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The Journal of Immunology, 2006, 176: 616-624.
Copyright © 2006 by The American Association of Immunologists

Molecular Characterization of Rat Leukocyte P-Selectin Glycoprotein Ligand-1 and Effect of Its Blockade: Protection from Ischemia-Reperfusion Injury in Liver Transplantation1

Sei-ichiro Tsuchihashi*, Constantino Fondevila*, Gray D. Shaw{dagger}, Meike Lorenz{dagger}, Kimberly Marquette{dagger}, Susan Benard{dagger}, Xiu-Da Shen*, Bibo Ke*, Ronald W. Busuttil* and Jerzy W. Kupiec-Weglinski2,*

* Dumont-University of California Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; and {dagger} Wyeth Research, Cambridge, MA 02140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-{alpha}, IL-1{beta}, IL-6, IFN-{gamma}, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Liver transplantation is a successful treatment for end-stage liver disease, but primary graft nonfunction or early dysfunction may occur, significantly affecting graft morbidity and mortality. Ischemia/reperfusion injury (IRI),3 an Ag-independent inflammatory component of organ "harvesting," remains an important factor in poor graft function. It causes up to 10% of early liver failure and can lead to the higher incidence of acute and chronic rejection (1). The mechanism of IRI involves microcirculatory flow disturbances caused by endothelial cell adhesion, leukocyte tethering to the endothelial cell with subsequent sequestration in tissue, activation of Kupffer cells leading to the production of oxygen-free radicals, inflammatory cytokines, and chemokines, resulting in sinusoidal endothelial cell death and ultimate hepatocyte damage (2, 3, 4, 5).

Cellular adhesion molecules, such as selectins, mediate the initial capture and support the rolling of leukocytes on sinusoidal endothelial cells in the initial phase of IRI (6). Selectins, a family of lectin-domain glycoproteins, are expressed on endothelial cells (P-selectin, E-selectin), platelets (P-selectin), and leukocytes (L-selectin) (7, 8). P-selectin is stored in {alpha} granules of platelets and Weibel-Palade bodies of endothelial cells and is rapidly translocated to the platelet and endothelial cells surface after reperfusion (9). In contrast to P-selectin, L-selectin is constitutively expressed on the surface of neutrophils and is the counterligand for P-selectin during early reperfusion (10). Selectins bind to sialyated and fucosylated glycoconjugates and express high affinity to a small subset of appropriately modified glycoproteins. The principal selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1), a homodimetric sialomucin found on the surface of most leukocytes that binds all three selectins (11). PSGL-1 is an essential ligand for primary tethering and rolling of leukocytes. Moreover, PSGL-1 is the only known high-affinity ligand for P-selectin. Although the gene and protein sequence of human and mouse PSGL-1 have been characterized (12), the structure of the rat PSGL gene and protein have not been reported.

The blockade of P-selectin and PSGL-1 protects the liver from IRI (13, 14, 15, 16, 17). We have also shown that blockade of the interaction between P-selectin and PSGL-1 with a recombinant soluble form of P-selectin glycoprotein ligand-1 (rPSGL-Ig) decreased portal resistance, increased bile production, and reduced polymorphonuclear leukocyte (PMN) infiltration in steatotic rat liver models of ex vivo cold ischemia followed by reperfusion. In the rat orthotopic liver transplant (OLT) model, rPSGL-Ig treatment before cold storage and before OLT increased animal survival (18). This effect correlated with improved liver function, depressed neutrophil activity, and decreased histological features of hepatocyte injury (19).

To more thoroughly understand the role of leukocyte adhesion events, we isolated the rat gene for PSGL-1 and generated a neutralizing Ab to the rat PSGL-1 protein. An outstanding question remains as to whether or not targeting PSGL-1 on circulating leukocytes with a blocking Ab would be an effective strategy in the setting of organ transplantation. This study is the first to analyze the effects of a neutralizing anti-PSGL-1 Ab in rat liver models of cold ischemia followed by reperfusion ex vivo via an isolated perfusion liver apparatus or in vivo via syngeneic OLT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation of the rat PSGL-1 sequence and generation of neutralizing Ab

The rat PSGL-1 coding region sequence was isolated from a rat liver {lambda} genomic library (Stratagene). The library was screened with a random primed probe from a PCR spanning the transmembrane and cytoplasm region of human PSGL-1. Plasmid pMT-PL85A encoding for full-length human PSGL-1 cDNA was used as a template to generate a 281 bp product with the following primers: 5'-CTA AGG GAG GAA GCT GTG CAG GG-3' + 5'-TGC CTG CTG GCC ATC CTA ATC TT-3'. From the three isolated phage clones, two clones showed identical restriction patterns and were positive in a Southern blot using a digoxigenin-labeled probe for PSGL-1. An ~3.5-kb XbaI fragment of the insert was subcloned into pUC18 for DNA sequencing. A soluble Ig fusion form of the mature N-terminal 47 aa of rat PSGL-1, rat47mutFc, was generated using PCR primers: 5'-ATA TAT TTA TTC TAG ACC ATG TTC CCA CAC TTC CTT CTG-3' + 5'-ATA TTT ATA TGC GGC CGC TGC TCT AAC ACG GCC ACC ATT G-3'. The resulting 285-bp product was digested with XbaI/NotI and ligated with pED.Fc vector fragment (20). The vector containing rat47mutFc coding sequence was transfected in stable fashion into Chinese hamster ovary (CHO) cells and supernatant from an expanded single clone was used as the source of recombinant material. Recombinant rat47mutFc protein was purified and used for generation and screening of neutralizing Ab, ratPSG-huG1. Epitope mapping was performed via peptide spot synthesis on cellulose membranes modified with polyethylene glycol. Following peptide synthesis, the membranes were washed in methanol for 10 min and in blocker (1% casein in TBS) for 10 min. Membranes were then incubated with 1 µg/ml ratPSGhuG1 in TBS for 1 h with gentle shaking. The membranes were washed in TBS and then probed with an HRP-conjugated anti-Fc Ab in blocker. After washing with TBS, bound protein was visualized using SuperSignal West reagent (Pierce) and a digital camera.

Animals

Male Lewis rats (LEW; 200–250 g) were obtained from Harlan Sprague Dawley. Rats were housed in the University of California, Los Angeles, animal facility under specific pathogen-free condition. Animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (Publication No. 86-23).

Ex vivo model of liver cold ischemia followed by reperfusion

Rats underwent isoflurane anesthesia and systemic heparinization. After skeletonization of the liver, the portal vein, bile duct, and inferior vena cava were cannulated, and the liver was flushed with 10 ml of University of Wisconsin (UW) solution. Livers were stored for 24 h at 4°C in UW and then perfused on an isolated perfusion rat liver apparatus. Before the perfusion, the apparatus was primed with perfusate consisting of rat whole blood diluted with Krebs-Ringer bicarbonate medium to a hematocrit of 15%. The perfusate was maintained at a pH of 7.4 and oxygenated with Hamilton silastic lung tubing (Fisher Scientific), which maintained the pO2 > 250 mmHg. The livers were perfused ex vivo for 2 h, with stable temperature (37°C), pressure (13 cm of H2O), and pH 7.4, as described (18, 19, 21, 22, 23, 24). Experimental animals were divided into two groups: anti-PSGL-1 group: blood donor animals received 1 mg/kg of anti-rat PSGL-1 Ab (Wyeth Pharmaceuticals) i.v. 30 min before blood harvest; control group, blood donor animals received 1 mg/kg control Ab (PSG3-G1; anti-human PSGL-1 Ab with inactive IgG1 Fc, Wyeth Pharmaceuticals), as did anti-PSGL-1 group. The endotoxin values for anti-PSGL-1 and control Abs were <0.27EU/mg.

Portal vein blood flow and pressure were recorded every 15 min, whereas bile output was monitored every 30 min. Blood was collected at 30-min intervals for serum glutamic-oxoaloacetic transaminase (sGOT) and glutamic-pyruvic transaminase (sGPT) levels, measured using an autoanalyzer (Antech Diagnostics). After 2 h of perfusion, a portion of liver was snap-frozen for myeloperoxidase (MPO) assay and was fixed in formalin for histological evaluation.

Syngeneic OLT model

OLT were performed using LEW livers that were stored for 24 h at 4°C in UW before being transplanted into syngeneic recipients with revascularization without hepatic artery reconstruction (25). In the anti-PSGL-1 group, livers were harvested from untreated rats, stored for 24 h at 4°C, and then transplanted into syngeneic recipients that were treated with anti-rat PSGL-1 Ab (1 mg/kg i.v.) 30 min before transplantation. In the control group, recipients received control Ab (1 mg/kg i.v.) as in the anti-PSGL-1 Ab. OLT recipients were followed for survival. Separate groups of rats were sacrificed at 6 and 24 h after OLT, and liver samples were collected for further analysis.

MPO assay

The presence of MPO, an enzyme specific for neutrophils, was used as an index of intrahepatic neutrophil accumulation (26). Briefly, the frozen tissue was thawed, weighed, and placed in 4 ml of iced 0.5% hexadecyltrimethylammonium bromide and 50 mM potassium phosphate buffer solution with the pH adjusted to 5. Each sample was then homogenized for 30 s 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 of MPO activity was defined as the quantity of enzyme degrading 1 µM peroxide/min at 25°C/g of tissue.

Histology

Liver specimens were fixed in 10% buffered formalin solution and embedded in paraffin. Sections were made at 5 µm and stained with H&E. The histological severity of liver IRI was graded using modified Suzuki’s criteria (27). 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 >60% lobular necrosis, is given a value of 4.

Immunohistology

Liver samples were embedded in Tissue-Tek OCT compound (Miles), snap-frozen in liquid nitrogen, and stored at –80°C. Five-micrometer sections were fixed in a cold acetone, and then endogenous peroxidase activity was inhibited with 0.3% H2O2 in PBS. 5% BSA was used for blocking. Primary Abs included P-selectin (CD62P; BD Pharmingen), ED-1 (macrophage/monocyte; Chemicon International), CD3 (T cell; BD Pharmingen), and OX-62 (dendritic cell (DC) marker; BD Pharmingen) was added at optimal dilutions. Bounded primary Abs were detected using peroxidase-conjugated goat anti-rabbit or anti-mouse secondary Abs (Dako Envision kit/HRP, diaminobenzidine; DakoCytomation). Negative controls included sections in which the primary Ab was replaced with dilution buffer or normal mouse serum. The sections were developed with 3,3'-diaminobenzidine tetrahydrochloride and evaluated blindly by counting the labeled cells in triplicate with 10 high-power fields/section.

Western blots

Liver tissue samples were homogenized in ice-cold PBSTDS buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% Triton-100, 5 mg/ml leupeptin, aprotinin, pepstain, and chymostain). The homogenates were centrifuged at 10,000 x g at 4°C; the resulting supernatants were mixed with sample buffer. Samples containing 40 µg of proteins were separated by SDS-electrophoresis on 10–20% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with specific primary Ab against Bcl-2, Bcl-xL, caspase-3, and {beta}-actin (Santa Cruz Biotechnology). After washing with PBS containing 0.05% Tween 20, the membranes were incubated with HRP-conjugated donkey anti-rabbit (Santa Cruz Biotechnology) secondary Ab. Immunoreactive bands were visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce). Relative quantities of protein were determined using a densitometer (Kodak Digital Science 1D Analysis Soft-ware) and presented in comparison to {beta}-actin expression.

RT-PCR

Total RNA was extracted from liver tissues specimens by the guanidium isothiocyanate method as described (28). Primers used in PCR were as follows: TNF-{alpha} sense (5'-CGA GTG ACA AGC CCG TAG-3') and TNF-{alpha} antisense (5'-GGA TGA ACA CGC CAG TCG-3'); IL-1{beta} sense (5'-CCA GGA TGA GGA CCC AAG CA-3') and IL-1{beta} antisense (5'-TCC CGA CCA TTG CTG TTT CC-3'); IL-6 sense (5'-CTT CCA GCC AGT TGC CTT C-3') and IL-6 antisense (5'-GAG AGC ATT GGA AGT TGG-3'); IFN-{gamma} sense (5'-CCC TCT CTG GCT GTT ACT GC-3') and IFN-{gamma} antisense (5'-CTC CTT TTC CGC TTC CTT AG-3'); IL-2 sense (5'-GCG CAC CCA CTT CAA GCC CT-3') and IL-2 antisense (5'-CCA CCA CAG TTG CTG GCT CA-3'); and {beta}-actin sense (5'-CTA TCG GCA ATG AGC GGT-3') and {beta}-actin antisense (5'-CTT AGG AGT TGG GGG TGG-3'). PCR conditions for each primer couple were as follows: TNF-{alpha}, 95°C for 45 s, 61°C for 45 s, and 72°C for 45 s during 35 cycles; IL-1{beta}, 95°C for 30 s, 63°C for 45 s, and 72°C for 30 s during 28 cycles; IL-6 and IFN-{gamma}, 95°C for 15 s, 60°C for 60 s, and 72°C for 30 s during 35 cycles; and IL-2, 95°C for 20 s, 53°C for 20 s, and 72°C for 30 s during 33 cycles. PCR products were analyzed in ethidium bromide-stained 2% agarose gel. The density was scanned with Kodak Digital Science 1D image analysis software (version 2.0; Eastman Kodak Scientific Imaging Systems).

Detection of apoptosis

A commercial in situ histochemical assay (Klenow-FragEL; Oncogene Research Products) was performed to detect DNA fragmentation in acetone-fixed cryostat OLTs. Biotinylated nucleotides were detected using streptavidin-HRP conjugate. counterstaining with methyl green aids in morphological evaluation/characterization of normal and apoptotic cells. Results were scored semiquantitatively by averaging the number of apoptotic cells/field at x200 magnification. Six fields per sample were evaluated.

Statistical analysis

All data are expressed as the mean ± SEM. Differences were analyzed by one-way ANOVA. Statistical calculations were made with the help of the StatView II Statistical Package (Abacus Concepts). A value of p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Isolation of the rat PSGL-1 gene

A phage library of rat genomic DNA was screened using the highly conserved segment of DNA encoding the human PSGL-1 transmembrane and cytoplasmic domains as a hybridization probe. Consistent with both the human and mouse PSGL-1 gene structure, the entire rat coding region is contained on a single exon (12, 29). The overall amino acid identity of rat PSGL-1 to the mouse and human sequences is 79 and 47%, respectively. The cleavage site for the paired basic amino acid-converting enzyme (furin/PACE) for the cleavage of the propeptide is preserved in the rat sequence (Fig. 1, bold letters). N-terminal sequencing of recombinant rat PSGL-1 produced in CHO cells reveals that the furin/PACE site is indeed cleaved and signal peptidase cleaves at between Ser-(–24) and Leu-(–25) (data not shown). The first nine N-terminal residues of the mature protein are identical between rat and mouse PSGL-1. The peptide region a critical binding determinant for P-selectin in human PSGL-1 is the anionic region containing three sulfated tyrosines at the N-terminal end of the molecule (Fig. 2). Intriguingly, there is considerable variation of the sequence in this anionic region between the three species. Similar to human PSGL-1, rat PSGL-1 contains three tyrosines each of which are predicted to be sulfated (30). Rat PSGL-1 also contains a T(E/D)PPE sequence corresponding to the threonine residue (Thr16) in human PSGL-1, previously identified as the attachment site for the sLex-modified O-linked glycan critical for P-selectin binding (20). Western blot and immunoprecipitation with mAbs specific for either human or mouse PSGL-1 failed to cross-react with recombinant forms of rat PSGL-1. Consequently, a soluble form of recombinant rat PSGL-1 containing the N-terminal 47 aa was used to screen a human phagemid scFv library (31). Reactive scFv clones were identified and converted into a full-length, intact, Ab using human {lambda} L chaín and human IgG1 H chain with alanine substitutions of Leu234 and Gly237 in the CH2 region to diminish Fc effector functions (32). Biacore binding experiments using recombinant rat PSGL-1 and rat P-selectin identified one neutralizing Ab with an IC50 of 0.31 nM, and this Ab was designated ratPSG-huG1. The reactivity of this Ab with native rat PSGL-1 was confirmed by FACS analysis using rat basophilic leukemia cell line, RBL. In vivo neutralization properties were confirmed by observing a marked decrease in leukocyte rolling by intravital microscopy in rat cremaster venules 10 min after i.v. injection of ratPSG-huG1 (data not shown).



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  		FIGURE 1. Rat PSGL-1 Gene. The nucleotide sequence and amino acid sequence of the entire coding exon are shown. The furin/PACE recognition site "RERR" and a single potential N-linked glycosylation site "NES" are boxed. The decameric repeat region is underlined. The putative transmembrane region is shown in bold letters and bold underlined. These sequences have been assigned GenBank accession no. AAM21052.

 


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  		FIGURE 2. Comparison of the mature N terminus of rat, mouse, and human PSGL-1 and the binding epitope of the neutralizing anti-rat PSGL-1 Ab. The sequence alignment is centered on the threonine residues (Thr18 in mature form of rat PSGL-1) known to be the attachment site of the sLex-bearing O-linked glycan in human and murine PSGL-1 (GenBank accession nos. U02297 and X91144, respectively). Sulfated tyrosines, O-linked threonines, and potential N-linked glycosylation sites are bolded and underlined. The binding epitope on rat PSGL-1 for the ratPSG-huG1 Ab was determined by peptide mapping and requires sulfated tyrosine. It is indicated by the bold line over the rat PSGL-1 sequence.

 
Effects of anti-PSGL-1 Ab in ex vivo model of liver cold ischemia followed by reperfusion

We monitored portal vein blood flow, bile production, and sGOT/GPT levels in rat livers that were stored for 24 h at 4°C in UW solution, and then perfused for 2 h on the isolated perfusion rat liver apparatus with perfusate consisting of whole blood obtained from rats that were treated with either control or anti-PSGL-1 Ab (1 mg/kg 30 min before blood harvest). As shown in Fig. 3, anti-PSGL-1 Ab adjunct significantly improved portal vein blood flow throughout the perfusion period, compared with controls (Fig. 3A; p < 0.05). Moreover, anti-PSGL-1 Ab group significantly increased bile production, compared with controls (Fig. 3B; p < 0.05). Ischemia and reperfusion (I/R)-induced hepatocyte injury, as measured by serum transaminase levels were also significantly reduced in anti-PSGL-1 Ab treatment group (Fig. 4, A and B; p < 0.05). Increasing Ab dose in the donor treatment regimen (up to 5 mg/kg) or combined blood donor plus liver graft treatment with anti-PSGL-1 Ab did not further improve the hepatocyte function. Moreover, unlike in our previous studies with rPSGL-Ig (19), selective ex vivo treatment of rat livers with anti-PSGL-1 Ab only, failed to ameliorate I/R-induced hepatocellular damage (data not shown).



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  		FIGURE 3. Effects of anti-PSGL-1 Ab in rat livers perfused for 2 h on the isolated perfusion apparatus after 24 h of cold ischemia. A, Anti-PSGL-1 Ab treatment significantly increased portal blood flow throughout the perfusion period, compared with controls. B, Bile production at 30-min intervals throughout the reperfusion period was also significantly higher in the group treated with anti-PSGL-1 Ab, compared with controls. These data represent the mean ± SEM of four to six independent perfusions for each group. *, p < 0.05 vs controls.

 


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  		FIGURE 4. Serum transaminase levels after 24 h of cold ischemia, followed by 2 h of ex vivo perfusion or OLTs. In the ex vivo model, the sGOT/GPT levels (IU/L) were measured in the blood samples taken at 30-min intervals during the perfusion. A, sGOT levels were lower at 90 and 120 min during the perfusion of the livers treated with anti-PSGL-1 Ab, compared with controls (*, p < 0.05). B, sGPT levels were lower at 30, 60, 90, and 120 min during the perfusion of the livers treated with anti-PSGL-1 Ab, compared with controls (*, p < 0.05). In the OLTs model, the sGOT/GPT levels (IU/L) were measured in the blood samples taken at 6 and 24 h after transplantation. At 6 and 24 h after transplantation, both sGOT (C) and sGPT (D) levels in anti-PSGL-1 Ab treatment groups were lower than those in the control groups (*, p < 0.05). These data represent the mean ± SEM of four to six independent experiments.

 
The hepatocyte injury was graded using the Suzuki’s criteria. In the control group, livers showed severe sinusoidal and vascular congestion with marked vacuolization change, focally associated with hepatocyte necrosis (Fig. 5A; score = 3.33 ± 0.70). In contrast, livers perfused with anti-PSGL-1 Ab showed minimal hepatocyte necrosis, and significantly less sinusoidal congestion, compared with control group (Fig. 5B; score = 0.67 ± 0.52, p < 0.01).



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  		FIGURE 5. Photomicrographs of representative rat livers after 24 h of cold ischemia and 2 h of reperfusion on the isolated perfusion rat liver apparatus or OLT harvested at 6 h. A, Control group with sinusoidal/vascular congestion and severe lobular distortion (Suzuki’s score = 3.33 ± 0.70). B, Anti-PSGL-1 Ab-treated group with minimal vascular congestion/vacuolar degeneration and preservation of lobular architecture (score = 0.67 ± 0.52, p < 0.01). C, Control OLTs group present extensive areas of necrosis and sinusoidal/vascular congestion at 6 h after transplantation (score = 6.67 ± 0.50). D, Anti-PSGL-1 Ab-treated OLT group present minimal vascular congestion, necrosis, and less lobular distortion (score = 1.17 ± 0.39; p < 0.01, compared with controls). (x100, H&E stain; n = 3–4/group).

 
We measured the MPO activity to determine the neutrophil infiltration in the liver tissue. The MPO activity was significantly reduced in the anti-PSGL-1 Ab treatment group, compared with control group (p < 0.05; Fig. 6A).



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  		FIGURE 6. Effects of anti-PSGL-1 Ab on neutrophil infiltration in rat livers cold stored for 24 h followed by 2 h of ex vivo perfusion or OLT. MPO assay (U/gm) was used to measure neutrophil infiltration in the liver tissue. A, At 2 h of ex vivo reperfusion, MPO levels were significantly lower in livers treated with anti-PSGL-1 Ab, compared with controls (*, p < 0.05). B, Six hours and 24 h posttransplant MPO levels were significantly lower in livers treated with anti-PSGL-1 Ab, compared with controls (*, p < 0.05). These data represent the mean ± SEM of four to six independent experiments.

 
Effects of anti-PSGL-1 Ab in rat liver model of cold ischemia followed by OLT

Anti-PSGL-1 Ab treatment prolongs OLT survival, improves hepatic function, and ameliorates hepatocellular injury. We next examined whether recipient treated with anti-PSGL-1 Ab would confer protection against hepatic IRI in the in vivo setting. OLT were performed that were cold stored for 24 h before the transplant into syngeneic rat recipients. Anti-PSGL-1 Ab treatment (1 mg/kg i.v. 30 min before reperfusion) significantly prolonged the animal survival from 20 to 80% at 2 wk post-OLT (p < 0.01; Fig. 7). This prolonged survival correlated with improved liver function, as measured by sGOT/GPT levels (Fig. 4, C and D).



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  		FIGURE 7. Effects of anti-PSGL-1 Ab on OLT survival. Rat livers were stored for 24 h at 4°C in UW solution and then transplanted to groups of control Ab-treated or anti-PSGL-1 Ab-treated recipients. The anti-PSGL-1 group significantly prolonged the animal survival from 20 to 80% at 2 wk post-OLT (p < 0.001; n = 10 rats/group).

 
The hepatic IRI in the OLT model was assessed using the Suzuki’s classification. The control group showed moderate to severe hepatocyte necrosis with disruption of lobular architecture and marked sinusoidal congestion at 6 h (Fig. 5C; Suzuki score = 6.67 ± 0.50). In contrast, the anti-PSGL-1 Ab treated group showed minimal necrosis and no evidence of sinusoidal congestion (Fig. 5D; Suzuki score = 1.17 ± 0.39; p < 0.01).

Anti-PSGL-1 Ab treatment decreases P-selectin expression, monocyte/macrophage, T cell, DC, and neutrophil infiltration in the liver. Fig. 8, A and B, shows immunohistochemical staining for P-selectin that mediates initial adhesive interactions of leukocytes with endothelium in inflammatory site. At 6 h after reperfusion, higher expression of P-selectin was observed in the liver of control group (Fig. 8A). In contrast, anti-PSGL-1 Ab group showed less expression of P-selectin (Fig. 8B).



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  		FIGURE 8. Immunohistochemical staining for P-selectin, ED-1, CD3, and OX-62 in rat livers harvested at 6 h after transplantation. Treatment with anti-PSGL-1 Ab decreased expression of P-selectin (B), ED-1 (D), CD3 (F), and OX-62 positive cells (H), compared with controls (A, C, E, and G). Representative of three OLTs is shown. Original magnification, x400.

 
To determine whether anti-PSGL-1 Ab treatment affected local leukocytes infiltration, we measured PMN infiltration using a MPO assay and macrophage (ED-1)/T cell (CD3)/DC (OX-62) infiltration by immunohistochemical staining. Indeed, MPO activity in the livers of anti-PSGL-1 Ab treated group at 6 h (3.42 ± 0.14 vs 1.92 ± 0.12, p < 0.01) and at 24 h (4.72 ± 0.70 vs 1.54 ± 0.36, p < 0.01) after transplantation was significantly decreased, compared with control group (Fig. 6B). In addition, anti-PSGL-1 therapy markedly decreased intragraft infiltration of ED-1 (11.4 ± 3.4 vs 36.2 ± 5.9; p < 0.001), CD3 (2.1 ± 1.1 vs 11.3 ± 2.2; p < 0.001), and OX-62 (3.7 ± 2.0 vs 10.6 ± 3.5; p < 0.01) positive cells, compared with control group (Fig. 8, C–H).

Anti-PSGL-1 Ab treatment decreases proinflammatory cytokine expression. As shown in Fig. 9, anti-PSGL-1 treatment significantly decreased intragraft expression of mRNA coding for macrophage-associated TNF-{alpha}, IL-1{beta}, and IL-6 at 6 and 24 h, compared with control OLTs (Fig. 9, A–C; p < 0.01). Furthermore, anti-PSGL-1 Ab reduced the expression of Th1-type IFN-{gamma} and IL-2 at 6 h and 24 h, compared with controls (Fig. 9, D and E; p < 0.01).



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  		FIGURE 9. Competitive template RT-PCR-assisted expression of proinflammatory cytokine gene at 6 and 24 h posttransplantation. Treatment with anti-PSGL-1 Ab decreased the expression of TNF-{alpha} (A), IL-1{beta} (B), IL-6 (C), IFN-{gamma} (D), and IL-2 (E), compared with controls (*, p < 0.01). Each bar graph represents the mean ± SEM of four to six independent experiments.

 
Anti-PSGL-1 Ab treatment leads to anti-apoptotic function. TUNEL assay was conducted to examine whether anti-PSGL-1 Ab treatment has an antiapoptotic effect on hepatic IRI. At 6 h after transplantation, the number of TUNEL-positive cells was markedly decreased in the anti-PSGL-1 group (2.94 ± 0.23; Fig. 10, B and C), compared with control (30.89 ± 3.11; p < 0.01; Fig. 10, A and C).



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  		FIGURE 10. TUNEL-assisted detection of apoptotic cells in rat OLTs at 6 h after transplantation. The frequency of TUNEL-positive cells in control group (A and C; 30.89 ± 3.11) was markedly increased, compared with those in anti-PSGL-1 group (B and C; 2.94 ± 0.23, *, p < 0.01). A minimum of six fields were evaluated per sample (magnification of x200); n = 4.

 
We then used Western blots to analyze the expression of antiapoptotic (Bcl-2/Bcl-xL) proteins and caspase-3. The relative intragraft expression levels of individual molecules were determined by densitometry, and expressed as ratios with the expression of {beta}-actin that was used as a housekeeping gene. As shown in Fig. 11, anti-PSGL-1 Ab treatment significantly up-regulated the expression of antiapoptotic Bcl-2 and Bcl-xL at 6 h (0.48 ± 0.08 and 0.31 ± 0.04, respectively) and 24 h (0.42 ± 0.05 and 0.30 ± 0.06, respectively), compared with controls (Bcl-2: 0.18 ± 0.09 and 0.21 ± 0.06, p < 0.01; Bcl-xL: 0.08 ± 0.02 and 0.13 ± 0.06, p < 0.01). The expression of cleaved caspase-3 in the anti-PSGL-1 group was significantly inhibited at 6 and 24 h (0.24 ± 0.06 and 0.21 ± 0.05), compared with control (0.63 ± 0.06 and 0.61 ± 0.03; p < 0.01).



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  		FIGURE 11. Western blot analysis of OLT expression of antiapoptotic (Bcl-2 and Bcl-xL) and caspase-3 proteins. Anti-PSGL-1 Ab treatment significantly up-regulated the expression of Bcl-2/Bcl-xL at 6 and 24 h, compared with controls. The expression of cleaved caspase-3 in anti-PSGL-1 Ab treatment group was significantly inhibited at 6 and 24 h, compared with controls. Representative of four separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report in this article the results of our studies on the protective effects of a targeting PSGL-1 on the surface of leukocytes with a new blocking Ab in rat liver models of cold ischemia followed by ex vivo reperfusion or syngeneic OLT. In ex vivo hepatic cold IRI model, blood donor treatment with anti-PSGL-1 Ab significantly improved portal venous flow, increased bile production, diminished neutrophil infiltration, and decreased hepatocellular damage. Pretreatment of recipient with a single dose of anti-PSGL-1 Ab: 1) improved liver function and hepatocyte integrity with resultant prolongation of graft survival; 2) decreased the expression of P-selectin; 3) prevented neutrophil, T cell, macrophage, and DC infiltration; 4) inhibited proinflammatory cytokine expression; and 5) reduced hepatic apoptosis, accompanied by increased expression of anti-apoptotic molecules and inhibition of caspase-3 activation.

Platelet-leukocyte-endothelial cell interactions play a central role in hepatic IRI. The accumulation of leukocytes to sites of tissue injury is mediated by three groups of cell adhesion molecules, i.e., the selectin family (P-, E-, and L-selectin), the {beta}2-integrin family (CD11/CD18), and the Ig superfamily (ICAM-1, PECAM-1) (2, 3). P-selectin, which is stored in the Weibel-Palade bodies of endothelial cell and {alpha} granules of the platelets (33), is expressed rapidly in early phase of reperfusion (14, 34). Indeed, P-selectin initiates the adhesion, rolling of leukocytes, and then recruits them to the injured site. Previous studies have shown that blockade of interaction between P-selectin and PSGL-1 can attenuate PMN adherence and infiltration during hepatic IRI. In a mouse partial hepatic warm IRI model, both P-selectin knockout (KO) mice and anti-P-selectin mAb treated mice showed significant decreases in liver enzyme levels and had less liver necrosis, compared with wild-type controls (14). Martinez et al. (16) reported that P-selectin KO mice showed significant decrease in liver enzyme levels and marked decrease in serum chemokines, compared with wild-type controls in a mouse partial hepatic warm IRI model. In addition, PMN infiltration was markedly decreased not only in the liver but also in the lung. Yadav et al. (13) also reported that P-selectin mediates hepatic IRI via PMN and platelet sequestration. In a total hepatic IRI model, survival was significantly improved in P-selectin KO mice, compared with wild-type controls. In our previous studies, we demonstrated that pretreatment of steatotic rat livers with rPSGL-Ig resulted in significant decrease in portal resistance, increased bile production, and reduced liver PMNs infiltration in the ex vivo cold ischemia model (18). In addition, treatment of rPSGL-Ig before storage and transplantation significantly prolonged survival of liver isograft (18). Treatment of fatty livers with rPSGL-Ig extended the survival of lean Zucker rat recipients from 40 to 90%. This effect correlated with suppressed macrophage/T cell infiltration, diminished expression of proinflammatory cytokines (TNF-{alpha}, IL-6, iNOS, IL-2, IFN-{gamma}), and decreased histological features of hepatocyte injury (19).

In this study, we showed that blood donor pretreatment with anti-PSGL-1 Ab significantly improved portal blood flow and bile production in the ex vivo cold ischemia followed by reperfusion model. Moreover, anti-PSGL-1 Ab treatment decreased MPO activity and exhibited well-preserved hepatic architecture with less congestion or necrosis both in an ex vivo and in OLT models. These results suggest that anti-PSGL-1 therapy inhibited early response of endothelial cells and the rolling of PMN including selectin-mediated loose adherence, which inhibited later firm adherence. The results of immunohistochemical staining in OLTs also suggests that anti-PSGL-1 therapy inhibited the interactions between other leukocytes and endothelial cells, because PSGL-1 is expressed on all these leukocytes and blockade of leukocyte-endothelial cell interaction diminished T cell, macrophage and DC infiltration in the graft itself. Interestingly, anti-PSGL-1 Ab treatment decreased the expression of P-selectin and this effect may also suppress leukocyte infiltration. In support of this concept, it has been reported that anti-PSGL-1 Ab suppressed the expression of P-selectin in the hepatic and renal tissues of rat warm IRI models and reduced the number of DCs in liver tissue after reperfusion (35). Whether P-selectin suppressive effects is a direct action of anti-PSGL-1 Ab or the reflection of the decreased levels of proinflammatory mediators that induce adhesion molecules is not clear.

TNF-{alpha}, IL-1{beta}, and IL-6 are generated mainly by Kupffer cells, and these cytokines can also recruit and activate both PMN (2, 3) and CD4+ T cells in the liver during the early phase of reperfusion (36). Activated CD4+ T cells produce Th1-type cytokines (IFN-{gamma} and IL-2), which amplify Kupffer cell activation and promote PMN recruitment into the liver. The blockade of leukocyte-endothelial cell interaction may decrease proinflammatory macrophage (TNF-{alpha}, IL-1{beta}, and IL-6) and Th1-type (IFN-{gamma}, IL-2) cytokines. In Zucker rat liver OLT model, rPSGL-Ig treatment significantly diminished expression of proinflammatory cytokine mRNA levels and suppressed infiltration of macrophages/T cells (19). Moreover, reduced proinflammatory cytokines by anti-PSGL-1 treatment correlated with diminished macrophage and T cell infiltration.

DCs, the most potent APCs, play an important role in the pathogenesis of hepatic IRI (35, 37). The potential mechanisms of DCs’ insult on the liver are that they migrate into the injured sinusoids, subsequently activate CD4+ T cells and allow them to release proinflammatory cytokines, which initiate and maintain the local inflammatory response and tissue injury accompanied with PMN and macrophage infiltration. In this study, we performed OX-62 immunohistochemical staining for the analysis of DC migration and found that DCs were significantly recruited into liver grafts of the control group but were reduced in the anti-PSGL-1 Ab treated group. Moreover, we found that DCs recruitment paralleled the infiltration of neutrophil, macrophage, and T cell into the graft. These results suggest that DCs also play an important role with other leukocytes (neutrophil, macrophage, and T cell) in the initiation and modulation of the inflammatory response in hepatic IRI and are related PSGL-1.

Apoptosis induced by cytokines such as TNF-{alpha}, IL-1, and IL-6 (38), Fas/Fas ligand interaction (39), and reactive oxygen species (40), has been identified as a key mechanism in hepatic IRI. Induction of apoptosis can be initiated by two major pathways to activate effector caspases, which lead to apoptotic cell death after reperfusion (38). The first pathway is mediated by reactive oxygen species, and cytokines (IL-1, IL-6, etc), which lead to disruption of mitochondrial membrane integrity resulting in the release of proapoptotic factors. In the second pathway, TNF-{alpha} or Fas ligand activates receptors (TNFR type 1 or Fas/CD95) resulting in the formation of a death complex, which lead to the activation of caspase-8 and then activate caspase-3. Blocking the early pathway of apoptosis can prevent hepatic injury and improve animal survival after reperfusion. In fact, inhibition of caspase-3 improves survival and reduces early graft injury after IRI in OLT (41). Furthermore, apoptosis is controlled through the expression of specific genes. Among these, the Bcl-2 gene family is the most important. The protein encoded by Bcl-2 gene has been implicated in prolongation of cell survival by blocking apoptosis and preventing hepatic IRI (42, 43). Although the protective effect of the blockade of leukocyte-endothelial cell interaction on the induction of apoptosis has been reported in liver injury models (17, 44, 45), the exact mechanism remains unclear. Khandoga et al. (45) reported that P-selectin KO mice prevented microvascular injury and apoptosis after hepatic warm I/R. The mechanism of antiapoptotic effect of P-selectin-PSGL-1 blockade was inhibition of caspase-3 activities. Indeed, anti-PSGL-1 Ab treatment significantly increased expression of anti-apoptotic Bcl-2 and Bcl-xL, which prevented expression of cleaved caspase-3 and decreased number of apoptotic cells in our OLT model. These results suggest that PSGL-1 mediates cell apoptosis by accumulation and activation of leukocytes that induce apoptotic factors. Interestingly, a recent report indicated that in activated lymphocytes, PSGL-1 mediated signaling may actually accelerate apoptosis and suppress GVHD (46). Inhibition of leukocytes and endothelial cells interaction may have important antiapoptotic and protective effects in hepatic IRI.

In conclusion, targeting leukocyte PSGL-1 with a blocking Ab that has diminished Fc effector function is an effective strategy to protect against hepatic IRI. PSGL-1 blockade prevented local inflammatory/apoptotic responses via reduced neutrophil, macrophage, T cell and DC infiltration. This resulted in the inhibition of proinflammatory macrophage/Th1-type cytokines and up-regulation of "protective" genes. Pretreatment with anti-PSGL-1 Ab is a simple and effective strategy, which provides the rationale for novel therapeutic approaches to maximize organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.


   Acknowledgments
 
We thank David Lowe of Cambridge Antibody Technologies for the screening of human scFv phagemid library.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
G. D. Shaw, M. Lorenze, K. Marquette, and S. Benard are employees of, and have stock or stock options with, Wyeth Research Institute.


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

1 This work was supported by National Institutes of Health Grants R01DK062357, AI23847, and AI42223 (to J.W.K.-W.) and the Dumont Research Foundation. Back

2 Address correspondence and reprint requests to Dr. Jerzy W. Kupiec-Weglinski, Dumont-University of California Transplant Center 77-120 CHS, Box 957054, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: jkupiec{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: IRI, ischemia/reperfusion injury; DC, dendritic cell; I/R, ischemia and reperfusion; KO, knockout; MPO, myeloperoxidase; OLT, orthotopic liver transplantation; PACE, paired basic amino acid-converting enzyme; PMN, polymorphonuclear leukocyte; PSGL-1, P-selectin glycoprotein ligand-1; sGPT, serum glutamic pyruvic transaminase; sGOT, serum glutamic oxaloacetic transaminase. Back

Received for publication July 26, 2005. Accepted for publication October 24, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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