The Protective Role of CD59 and Pathogenic Role of Complement in Hepatic Ischemia and Reperfusion Injury
2011; Elsevier BV; Volume: 179; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.08.040
ISSN1525-2191
AutoresJinyan Zhang, Weiguo Hu, Wei Xing, Tao You, Junming Xu, Xuebin Qin, Zhihai Peng,
Tópico(s)Renal Transplantation Outcomes and Treatments
ResumoHepatic ischemia-reperfusion injury (IRI) is a major factor influencing graft outcome in liver transplantation, but its mechanism is not well defined. Although complement, including the membrane attack complex (MAC), a terminal product of complement activation, is thought to be involved in the multiple reactions subsequent to the ischemia-reperfusion (IR) process, the role of MAC in the pathogenesis of hepatic IRI requires further investigation. We used a warm ischemia-reperfusion injury model in mice and a syngeneic orthotopic liver transplantation model in rats to define the role of complement, including MAC, in hepatic IR. CD59-deficient mice had more severe liver dysfunction, evidenced by increased aspartate aminotransferase levels and increased injury of liver parenchymal and nonparenchymal cells than did CD59-sufficient mice during warm hepatic IR. Furthermore, complement depletion in CD59-deficient mice by pretreatment with cobra venom factor (CVF) or the genetic introduction of C3 deficiency partially protected against development of the severe liver dysfunction that occurred in CD59-deficient mice. Severity of liver dysfunction correlated with MAC deposition, apoptotic cells, and increased inflammatory mediators such as tumor necrosis factor α. Moreover, depletion of complement with CVF in orthotopic liver transplantation recipient rats attenuated IRI of the donor livers. Taken together, these results highlight the protective role of CD59 and pathogenic role of complement, including MAC, in the pathogenesis of hepatic IRI. Hepatic ischemia-reperfusion injury (IRI) is a major factor influencing graft outcome in liver transplantation, but its mechanism is not well defined. Although complement, including the membrane attack complex (MAC), a terminal product of complement activation, is thought to be involved in the multiple reactions subsequent to the ischemia-reperfusion (IR) process, the role of MAC in the pathogenesis of hepatic IRI requires further investigation. We used a warm ischemia-reperfusion injury model in mice and a syngeneic orthotopic liver transplantation model in rats to define the role of complement, including MAC, in hepatic IR. CD59-deficient mice had more severe liver dysfunction, evidenced by increased aspartate aminotransferase levels and increased injury of liver parenchymal and nonparenchymal cells than did CD59-sufficient mice during warm hepatic IR. Furthermore, complement depletion in CD59-deficient mice by pretreatment with cobra venom factor (CVF) or the genetic introduction of C3 deficiency partially protected against development of the severe liver dysfunction that occurred in CD59-deficient mice. Severity of liver dysfunction correlated with MAC deposition, apoptotic cells, and increased inflammatory mediators such as tumor necrosis factor α. Moreover, depletion of complement with CVF in orthotopic liver transplantation recipient rats attenuated IRI of the donor livers. Taken together, these results highlight the protective role of CD59 and pathogenic role of complement, including MAC, in the pathogenesis of hepatic IRI. Liver transplantation is a routine and powerful approach for treatment of patients with acute or chronic liver failure of various causes.1Parker B.M. Cywinski J.B. Alster J.M. Irefin S.A. Popovich M. Beven M. Fung J.J. Predicting immunosuppressant dosing in the early postoperative period with noninvasive indocyanine green elimination following orthotopic liver transplantation.Liver Transpl. 2008; 14: 46-52Crossref PubMed Scopus (9) Google Scholar Nevertheless, hepatic ischemia-reperfusion (IR) remains a major deleterious factor influencing graft outcome in organ transplantation. The incidence of primary graft failure (5% to 15%) and initial poor function (10% to 25%) is strongly dependent on the extent of ischemia-reperfusion injury (IRI).2Ploeg R.J. D'Alessandro A.M. Hoffmann R.M. Eckhoff D. Isaacs R. Knechtle S.J. Pirsch J.D. Stegall M.D. Kalayoglu M. Belzer F.O. Impact of donor factors and preservation on function and survival after liver transplantation.Transplant Proc. 1993; 25: 3031-3033PubMed Google Scholar, 3Katz E. Mor E. Patel T. Theise N. Emre S. Schwartz M.E. Miller C.M. Association between preservation injury and early rejection in clinical liver transplantation: fact or myth?.Transplant Proc. 1993; 25: 1907-1908PubMed Google Scholar, 4Lemasters J.J. Thurman R.G. Reperfusion injury after liver preservation for transplantation.Annu Rev Pharmacol Toxicol. 1997; 37: 327-338Crossref PubMed Google Scholar IRI also initiates later graft failure by triggering irreversible intrahepatic biliary tract injury (ischemic-type biliary lesion) or by promoting rejection through activation of innate immunity.5Buis C.I. Hoekstra H. Verdonk R.C. Porte R.J. Causes and consequences of ischemic-type biliary lesions after liver transplantation.J Hepatobiliary Pancreat Surg. 2006; 13: 517-524Crossref PubMed Scopus (186) Google Scholar At present, because of the shortage of organs for transplantation, the donor pool has been expanded by utilization of marginal organs from old donors or non-heart-beating donors, as well as grafts with prolonged cold storage, and even allografts donated after cardiac death. It is conceivable that grafts from such donors could cause severe liver injury, because they have usually experienced a long ischemia time. To improve the outcome of liver transplantation, therefore, it is imperative to better understand the mechanisms involved in IRI and to design novel therapeutic strategies for prevention of IRI. Ischemia-reperfusion injury is characterized by the presence of activated polymorphonuclear leukocytes, oxygen radical formation,6Zweier J.L. Talukder M.A. The role of oxidants and free radicals in reperfusion injury.Cardiovasc Res. 2006; 70: 181-190Crossref PubMed Scopus (588) Google Scholar and cytokine release.7Motoki A. Adachi N. Liu K. Takahashi H.K. Nishibori M. Yorozuya T. Arai T. Nagaro T. Suppression of ischaemia-induced cytokine release by dimaprit and amelioration of liver injury in rats.Basic Clin Pharmacol Toxicol. 2008; 102: 394-398Crossref PubMed Scopus (10) Google Scholar, 8Mbachu E.M. Klein L.V. Rubin B.B. Lindsay T.F. A monoclonal antibody against cytokine-induced neutrophil chemoattractant attenuates injury in the small intestine in a model of ruptured abdominal aortic aneurysm.J Vasc Surg. 2004; 39: 1104-1111Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar The process that temporarily blocks blood supply followed by blood reperfusion to the living donor after the transplantation causes attraction, activation, adhesion, and migration of neutrophils at the site of donor organ, thereby leading to both local tissue and remote organ damage.9Watts J.A. Kline J.A. Bench to bedside: the role of mitochondrial medicine in the pathogenesis and treatment of cellular injury.Acad Emerg Med. 2003; 10: 985-997Crossref PubMed Google Scholar Recently, clinical and experimental studies in several organ systems have shown that IR also results in excessive activation of the complement system, which is indicative of the critical role of complement in the IRI.10Karpel-Massler G. Fleming S.D. Kirschfink M. Tsokos G.C. Human C1 esterase inhibitor attenuates murine mesenteric ischemia/reperfusion induced local organ injury.J Surg Res. 2003; 115: 247-256Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 11Atkinson C. Song H. Lu B. Qiao F. Burns T.A. Holers V.M. Tsokos G.C. Tomlinson S. Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (144) Google Scholar, 12Jha P. Sohn J.H. Xu Q. Nishihori H. Wang Y. Nishihori S. Manickam B. Kaplan H.J. Bora P.S. Bora N.S. The complement system plays a critical role in the development of experimental autoimmune anterior uveitis.Invest Ophthalmol Vis Sci. 2006; 47: 1030-1038Crossref PubMed Scopus (54) Google Scholar The complement system, an important mediator of innate immune defense and inflammation, is activated through three different cascades: the classical, alternative, and lectin pathways.13Tomlinson S. Complement defense mechanisms.Curr Opin Immunol. 1993; 5: 83-89Crossref PubMed Scopus (94) Google Scholar, 14Qin X. Gao B. The complement system in liver diseases.Cell Mol Immunol. 2006; 3: 333-340PubMed Google Scholar All three activation pathways converge at the C3 level, forming the membrane attack complex (MAC). The MAC forms a macromolecular pore capable of inserting itself into cell membranes and lysing heterologous cells, including bacteria and viruses.15Nicholson-Weller A. Halperin J.A. Membrane signaling by complements C5b-9, the membrane attack complex.Immunol Res. 1993; 12: 244-257Crossref PubMed Scopus (131) Google Scholar MAC formation in autologous cell membrane plays multiple and complex functions.16Benzaquen L.R. Nicholson-Weller A. Halperin J.A. Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells.J Exp Med. 1994; 179: 985-992Crossref PubMed Scopus (212) Google Scholar Sublytic MAC in endothelial and smooth muscle cells is also an important mediator of cellular signals that trigger mitogenic effects17Acosta J. Qin X. Halperin J. Complement and complement regulatory proteins as potential molecular targets for vascular diseases.Curr Pharm Des. 2004; 10: 203-211Crossref PubMed Scopus (55) Google Scholar and release growth factors such as basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), as well as cytokines such as IL-1 and monocyte chemotactic protein-1 (MCP-1).18Fosbrink M. Niculescu F. Rus V. Shin M.L. Rus H. C5b-9-induced endothelial cell proliferation and migration are dependent on Akt inactivation of forkhead transcription factor FOXO1.J Biol Chem. 2006; 281: 19009-19018Crossref PubMed Scopus (71) Google Scholar, 19Kilgore K.S. Schmid E. Shanley T.P. Flory C.M. Maheswari V. Tramontini N.L. Cohen H. Ward P.A. Friedl H.P. Warren J.S. Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kB activation.Am J Pathol. 1997; 150: 2019-2031PubMed Google Scholar To protect autologous cells from MAC-mediated attack, an array of complement regulators have evolved to restrict complement activation, including CD59. A glycosyl-phosphatidylinositol (GPI)-linked membrane protein, CD59 strongly restricts MAC formation by preventing C9 incorporation and polymerization.20Morgan B.P. Regulation of the complement membrane attack pathway.Crit Rev Immunol. 1999; 19: 173-198Crossref PubMed Google Scholar, 21Holt D.S. Botto M. Bygrave A.E. Hanna S.M. Walport M.J. Morgan B.P. Targeted deletion of the CD59 gene causes spontaneous intravascular hemolysis and hemoglobinuria.Blood. 2001; 98: 442-449Crossref PubMed Scopus (122) Google Scholar, 22Qin X. Krumrei N. Grubissich L. Dobarro M. Aktas H. Perez G. Halperin J.A. Deficiency of the mouse complement regulatory protein mCd59b results in spontaneous hemolytic anemia with platelet activation and progressive male infertility.Immunity. 2003; 18: 217-227Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Experimental and clinical evidence indicates that IR triggers complement activation in several organs.23Diepenhorst G.M. van Gulik T.M. Hack C.E. Complement-mediated ischemia-reperfusion injury lessons learned from animal and clinical studies.Ann Surg. 2009; 249: 889-899Crossref PubMed Scopus (121) Google Scholar Recently, using a novel inhibitor CR2-Crry to inhibit MAC formation and C3 activation at the site of complement activation, He et al24He S. Atkinson C. Evans Z. Ellett J.D. Southwood M. Elvington A. Chavin K.D. Tomlinson S. A role for complement in the enhanced susceptibility of steatotic livers to ischemia and reperfusion injury.J Immunol. 2009; 183: 4764-4772Crossref PubMed Scopus (36) Google Scholar demonstrated that an enhanced susceptibility to IRI of steatotic livers was associated with complement activation in a mouse model. Additionally, C6 deficiency in rats protects against ischemia damage in rat orthotopic liver transplantation (OLT) recipients, which indicates the role of MAC in the pathogenesis of hepatic IRI.25Fondevila C. Shen X.D. Tsuchihashi S. Uchida Y. Freitas M.C. Ke B. Busuttil R.W. Kupiec-Weglinski J.W. The membrane attack complex (C5b-9) in liver cold ischemia and reperfusion injury.Liver Transpl. 2008; 14: 1133-1141Crossref PubMed Scopus (44) Google Scholar, 26Yamada K. Miwa T. Liu J. Nangaku M. Song W.C. Critical protection from renal ischemia reperfusion injury by CD55 and CD59.J Immunol. 2004; 172: 3869-3875PubMed Google Scholar Nonetheless, the protective role of CD59 and the pathogenic role of complement, including MAC, in the pathogenesis of IRI still require further investigation. To assess the role of CD59 and MAC in warm IRI, we used mCd59a and mCd59b double-knockout mouse model27Qin X. Hu W. Song W. Grubissich L. Hu X. Wu G. Ferris S. Dobarro M. Halperin J.A. Generation and phenotyping of mCd59a and mCd59b double-knockout mice.Am J Hematol. 2009; 84: 65-70Crossref PubMed Scopus (20) Google Scholar and our own mCd59a, mCd59b, and C3 triple-knockout mouse model, together with a complement depletion method [ie, preadministration of mCd59ab−/− mice with cobra venom factor (CVF)]. Furthermore, to extend the present study to a clinically relevant context of liver transplantation, rats with complement deficiency induced by CVF were used to investigate the role of complement activation in IRI after syngeneic OLT. All animals were housed in a specific pathogen-free facility and were confirmed to be negative for common murine viral pathogens by routine sera analysis. All mice on a C57BL/6 (B6) genetic background were 8 to 12 weeks old, with 20 to 30 g body weight. mCd59a and mCd59b double-knockout mice (mCd59ab−/−) and mCd59a, mCd59b, and mC3 triple-knockout mice (mCd59ab−/−/mC3−/−) in a B6 background were generated at the Laboratory for Translational Research at Harvard Medical School as previously described by Qin et al.27Qin X. Hu W. Song W. Grubissich L. Hu X. Wu G. Ferris S. Dobarro M. Halperin J.A. Generation and phenotyping of mCd59a and mCd59b double-knockout mice.Am J Hematol. 2009; 84: 65-70Crossref PubMed Scopus (20) Google Scholar In the rat experiment, all adult male Wistar rats (200 to 250 g body weight) were purchased from the Animal Facility of Shanghai Jiao Tong University. The mouse experiments were conducted at the Laboratory for Translational Research at Harvard Medical School and the rat experiments at the Department of General Surgery of Shanghai First People's Hospital at Shanghai Jiao Tong University. The protocols for hepatic IRI were approved by the respective animal committees of Harvard Medical School and Shanghai Jiao Tong University. The hepatoma cell line Hep3B was obtained from the Shanghai cell bank of the Chinese Academy of Sciences. Cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L l-glutamine, and 1% penicillin/streptomycin (Gibco; Invitrogen, Carlsbad, CA). Mice were anesthetized by mixed isoflurane (Baxter International, Deerfield, IL) inhalation (5%) with oxygen at 3 L/min, and were subjected to total warm hepatic ischemia as described previously.28Evans Z.P. Ellett J.D. Schmidt M.G. Schnellmann R.G. Chavin K.D. Mitochondrial uncoupling protein-2 mediates steatotic liver injury following ischemia/reperfusion.J Biol Chem. 2008; 283: 8573-8579Crossref PubMed Scopus (58) Google Scholar After 45 minutes of hepatic ischemia, surviving mice were sacrificed at 6, 12, or 24 hours after reperfusion. Pretreatment with CVF (Quidel, San Diego, CA) is a well established method to deplete complement for assessing the role of complement in animal models of human diseases.29Vriesendorp F.J. Flynn R.E. Malone M.R. Pappolla M.A. Systemic complement depletion reduces inflammation and demyelination in adoptive transfer experimental allergic neuritis.Acta Neuropathol. 1998; 95: 297-301Crossref PubMed Scopus (23) Google Scholar Thus, CVF was used for induction of complement depletion in vivo for mCd59ab+/+ and mCD59ab−/− mice with injection of a single dose (1.5 μg/g i.p.) at 24 hours before hepatic IR. To deplete complement in the rats, CVF was administered (1 g/kg body weight, i.p.) to recipient rats 24 hours before syngeneic OLT. Donor rats underwent isoflurane inhalation anesthesia. After a flushing via the cannulated portal vein with 20 mL of heparinized University of Wisconsin solution (DuPont Pharmaceuticals, Wilmington, DE), the liver was isolated and stored at 4°C for 2 hours before transplantation. A cuff was used for revascularization for the portal vein and inferior vena cava after the superior vena cava was reconstructed with 6–0 running suture (Prolene; Ethicon, Somerville, NJ) in rat nonarterialized OLT.30Li J. Dahmen U. Dirsch O. Shen K. Gu Y. Broelsch C.E. Modified sleeve anastomosis for reconstruction of the hepatic artery in rat liver transplantation.Microsurgery. 2002; 22: 62-68Crossref PubMed Scopus (23) Google Scholar The abdomen was closed in a double layer using 5-0 USP suture (Monocryl; Ethicon), and 1 mL sterile lactated Ringer's solution (Baxter International) was administered subcutaneously to compensate for operative fluid loss. Blood aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined spectraphotometrically using plasma prepared from collected blood, as described previously.31Rochling F.A. Evaluation of abnormal liver tests.Clinical Cornerstone. 2001; 3: 1-12Crossref PubMed Scopus (43) Google Scholar AST and ALT values were expressed in international units per liter (IU/L). Plasma TNF-α, IL-6, IL-12, and MIP-2 levels were determined with a Bio-Plex multiplex cytokine assay (Bio-Rad Laboratories, Hercules, CA), using a protocol described previously.32Bjerre M. Hansen T.K. Flyvbjerg A. Tønnesen E. Simultaneous detection of porcine cytokines by multiplex analysis: development of magnetic bioplex assay.Vet Immunol Immunopathol. 2009; 130: 53-58Crossref PubMed Scopus (23) Google Scholar sP-selectin and plasma Von Willebrand factor (vWF) activity were measured using an ELISA kit (R&D Systems, Minneapolis, MN), as described previously.33Hu W. Ferris S.P. Tweten R.K. Wu G. Radaeva S. Gao B. Bronson R.T. Halperin J.A. Qin X. Rapid conditional targeted ablation of cells expressing human CD59 in transgenic mice by intermedilysin.Nat Med. 2008; 14: 98-103Crossref PubMed Scopus (31) Google Scholar, 34Hu W. Jin R. Zhang J. You T. Peng Z. Ge X. Bronson R.T. Halperin J.A. Loscalzo J. Qin X. The critical roles of platelet activation and reduced NO bioavailability in fatal pulmonary arterial hypertension in a murine hemolysis model.Blood. 2010; 116: 1613-1622Crossref PubMed Scopus (56) Google Scholar To monitor the kinetics of complement depletion, serum C3 levels of mice with and without CVF treatment were measured by ELISA using F(ab′)2 fragments of goat anti-mouse C3 as capture antibody and peroxidase-conjugated goat anti-mouse C3 as the second antibody.35Wu G. Chen T. Shahsafaei A. Hu W. Bronson R.T. Shi G.P. Halperin J.A. Aktas H. Qin X. Complement regulator CD59 protects against angiotensin II-induced abdominal aortic aneurysms in mice.Circulation. 2010; 121: 1338-1346Crossref PubMed Scopus (41) Google Scholar Optical density (OD414) was determined in a 96-well plate by means of an ELISA plate reader. All samples were tested in duplicate. Liver tissue were fixed in 10% formalin and processed for paraffin embedding. Sections of 4-μm thickness were cut for H&E staining. A pathologist (W.X.), in a blinded fashion, evaluated and graded the severity of liver damage in the sections based on sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis, and neutrophil infiltration, as described previously.36Serafín A. Roselló-Catafau J. Prats N. Xaus C. Gelpí E. Peralta C. Ischemic preconditioning increases the tolerance of fatty liver to hepatic ischemia-reperfusion injury in the rat.Am J Pathol. 2002; 161: 587-601Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar In brief, a grade of 0 indicates minimal or absent necrosis; grade 1 indicates individual mild injury with cytoplasm vacuolization and focal nuclear pyknosis; grade 2 indicates moderate to severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, and loss of intercellular borders; and grade 3 indicates severe necrosis with disintegration of hepatic cords, hemorrhage, and neutrophil infiltration. Ten high-powered fields per section were analyzed in relation to the central vein to determine the percentage of necrotic cells. In situ apoptosis was evaluated by TUNEL staining (Roche Applied Science, Indianapolis, IN) and immunohistochemistry staining with anti-active caspase-3 antibodies according to the manufacturer's instructions. Fluorescent staining results were analyzed and evaluated using Image-Pro Plus version 4.5 software (Media Cybernetics, Silver Spring, MD). The percentage of TUNEL-positive cells served as the apoptosis index. The apoptosis rate confirmed by immunohistochemistry staining with anti-active caspase-3 antibodies was calculated as the number of activated caspase-3 stained cells per 1000 hepatocytes. Frozen sections were stained with rabbit anti-rat C9, which cross-reacts with mouse C9 (kindly provided by Dr. B. Paul Morgan, University of Wales), as we have described previously.35Wu G. Chen T. Shahsafaei A. Hu W. Bronson R.T. Shi G.P. Halperin J.A. Aktas H. Qin X. Complement regulator CD59 protects against angiotensin II-induced abdominal aortic aneurysms in mice.Circulation. 2010; 121: 1338-1346Crossref PubMed Scopus (41) Google Scholar, 37Wu G. Hu W. Shahsafaei A. Song W. Dobarro M. Sukhova G.K. Bronson R.R. Shi G.P. Rother R.P. Halperin J.A. Qin X. Complement regulator CD59 protects against atherosclerosis by restricting the formation of complement membrane attack complex.Circ Res. 2009; 104: 550-558Crossref PubMed Scopus (98) Google Scholar Hep3B cells were used because this cell line has been demonstrated to highly express human CD59.38Halme J. Sachse M. Vogel H. Giese T. Klar E. Kirschfink M. Primary human hepatocytes are protected against complement by multiple regulators.Mol Immunol. 2009; 46: 2284-2289Crossref PubMed Scopus (22) Google Scholar We followed the same protocol as described previously38Halme J. Sachse M. Vogel H. Giese T. Klar E. Kirschfink M. Primary human hepatocytes are protected against complement by multiple regulators.Mol Immunol. 2009; 46: 2284-2289Crossref PubMed Scopus (22) Google Scholar, 39Spiller O.B. Criado-García O. Rodríguez De Córdoba S. Morgan B.P. Cytokine-mediated up-regulation of CD55 and CD59 protects human hepatoma cells from complement attack.Clin Exp Immunol. 2000; 121: 234-241Crossref PubMed Scopus (65) Google Scholar to culture the cells and to perform complement-mediated cytotoxicity assay. Briefly, 105 cells were treated with anti-hCD59 antibody (Bric 229 clone) for 40 minutes. After three washings with PBS, the cells were incubated at 37°C for 1 hour with a rabbit anti-human asialoglycoprotein receptor (ASGPR) polyclonal antibody (Abcam, Cambridge, UK) for activating the complement classical pathway, and 25% human serum or heat-inactivated human serum as a source of complement. We used cells cultured in the medium as untreated control. Supernatant (20 μL) was collected from each well after centrifugation and processed with 150 μL europium solution (DELFIA; PerkinElmer-Wallac, Turku, Finland) for fluorescent staining. The fluorescence was measured with a 1420 multilabel counter (PerkinElmer-Wallac). Comparisons between two groups were performed using an unpaired t-test. Comparisons across multiple groups and various time points were analyzed using analysis of variance followed by a Fisher's protected least significant difference (PLSD) post hoc test. P ≤ 0.05 was considered statistically significant. All data were analyzed using SigmaStat version 3.5 software (SPSS, Chicago, IL). Values are presented as means ± SEM. To define the protective role of CD59 and pathogenic role of MAC in the pathogenesis of IRI, we used mCd59ab−/− mice and an established murine IR model. AST levels, a marker for liver function routinely tested in clinical laboratories, were used to monitor the dynamic change of liver function. At 12 hours after warm reperfusion, mCd59ab−/− mice had significantly higher levels of AST, compared with CD59-sufficient wild-type mice (P < 0.05; Figure 1A). Also, levels of AST and ALT in mCd59ab−/− mice tended to be higher than those of mCd59ab+/+ mice, although the difference did not reach statistical significance at 6 hours or 24 hours after reperfusion (Figure 1A; see also Supplemental Figure S1A at http://ajp.amjpathol.org). These results indicate that CD59 protects against hepatic IRI. In addition to its anti-MAC role, CD59 has a complement-independent function in regulating activity of NK, B, and T cells,37Wu G. Hu W. Shahsafaei A. Song W. Dobarro M. Sukhova G.K. Bronson R.R. Shi G.P. Rother R.P. Halperin J.A. Qin X. Complement regulator CD59 protects against atherosclerosis by restricting the formation of complement membrane attack complex.Circ Res. 2009; 104: 550-558Crossref PubMed Scopus (98) Google Scholar, 40Longhi M.P. Williams A. Wise M. Morgan B.P. Gallimore A. CD59a deficiency exacerbates influenza-induced lung inflammation through complement-dependent and -independent mechanisms.Eur J Immunol. 2007; 37: 1266-1274Crossref PubMed Scopus (26) Google Scholar, 41Sivasankar B. Donev R.M. Longhi M.P. Hughes T.R. Davies R. Cole D.S. Morgan B.P. Marchbank K.J. CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens.Mol Immunol. 2007; 44: 2978-2987Crossref PubMed Scopus (6) Google Scholar which may also contribute to hepatic IRI. To define the underlying mechanism by which the deficiency of CD59 accelerates the IRI, we generated mCd59ab−/−/mC3−/− mice by crossing mC3 C3-deficient mice (mC3−/−) with mCd59ab−/− mice. Absence of C3 in mCd59ab−/−/C3−/− functionally confirmed the C3 deficiency in mCd59ab−/− (Figure 1B). At 12 hours after warm IR, the mCd59ab−/−/C3−/− had a significantly lower level of AST, compared with mCd59ab−/− mice. At 6 and 24 hours after reperfusion, AST levels tended to be lower in mCd59ab−/−/C3−/− mice, compared with mCd59ab−/− mice (Figure 1A). Furthermore, there were no significant difference at the level of AST between mCd59ab−/−/C3−/− and mCd59ab+/+ mice (Figure 1A). These data indicate that the accelerated IRI in CD59-deficient mice is complement-dependent and that the MAC contributes to the development of IRI. Because CD59 is a key regulator for the formation of MAC,12Jha P. Sohn J.H. Xu Q. Nishihori H. Wang Y. Nishihori S. Manickam B. Kaplan H.J. Bora P.S. Bora N.S. The complement system plays a critical role in the development of experimental autoimmune anterior uveitis.Invest Ophthalmol Vis Sci. 2006; 47: 1030-1038Crossref PubMed Scopus (54) Google Scholar we analyzed MAC deposition through immunofluorescence assay for C9. Consistently, we observed significantly increased MAC deposition in mCd59ab−/− than in mCd59ab+/+ or mCd59ab−/−/C3−/− mice (Figure 2B). Histological analysis of liver tissues revealed much more evident inflammation of mCd59ab−/− livers, with remarkable hepatocyte swelling and vacuolization, sinusoidal congestion, spotty necrosis in centrilobular areas, and diffuse neutrophil infiltration in the portal spaces, compared with mCd59ab+/+ or mCd59ab−/−/C3−/− mice (Figure 2, A and C). These results highlight the critical roles of MAC in the pathogenesis of hepatic IRI. The inflammatory response to hepatic IR is associated with an increase in cytokine production.42Pevni D. Frolkis I. Shapira I. Schwartz D. Schwartz I. Chernichovski T. Nesher N. Uretzky G. Cardioplegic ischemia or reperfusion: which is a main trigger for tumor necrosis factor production.Int J Cardiol. 2008; 127: 186-191Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar The biphasic pattern of hepatic IRI was characterized by Kupffer cell activation and release of proinflammatory cytokines in the early phase, followed by massive neutrophil infiltration and further production of the inflammatory mediators in the late phase. To investigate the molecular mechanisms by which MAC may be involved in IRI, we measured plasma levels of TNF-α, IL-12, IL-6, and MCP-1, the cytokines commonly elevated in liver damage,43Libert C. Wielockx B. Grijalba B. Libert C. Wielockx B. Grijalba B. Van Molle W. Kremmer E. Colten H.R. Fiers W. Brouckaert P. The role of complement activation in tumor necrosis factor-induced lethal hepatitis.Cytokine. 1999; 11: 617-625Crossref PubMed Scopus (12) Google Scholar, 44Mollnes T.E. Song W.C. Lambris J.D. Complement in inflammatory tissue damage and disease.Trends Immunol. 2002; 23: 61-64Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar at 6 and 12 hours after reperfusion. At the 12-hour time point, plasma TNF-α levels of mCd59ab−/− mice were significantly higher than those of mCd59ab+/+ and mCd59ab−/−/C3−/− (Figure 3). Furthermore, the plasma IL-6 levels of mCd59ab−/− mice tended to be higher than those of mCd59ab+/+ and mCd59ab−/−/C3−/− (Figure 3, A and B). There were no significant differences for levels of IL-12 and MCP-1 among the groups (data not shown). These results indicate that increased TNF-α and IL-6 may be associated with the development of MAC-accelerated hepatic IRI. We also measured plasma levels of vWF and sP-selection, the biomarkers for endothelial dysfunction and platel
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