (3), and microcirculatory flow disturbances (4). A subacute inflammatory phase ensues, leading to cellular damage mediated primarily by neutrophils (5,6). Although these cell events peak at different times, the pathogenesis of hepatic I/R seems to involve a continuum of interrelated processes, which all culminate in hepatocellular dysfunction (1). Moreover, there is a growing body of evidence that host T cells also participate in hepatic I/R injury. For instance, both cyclosporine A and FK 506, which are potent T-cell-deactivating agents, may decrease reperfusion injury after transplantation or warm ischemia, as compared with untreated controls (5,7). Increased accumulation of CD4+ T cells and expression of Th1-type cytokines were also observed in postischemic experimental livers (8).
CD154 (CD40 ligand), a member of the tumor necrosis factor (TNF) gene family, is expressed predominantly on mature activated CD4+ T cells and other cell types but not on resting T cells (9). The interaction between CD154 and CD40, a glycoprotein receptor on antigen presenting cells (APCs), provides a second costimulatory signal that is essential for the development of both cellular and humoral immune responses to T-dependent Ags (10). The efficacy of CD154-targeted therapy to prevent acute and chronic rejection, and to induce tolerance in some transplantation models, has been well established (11). However, the role of CD154-CD40 costimulation pathway in I/R injury has not been studied before. Here, we report on our studies in which we first investigated the involvement of T cells and then the role of CD154-CD40 T-cell costimulation signals in the pathophysiology of warm I/R injury in a mouse liver model.
MATERIALS AND METHODS
Male B6/129 (B6) wild-type (WT) mice, T-cell-deficient athymic (nu/nu; B6) mice, as well as CD154 knockout (KO) mice (B6; 8–12 weeks of age; intercrossed at least 10 generations) were used (Jackson Laboratory, Bar Harbor, ME). Animals were housed in the UCLA animal facilities under specific pathogen-free conditions and according to National Institutes of Health guidelines.
Hepatic I/R Injury Model
A warm hepatic I/R model was established as described (8), with some modifications. Briefly, after a midline laparotomy, mice were injected with heparin (100 μg/Kg), and an atraumatic clip was used to interrupt the arterial and the portal venous blood supply to the cephalad lobes of the liver. After 90 min of partial hepatic warm ischemia, the clip was removed, initiating hepatic reperfusion. Mice were killed after 4 h or 20 h of reperfusion; liver tissue and peripheral blood samples were collected for future analysis. The extent and severity of hepatic I/R injury were assessed in groups of WT, nu/nu, and CD154 KO mice, as well as in WT recipients that were pretreated with CD154 (MR1) monoclonal antibody (mAb) (0.25 mg/mouse intraperitoneally (IP) at day −2 and −1). Sham WT controls underwent the same procedure but without vascular occlusion.
To elucidate the functional significance of heme oxygenase-1 (HO-1) expression, we treated mice with a competitive HO inhibitor, tin protoporphyrin (SnPP; Porphyrin Products, Logan, UT). SnPP was diluted in 100 mM NaOH to a stock solution of 50 mM and kept at −70°C until used. Light exposure was limited as much as possible. SnPP was administered twice IP (30 μM/kg) one day before the experiment (day −1) and then at the time of hepatic warm ischemia, as described (12).
Assessment of Hepatocyte Function
Serum alanine aminotransferase (sALT) levels, as indicator of hepatocellular injury, were measured in blood samples obtained 4 h or 20 h after reperfusion in the warm hepatic I/R model. Measurements of sALT were made using an auto analyzer by ANTECH Diagnostics (Los Angeles, CA).
Assessment of Neutrophil Infiltration
The presence of myeloperoxidase (MPO), an enzyme specific for neutrophils (and some macrophages), was used as an index of intrahepatic neutrophil accumulation (13). Briefly, the frozen tissue was thawed, weighed, and placed in 4 ml iced 0.5% hexadecyltrimethyl-ammonium bromide and 50 mM potassium phosphate buffer solution with the pH adjusted to 5. Each sample was then homogenized for 30 sec and centrifuged at 15,000 rpm for 15 min at 4°C. Supernatants were then mixed with hydrogen peroxide-sodium acetate and tetramethyl-benzidine solutions. The change in absorbance was measured by spectrophotometry at 655 nm. One unit (AU) of MPO activity was defined as the quantity of enzyme degrading 1 μM peroxide per minute at 25°C per gram of tissue.
Liver specimens were fixed in a 10% buffered formalin solution and embedded in paraffin. Liver sections (4 μm) were stained with hematoxylin and eosin and then analyzed blindly. The histological severity of I/R injury was graded using previously published Suzuki’s criteria (5), with modifications. In this classification, sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration are graded from 0 to 4. No necrosis, congestion, or centrilobular ballooning is given a score of 0, whereas severe congestion and ballooning degeneration as well as greater than 60% lobular necrosis is given a value of 4.
Adoptive Transfer Studies
Spleen cell suspensions were obtained from separate groups of WT and CD154 KO mice. Freshly isolated spleens were placed in Roswell Park Memorial Institute (RPMI) 1640 media containing 5% fetal calf serum, and the splenocytes were separated by dissection and scraping through a metal mesh. Cell aggregates were removed by filtration through a nylon screen mesh. Splenocytes were collected by centrifugation, and red blood cells were removed by hypertonic lysis with NH4Cl. Nonadherent, nylon, wool-enriched spleen T-cell fractions (>80% purity by fluorescence-activated cell sorter [FACS] analysis) were obtained, as described (8). These were then adoptively transferred (20×106) via portal vein into groups of nu/nu mice at 24 h before the induction of hepatic I/R injury.
Western Blot Analysis
Protein was extracted from liver tissue samples with PBSTDS buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, and 1% triton X-100, pH 7.2). 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 and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The gel was stained with Coomassie blue to document equal protein loading. The membrane was blocked with 3% dry milk and 0.1% Tween 20 (USB, Cleveland, OH) in PBS and incubated with rabbit anti-rat (HO-1 polyclonal antibody (Ab) (Sangstat Corp, Fremont, CA). The filters were washed and incubated with horseradish peroxidase donkey anti-rabbit Ab (Amersham, Arlington Heights, IL). Relative quantities of HO-1 protein were determined using a densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY).
Data are expressed as mean±SE. Data were analyzed with an unpaired two-tailed Student t test. P <0.05 was considered to be statistically significant.
T-cell Deficiency or CD154 Blockade Decrease Hepatic I/R Injury
Ninety minutes of hepatic warm ischemia followed by 4 h of reperfusion significantly increased sALT levels (IU/l) in WT mice as compared with sham-operated controls (1129±285 and 24±4, respectively;P <0.001;Fig. 1). However, unlike in T- cell-competent (WT) animals, sALT levels were dramatically reduced in T-cell-deficient (nu/nu) mice (352±108;P <0.0005). By 20 h, sALT activities decreased in both WT and nu/nu groups (142±36 and 72±30 IU/l, respectively; n=5 per group; data not shown).
Similarly, the disruption of CD154 T-cell costimulation pathway in CD154 KO mice or its blockade in WT hosts conditioned with MR1, anti-mouse CD154 mAb, significantly diminished I/R-induced hepatocellular injury (sALT at 4 h 173±42 and 167±29 IU/l, respectively;P <0.002).
The local neutrophil accumulation at 4 h of reperfusion, as analyzed by MPO enzymatic activity (IU/g) in ischemic lobes, increased from 0.47±0.08 in sham-operated controls to 4.2±0.6 in the WT group (Fig. 2;P <0.0001). MPO activity in nu/nu mice (3.0±0.8) was comparable with that in WT animals. In contrast, the lack of CD154 T-cell costimulation in CD154 KO mice or its blockade in WT mice treated with MR1 mAb diminished MPO activity to 1.9±0.3 and 1.7±0.01, respectively (P <0.02, as compared with WT).
Groups of animals subjected to 90 min of warm hepatic ischemia, followed by 4 h of reperfusion, were then killed and their livers evaluated for the severity of histological features of I/R injury by Suzuki’s criteria (5). The ischemic lobes in T-cell-competent WT mice revealed significant edema, extensive centrilobular ballooning, and hepatocellular necrosis (30–60%) in association with sinusoidal and central vein congestion (Fig. 3A; score=4.2±0.6). In contrast, livers harvested from T-cell-deficient mice, CD154 KO mice, or from WT hosts treated with MR1 mAb exhibited overall good preservation of lobular architecture without edema, central vein, or sinusoidal congestion and an absence of centrilobular ballooning or necrosis (Fig. 3B–D; score=1.0±0.3, 1.2±0.2, and 1.1±0.4, respectively).
Adoptive Transfer of WT but not CD154-Deficient T Cells Restores Hepatic I/R Injury
To evaluate the role of T cells in hepatic I/R injury, we reconstituted T-cell-deficient mice with spleen T cells (20×106) from WT or CD154 KO mice. Indeed, as shown in Figure 1, sALT levels (IU/l) in nu/nu mice reconstituted with WT cells increased significantly after hepatic warm ischemia and reperfusion, as compared with untreated nu/nu counterparts (687±140 and 352±108, respectively;P <0.05). Interestingly, unlike WT cells, CD154-deficient T cells failed to restore I/R injury after adoptive transfer into nu/nu mice (sALT=182±16 IU/l). MPO enzymatic activity (IU/g) in ischemic lobes increased from 3.0±0.8 in untreated nu/nu mice to 4.55±0.8 after infusion of WT cells (P <0.5); it decreased to 1.81±0.5 after administration of CD154-deficient spleen cells (Fig. 2). The ability of T cells from WT but not CD154 KO mice to restore I/R injury following adoptive transfer into nu/nu mice was also confirmed by histological evaluation of the ischemic hepatic lobes (not shown).
The Disruption of CD154 Signaling Triggers Intrahepatic HO-1 Overexpression
We evaluated HO-1 protein expression in livers subjected to 90 min of warm ischemia and reperfusion by Western blots. The relative expression levels in absorbance units (AU) were analyzed by densitometry. As shown in Figure 4, improved hepatic function at 4 h in CD154 KO recipients, WT mice treated with CD154 mAb, or nu/nu mice infused with CD154-deficient cells resulted in consistently enhanced expression of HO-1 (lines 5–7; 1.8–2.1 AU). In contrast, the corresponding liver samples in WT mice and nu/nu mice that remained untreated or received WT cells showed markedly diminished HO-1 levels (lines 2–4; 0.1–0.3 AU) compared with sham controls (line 1; 0.1 AU).
Abrogation of HO-1 Restores Hepatic I/R Injury in CD154-Deficient Mice
To establish a functional link between the disruption of CD154 signaling and HO-1 expression, we treated a group of CD154 KO mice (n=3) with SnPP, a known HO-1 inhibitor. Interestingly, SnPP treatment, according to the protocol that abrogates HO-1 expression/HO-activity (12), restored the hepatic damage in CD154 KO mice seen after 90 min of warm ischemia followed by 4 h of reperfusion (data not shown). Indeed, sALT levels (IU/l) after HO-1 depression in CD154 KO mice were comparable with those in WT controls (1187±310 and 1129±285, respectively) yet were significantly higher as compared with those in otherwise untreated CD154 KO counterparts (173±42;P <0.05).
This study confirms that T cells mediate hepatic I/R injury in mice and provides the first evidence that CD154-CD40 T-cell costimulation pathway is essential in the pathogenesis of hepatic reperfusion injury following warm ischemia. Moreover, the beneficial effect of CD154 blockade upon the hepatic I/R insult results and depends on local intrahepatic HO-1 overexpression. Indeed, HO-1 induction has been shown to be an important adaptive mechanism that protects cells from the stress, in particular inflammation or ischemia (14).
In our model of a 90 min warm ischemia, liver function assessed by sALT levels at 4 h of reperfusion was significantly improved in T- cell-deficient mice, as compared with T-cell-competent controls (data supported by histological Suzuki’s scores of the ischemic hepatic lobes). By 20 h, sALT activities decreased sharply in both WT and nu/nu groups. Our findings, however, contradict another study in a similar warm ischemia model in which liver function was equally deteriorated in nu-nu and WT mice at 3–6 h post lobar injury (8). In the latter work, T-cell-dependent injury was most pronounced at 20 h, returning to baseline by 36 h. Although not fully understood at present, putative strain-specific differences may have contributed to distinct kinetics and severity patterns of hepatic insult suffered by Balb/c vs. B6 mice in our study. However, regardless of these disparities, our present data are in agreement with recently published reports in which reconstitution of T-cell-deficient mice with T-cell-enriched splenocytes restored features of I/R injury in hepatic (8,15), intestinal (16), and renal (17) murine models in a manner similar to that noted in WT counterparts. As little as 20×106 WT cells were sufficient to restore hepatic dysfunction and damage in our study. Although the purity of T cells was always verified by FACS analysis before the transfer, we cannot exclude a possibility that contaminating cells may have contributed to the restoration of hepatic I/R injury. However, the inability of cells from CD154-deficient mice to recreate injury minimizes the putative role of contaminating cells. All these findings are consistent with an emerging paradigm on the pivotal role of T cells in the pathophysiology of I/R injury (18).
Unlike in the other system (5,6,8), we have detected comparable neutrophil levels in WT and T-cell-deficient mice, as assessed by MPO assay. Although we are aware that MPO technique may also detect macrophages (19), our finding is consistent with recent studies in rat models of hepatic cold ischemia followed by isotransplantation in which cytoprotection rendered by Ad-HO-1 gene transfer was accompanied by decreased macrophage infiltration despite elevated MPO activity, comparable with Ad-β-Gal controls (20). Our present data complement those from murine renal (17,21) and lung (22) inflammatory models and suggest important pathophysiological dissociation of cytoprotection from the neutrophil influx.
This report is the first to document the importance of CD154 costimulation signals in the mechanism of T-cell-mediated I/R hepatic injury. Indeed, disruption of CD154-CD40 pathway (in KO mice) or its blockade in WT recipients (after MR1 mAb treatment), virtually prevented hepatic I/R insult as analyzed by sALT/MPO activities and confirmed by histological Suzuki’s criteria. Moreover, in contrast to WT cells, CD154-deficient cells failed to restore I/R injury after transfer into nu/nu mice. Others have shown that CTLA4Ig-mediated blockade of CD28-B7 pathway protected rats from cold renal I/R injury (23), whereas transfer of CD28-deficent cells failed to restore renal insult in T-cell-deficient mice (17). Therefore, both CD28 and CD154 costimulation pathways play a role in the pathophysiology of I/R injury suffered by solid organs. However, the question remains as to how T cells become activated during I/R insult, which by definition is an Ag-independent event. Indeed, liver sinusoidal endothelial cells (LSEC) constitutively express all molecules necessary for Ag presentation (CD54, CD80, CD86, MHC class I/II, and CD40) and can function as APCs for CD4+ and CD8+ T cells (24,25). LSEC do not need to undergo maturation for acquisition of APC function but can efficiently undergo endocytosis and can present Ag to T cells outside lymphatic environment(25,26). Moreover, ischemic liver-infiltrating T lymphocytes express a panel of cytokines, chemokines, and adhesion molecules that may lead to an increased adhesion of T cells to LSEC. As the liver is involved in clearance of foreign Ags and toxic products from the gastrointestinal tract, it might be possible that LSEC present “foreign” Ag and activate T-lymphocytes during warm hepatic I/R injury. Alternatively, Ag-independent T-cell activation due to preexisting hypoxia (27) or via direct activation of Kupffer cells (28) may also contribute to the “danger” signals that, instead of a classic adaptive immune role, may function in a more innate manner (29).
HO-1, also known as hsp32, is a rate limiting enzyme in the degradation of heme, which catalyzes the conversion of heme into biliverdin, carbon monoxide (CO), and free iron (14). HO-1 is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. The overexpression of this enzyme exerts potent cytoprotective effects, consistent with our recent studies in rat liver (20,30,31) and cardiac (32) models of I/R injury. In our present study, HO-1 protein expression was almost absent at the conclusion of the 4 h reperfusion period in ischemic hepatic lobes of WT and nu/nu mice with or without WT cell transfer. In contrast, ischemic lobes in CD154-deficient mice, WT litter mates treated with CD154 mAb, or T-cell-deficient mice infused with CD154-deficient cells all strongly overexpressed HO-1. Our previous studies (31) have identified macrophages and Kupffer cells as the prime hepatic HO-1 producers. Interestingly, abrogation of HO-1 expression in this study fully restored hepatic I/R insult in CD154-deficient recipients, further documenting the key role of operational HO-1-mediated cytoprotection during I/R injury.
In conclusion, this study confirms the importance of T cells and documents for the first time the key role of CD154 costimulation signaling in the mechanism of hepatic I/R injury. We also demonstrate that CD154 blockade facilitates and depends on HO-1-mediated cytoprotection. Our data provide the rationale for human trials to target CD154-CD40 costimulation in hepatic I/R injury, particularly in the transplant patient.
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