Anti-CD95-induced Lethality Requires Radioresistant FcγRII+ Cells
2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês
10.1074/jbc.m211229200
ISSN1083-351X
AutoresSatoshi Jodo, John T. Kung, Sheng Xiao, Derek V. Chan, Seiichi Kobayashi, Masatoshi Tateno, Robert Lafyatis, Shyr‐Te Ju,
Tópico(s)Cell death mechanisms and regulation
ResumoThe Jo2 anti-mouse CD95 monoclonal antibody induces lethality in mice characterized by hepatocyte death and liver hemorrhage. Mice bearing a defect in Fas expression or in the Fas-mediated apoptotic pathway are resistant to Jo2. Here we show that FcγRII knockout mice or mice with monoclonal antibody-blocked FcγRII are also resistant to Jo2. The critical FcγRII+ cells are radioresistant and could not be reconstituted with splenic cells. Death of sinusoidal lining cells and destruction of sinusoids were observed, consistent with the characteristic liver hemorrhage and the selective FcγRII expression in sinusoidal lining cells but not hepatocytes. Hemorrhage developed coincident with hepatocyte death and the sharp rise of serum alanine aminotransferase and alanine aminotransferase. Invariably, moribund mice showed severe liver hemorrhage and destruction of sinusoids. The data demonstrate a novel mechanism by which the destruction of liver sinusoids, induced by the Jo2-mediated co-engagement of Fas and FcγRII, leads to severe hemorrhage and lethal fulminant hepatitis. The Jo2 anti-mouse CD95 monoclonal antibody induces lethality in mice characterized by hepatocyte death and liver hemorrhage. Mice bearing a defect in Fas expression or in the Fas-mediated apoptotic pathway are resistant to Jo2. Here we show that FcγRII knockout mice or mice with monoclonal antibody-blocked FcγRII are also resistant to Jo2. The critical FcγRII+ cells are radioresistant and could not be reconstituted with splenic cells. Death of sinusoidal lining cells and destruction of sinusoids were observed, consistent with the characteristic liver hemorrhage and the selective FcγRII expression in sinusoidal lining cells but not hepatocytes. Hemorrhage developed coincident with hepatocyte death and the sharp rise of serum alanine aminotransferase and alanine aminotransferase. Invariably, moribund mice showed severe liver hemorrhage and destruction of sinusoids. The data demonstrate a novel mechanism by which the destruction of liver sinusoids, induced by the Jo2-mediated co-engagement of Fas and FcγRII, leads to severe hemorrhage and lethal fulminant hepatitis. monoclonal antibody Fas ligand alanine aminotransferase aspartate aminotransferase d-galactosamine knockout lipopolysaccharide soluble FasL vesicle preparation tumor necrosis factor-α terminal dUTP nick-end labeling Cross-linking of CD95 (Fas), a type I transmembrane protein with a cytoplasmic death domain (1Watanabe-Fukunaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2696) Google Scholar), induces cells to undergo apoptosis (2Yonehara S. Ishii A. Yonehara M. J. Exp. Med. 1989; 169: 1747-1756Crossref PubMed Scopus (1421) Google Scholar). Jo2, a hamster-derived mAb1directed against mouse Fas, is lethal when administered into mice either intravenously or intraperitoneally (3Ogasawara J. Fukunaga-Watanabe R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1800) Google Scholar). Mice defective in either Fas expression or Fas-mediated apoptotic pathways are resistant to Jo2-induced lethality (4Yin X.M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar, 5Woo M.A. Hakem A. Elia A.J. Jakem R. Duncan G.S. Patterson B.J. Mak T.W. J. Immunol. 1999; 163: 4909-4916PubMed Google Scholar). Jo2-induced lethality is associated with extensive hepatocyte death and marked elevation of serum AST and ALT (3Ogasawara J. Fukunaga-Watanabe R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1800) Google Scholar). Injection of a caspase inhibitor protects mice against Jo2-induced apoptosis of hepatocytes and lethality (6Rodriguez I. Matsuura K. Ody C. Nagata S. Vassalli P. J. Exp. Med. 1996; 184: 2067-2072Crossref PubMed Scopus (271) Google Scholar). These observations suggest that hepatocytes are the primary target and that the Jo2-induced death of hepatocytes is the cause of lethality. However, freshly isolated hepatocytes are highly resistant to Jo2, displaying weak apoptosis only after prolonged incubation in the presence of cycloheximide, which facilitates Fas-mediated apoptosis (7Nishimura Y. Kirabayashi Y. Matsuzaki Y. Musette P. Ishii A. Nakauchi H. Inoue T. Yonehara S. Int. Immunol. 1996; 9: 307-316Crossref Scopus (64) Google Scholar, 8Feng G. Kaplowitz N. J. Clin. Invest. 2000; 105: 329-339Crossref PubMed Scopus (71) Google Scholar, 9Huang D.C.S. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (274) Google Scholar). Thus, whereas lethality could be induced within 2 h after application, death of hepatocytes in in vitro experiments could only be induced after 12 h of treatment or more (3Ogasawara J. Fukunaga-Watanabe R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1800) Google Scholar, 7Nishimura Y. Kirabayashi Y. Matsuzaki Y. Musette P. Ishii A. Nakauchi H. Inoue T. Yonehara S. Int. Immunol. 1996; 9: 307-316Crossref Scopus (64) Google Scholar, 8Feng G. Kaplowitz N. J. Clin. Invest. 2000; 105: 329-339Crossref PubMed Scopus (71) Google Scholar, 9Huang D.C.S. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (274) Google Scholar). In addition, direct binding of Jo2 to hepatocytes in vivo has not been reported. Like hepatocyte damage, massive hemorrhage in the liver invariably accompanies Jo2-induced lethality (3Ogasawara J. Fukunaga-Watanabe R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1800) Google Scholar). Jo2-induced hemorrhage is highly specific, restricted only in the liver but not in other tissues whose cells often express a higher level of Fas than hepatocytes. Thus, the selective toxicity of Jo2 toward liver cannot be explained either by its Fas expression level or by the in vitro sensitivity of hepatocytes. Conversely, a direct attack of hepatocytes by Jo2 cannot explain the induction of severe hemorrhage in liver. Jo2 but not other anti-Fas mAb effectively induces lethality in mice (7Nishimura Y. Kirabayashi Y. Matsuzaki Y. Musette P. Ishii A. Nakauchi H. Inoue T. Yonehara S. Int. Immunol. 1996; 9: 307-316Crossref Scopus (64) Google Scholar). Still, the apparent liver toxicity of Jo2 has raised a serious concern regarding the use of Fas cross-linking agents as therapeutics. We noted that Jo2-mediated cytotoxicity is critically dependent on target FcγR expression. Both hepatocytes and sinusoidal lining cells express Fas, but only the latter express FcγR (10Ravetch J.V. Bolland S. Annu. Rev. Immunol. 2001; 19: 275-290Crossref PubMed Scopus (1362) Google Scholar, 11Muro H. Shirasawa H. Kosugi I. Nakamura S. Am. J. Pathol. 1993; 143: 105-120PubMed Google Scholar, 12Takezawa R.-I. Watanabe Y. Akaike T. J. Biochem. (Tokyo). 1995; 118: 1175-1183Crossref PubMed Scopus (43) Google Scholar). Because a co-engagement of Fas and FcγR can facilitate binding and strengthen the subsequent signaling process, we asked the question of whether the Jo2-induced lethality requires co-engagement of Fas and FcγR. We found that Jo2-induced lethality is critically dependent on host FcγRII expression. This observation and additional studies described herein provide strong evidence for a novel mechanism for Jo2-induced hepatic injury in which hemorrhage and hepatocyte death are secondary to the killing of sinusoidal lining cells, which are the primary target of Jo2 and the deciding factor for Jo2-induced lethality. In addition, this study revives the possibility of using Fas cross-linking agents that lack the FcγRII binding activity as useful therapeutic agents. Cytotoxicity assays against the FcγR+ A20 and the FcγR− variant IIA1.6 target cells (obtained from Dr. C. J. Janeway, Yale University, New Haven, CT) were conducted as described previously (13Jodo S. Hohlbaum A. Xiao S. Chan D. Strehlow D. Sherr D.H. Marshak-Rothstein A. Ju S.-T. J. Immunol. 2000; 165: 5487-5494Crossref PubMed Scopus (33) Google Scholar). Target cells (2 × 104), labeled with Na251CrO4 (PerkinElmer Life Sciences), were cultured with various doses of Jo2 (BD Biosciences) or FasL vesicle preparation (FasL VP) (13Jodo S. Hohlbaum A. Xiao S. Chan D. Strehlow D. Sherr D.H. Marshak-Rothstein A. Ju S.-T. J. Immunol. 2000; 165: 5487-5494Crossref PubMed Scopus (33) Google Scholar, 14Jodo S. Xiao S. Hohlbaum A. Strehlow D. Marshak-Rothstein A. Ju S.-T. J. Biol. Chem. 2001; 276: 39938-39944Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) in 0.2 ml in individual wells of a 96-well plate. Supernatants were removed at 5 h after culture, and radioactivity (counts/min) was determined. Background release was determined by culturing cells alone. Cytotoxicity is expressed as % specific 51Cr release, which is determined by the formula: 100% × (experimental release − background release)/(total cpm released by 0.5% Nonidet P-40 − background release). Assays were carried out in duplicate, and the experiment was repeated three times. The amount of human FasL protein present in FasL VP was determined using a capture enzyme-linked immunosorbent assay kit (Oncogene, Boston, MA) (14Jodo S. Xiao S. Hohlbaum A. Strehlow D. Marshak-Rothstein A. Ju S.-T. J. Biol. Chem. 2001; 276: 39938-39944Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). B6, B6;129SF2/J, B6;129S-Fcgr2 tm1Rav (FcγRII KO), C57BL/6-Fcgr3 tm1Sjv (FcγRIII KO), and B6.MRL-Fas lpr mice were obtained from The Jackson Laboratory, Bar Harbor, ME. In accordance with IACUC guidelines, mice were treated under various conditions as described in the legends of the figures and tables. To determine the lethality, mice were injected intraperitoneally with various doses of Jo2 or were untreated. Mice were euthanized at the time that the Jo2-treatedB6 mice became moribund. Tissues were fixed with 10% paraformaldehyde and photographed. Sera were collected and assayed for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using a commercial kit (Sigma). In some experiments, mice were euthanized at various times after injection of Jo2. Livers were fixed and examined for apoptotic cells using the in situ apoptosis detection kit (Trevigen, Inc., Gaithersburg, MD). To determine the role of FcγR and target radiosensitivity in the Jo2-induced lethal fulminant hepatitis, B6 and FcγRII KO mice were irradiated with 600 rads and used 4 days later. In some experiments, mice were reconstituted 24 h before the injection of Jo2 withB6 splenic cells (108/mouse) by intravenous injection of single cell suspension prepared from B6 spleen cells. In other experiments, mice were injected intravenously with purified, FcγR-specific 2.4G2 or control rat Ig (Jackson ImmunoResearch, Atlanta, GA) 2 h before the intraperitoneally injection of Jo2. Both B6 and FcγRII KO mice, 3 mice per group, were injected with d-Gal (0.5 g/kg body weight) plus bacterial LPS (50 μg/kg body weight) intraperitoneally as described (15Leist M. Gantner F. Boblinger I. Tiegs G. Am. J. Pathol. 1995; 146: 1220-1234PubMed Google Scholar, 16Morikawa A. Sugiyama T. Kato Y. Koide N. Jiang G.Z. Takahashi K. Tamada Y. Yokochi T. Infect. Immun. 1996; 64: 734-738Crossref PubMed Google Scholar). Neither agent alone induced lethality. Mice were observed for 24 h. Sera and tissues of moribund mice were collected. Samples of control mice were collected at the same times when moribund mice were euthanized. We tested Jo2 anti-Fas mAb for FcγR-dependent cytotoxicity using 51Cr-labeled, FcγR+ A20 and FcγRII− variant IIA1.6 cells (derived from A20) as targets in a 5-h cytotoxicity assay (Fig.1). Jo2 effectively killed FcγR+ A20 cells but not IIA1.6 cells (Fig.1 a). Both targets were equally sensitive to FasL VP, the apoptosis-inducing membrane vesicles that induce cell death independent of target FcγR expression (Fig. 1 b). In addition, the killing of FcγR+ A20 targets by Jo2 was inhibited by 2.4G2 mAb that blocks Fc binding to FcγRII and FcγRIII (17Ravetch J.V. Luster A.D. Weinshank R. Kochan J. Pavlovec A. Portnoy D.A. Hulmes J. Pan Y.C. Unkeless J.C. Science. 1986; 234: 718-725Crossref PubMed Scopus (266) Google Scholar, 18Araujo-Jorge T. Rivera M.T. el Bouhdidi A. Daeron M. Carlier Y. Infect. Immun. 1993; 61: 4925-4928Crossref PubMed Google Scholar). The control rat IgG2b did not inhibit the killing (Fig. 1 c). In addition, 2.4G2 did not inhibit FasL VP-mediated killing (Fig.1 d). These results indicate that the acute cytotoxicity of Jo2 depends on FcγR binding. Because 2.4G2 blocked both FcγRII and FcγRIII, we determined whether Jo2 induces lethality in FcγRII KO mice (B6;129S-Fcgr2 tm1Rav) or FcγRIII KOmice (C57BL/6-Fcgr2 tm1Sjv). Control groups included B6129SF2/J, B6, and B6.MRL-Fas lpr mice. FcγRII KO mice were completely resistant to Jo2, including those treated with a high dose of Jo2 (5 mg/kg body weight, Table I). In contrast, almost all of the control B6;129SF2/Jmice died even when treated with a lower dose of Jo2 (1.5 mg/kg body weight). In addition, all of the FcγRIII KO mice died after injection of a lower dose of Jo2.B6.MRL-Fas lpr mice were resistant to the high dose of Jo2 (5 mg/kg body weight). Thus, Jo2-induced lethality requires the presence of FcγRII+ and Fas+ cells and correlates with FcγR-dependent cytotoxicity in vitro.Table IThe Jo2-induced lethality depends on host FcγRII expressionStrainJo2 dosageMortalitymg/kg·body weightdeath/totalB61.53/4B653/3B6;129SF2/J1.54/4FcγRII KO1.50/4FcγRII KO50/2FcγRIII KO1.53/3B6.MRL-Faslpr50/3Mice, 5–7 weeks old, were weighed and injected, intraperitoneally, with various doses of Jo2. Mortality was determined within 24 h after injection. The fatality was usually observed within 7 h. All survived mice were followed up for additional 3 days and all remained alive. Open table in a new tab Mice, 5–7 weeks old, were weighed and injected, intraperitoneally, with various doses of Jo2. Mortality was determined within 24 h after injection. The fatality was usually observed within 7 h. All survived mice were followed up for additional 3 days and all remained alive. To exclude the possibility that FcγRII KO mice are inherently resistant to death-inducing regimens that cause liver damage, they were tested with a bacterial peritonitis/endotoxin shock model in which lethality is induced by injecting d-Gal and LPS (15Leist M. Gantner F. Boblinger I. Tiegs G. Am. J. Pathol. 1995; 146: 1220-1234PubMed Google Scholar, 16Morikawa A. Sugiyama T. Kato Y. Koide N. Jiang G.Z. Takahashi K. Tamada Y. Yokochi T. Infect. Immun. 1996; 64: 734-738Crossref PubMed Google Scholar). The molecule responsible for the lethality is TNF-α (19Tiegs G. Wolter M. Wendel A. Biochem. Pharmacol. 1989; 38: 627-631Crossref PubMed Scopus (301) Google Scholar), which, like Jo2, induces caspase-dependent fulminant hepatic failure (20Bahjat F.R. Dharnidharka V.R. Fukuzuka K. Morel L. Crawford J.M. Clare-Salzler M.J. Moldawer L.L. J. Immunol. 2000; 165: 6559-6567Crossref PubMed Scopus (28) Google Scholar). In contrast to Jo2 treatment, however, FcγRII KO,B6;129SF2/J, and B6 mice were equally sensitive to this regimen and died within 7 h after treatment (Table II). Thus, FcγRII KOmice are specifically resistant to Jo2-induced lethality.Table IIJo2 elevates serum transaminase of B6 but not FcγRII KO miceStrainTreatmentMortalityASTALTdeath/totalB6Not treated0/346 ± 1039 ± 27B6Jo25/55070 ± 23694520 ± 2829FcγRII KOJo20/386 ± 1770 ± 18B6d-Gal/LPS3/32017 ± 15042340 ± 1502FcγRII KOd-Gal/LPS6/73283 ± 19566332 ± 1201B6;129SF2/Jd-Gal/LPS3/43163 ± 9783400 ± 2208Mice were either untreated, injected with Jo2 (3 mg/kg. body weight, intraperitoneally), or treated with d-Gal/LPS as described under “Materials and Methods.” Mortality developed within 3–7 h after injection. All moribund mice displayed severe hemorrhage in the liver. Sera were collected from moribund mice. Sera of viable mice were collected at 7 h after treatment. The serum levels (units/ml) of AST and ALT were determined with a commercial kit. Open table in a new tab Mice were either untreated, injected with Jo2 (3 mg/kg. body weight, intraperitoneally), or treated with d-Gal/LPS as described under “Materials and Methods.” Mortality developed within 3–7 h after injection. All moribund mice displayed severe hemorrhage in the liver. Sera were collected from moribund mice. Sera of viable mice were collected at 7 h after treatment. The serum levels (units/ml) of AST and ALT were determined with a commercial kit. The serum levels of ALT and AST increased dramatically in Jo2-susceptible mice but remained normal in Jo2-treated FcγRII KO mice (Table II). However, theFcγRII KO mice that succumbed to d-Gal/LPS treatment showed high levels of serum AST and ALT. Although the lethality correlated with hepatocyte damage as indicated by high serum transaminase activities, a complete concordance between hemorrhage in liver and lethality was also observed. Extensive hemorrhage in the liver was observed in Jo2-treated B6 mice andd-Gal/LPS-treated FcγRII KO mice but not in Jo2-treated FcγRII KO mice (Fig.2). Hemorrhage was selectively induced in liver but not in kidney (Fig. 2 a), spleen, thymus, intestine, or heart. Livers from B6 mice taken at 2 h after treatment showed congestion, infiltration of erythrocytes, distorted sinusoids, and hepatocyte death, whereas livers from Jo2-treated FcγRII KO mice displayed normal structure, similar to those observed in untreated mice (Fig. 2, b andc). These observations indicate that anFcγRII-dependent event that mediates lethality by Jo2 must occur before the death of hepatocytes and the hemorrhage in the liver. In the hematopoietic system, FcγRII is abundantly expressed on macrophages, B cells, neutrophils, eosinophils, and platelets (10Ravetch J.V. Bolland S. Annu. Rev. Immunol. 2001; 19: 275-290Crossref PubMed Scopus (1362) Google Scholar, 17Ravetch J.V. Luster A.D. Weinshank R. Kochan J. Pavlovec A. Portnoy D.A. Hulmes J. Pan Y.C. Unkeless J.C. Science. 1986; 234: 718-725Crossref PubMed Scopus (266) Google Scholar, 18Araujo-Jorge T. Rivera M.T. el Bouhdidi A. Daeron M. Carlier Y. Infect. Immun. 1993; 61: 4925-4928Crossref PubMed Google Scholar). In the liver, FcγRII is found on the sinusoidal lining cells but not on hepatocytes, and FcγRIII expression on the sinusoidal lining cells is controversial (11Muro H. Shirasawa H. Kosugi I. Nakamura S. Am. J. Pathol. 1993; 143: 105-120PubMed Google Scholar, 12Takezawa R.-I. Watanabe Y. Akaike T. J. Biochem. (Tokyo). 1995; 118: 1175-1183Crossref PubMed Scopus (43) Google Scholar). Because many FcγRII+ hematopoietic cells are radiosensitive, mice were treated with 600 rads of γ-irradiation and tested 4 days later for susceptibility to Jo2. Although irradiation eliminated more than 98% of the spleen and blood leukocytes, the mice remained susceptible to Jo2. Conversely, irradiated FcγRII KO mice remained resistant to Jo2 (TableIII). Irradiated FcγRII KO mice that had been reconstituted with B6 splenic cells 24 h earlier remained resistant to Jo2. In addition, we blocked FcγR in irradiated B6 mice with 2.4G2 and 2 h later injected Jo2. FcγR blockade prevented Jo2-induced death of irradiated mice. In contrast, irradiated mice that were either untreated or treated with normal rat Ig succumbed to Jo2 within 4–16 h (Table III). These observations indicate that under the experimental conditions, the critical cells controlling mouse susceptibility to Jo2 were radioresistant FcγRII+ cells.Table IIIAnti-FcγR mAb 2.4G2 blocks Jo2-induced lethalityStrainBlocking antibodyB6 splenic cellsMortalitydeath/totalB6NoneNone4/6B6Rat Ig (0.3 mg, intravenously)None3/4B62.4G2 (0.3 mg, intravenously)None0/5FcγRII KONoneNone0/3FcγRII KO aIrradiated FcγRII KO mice that had been reconstituted with B6 spleen cells 24 h earlier before the injection of Jo2.None108/mouse (intravenously)0/3Mice were irradiated with 600 rads and used 4 days later. Some mice were injected intravenously with 2.4G2 or control rat Ig 2 h before the injection of Jo2 (3 mg/kg body weight, intraperitoneally).a Irradiated FcγRII KO mice that had been reconstituted with B6 spleen cells 24 h earlier before the injection of Jo2. Open table in a new tab Mice were irradiated with 600 rads and used 4 days later. Some mice were injected intravenously with 2.4G2 or control rat Ig 2 h before the injection of Jo2 (3 mg/kg body weight, intraperitoneally). Because FcγR express in sinusoidal lining cells but not hepatocytes, the critical requirement of FcγRII for Jo2-induced lethality strongly suggests that the primary targets of Jo2 are sinusoidal lining cells. As sinusoidal lining cells separate blood from liver mesenchyma, destruction of sinusoids should lead to hemorrhage. According to this interpretation, hepatocyte death is secondary to sinusoid destruction. Consequently, hepatocyte death should be observed around damaged sinusoids but not in areas where sinusoids remain intact. TUNEL assays indicated that the sinusoidal lining cells become apoptotic at 1 h after injection of Jo2 (Fig.3, top versus middle panels). At 2 h after injection, focal hemorrhage and more cells, including apoptotic hepatocytes, became apparent around the damaged sinusoids (Fig. 3, bottom panel). Jo2-induced toxicity of hepatocytes is prevalent around damaged sinusoids. By contrast, hepatocytes around areas where destruction of sinusoids was not induced did not become apoptotic. The data suggest that the FcγRII-dependent destruction of sinusoids is the primary mechanism for Jo2-induced lethality by causing severe hemorrhage in liver and hepatocyte apoptosis. Our study supports a model in which the critical targets of Jo2 are the sinusoidal lining cells that co-express FcγRII and Fas. As sinusoidal lining cells are the gateway for blood components to enter liver parenchyma, our data indicate that Jo2 travels through the blood, encounters sinusoidal lining cells, and delivers a Fas-mediated apoptotic signal that is dependent on target FcγRII expression. Under in vitro conditions, we showed that the co-engagement of Fas and FcγRII provided a strong apoptotic signal, and presumably the FcγR-engaged Jo2 could enhance binding and resist endocytosis. Under in vivo conditions and within 1 h of application, Jo2 induces the detachment of sinusoidal lining cells and inhibits their microvascular perfusion function (21Wanner G.A. Mica L. Wanner-Schmid E. Kolb S.A. Hentze H. Trentz O. Ertel W. FASEB J. 1999; 13: 1239-1248Crossref PubMed Scopus (48) Google Scholar, 22.Deleted in proof.Google Scholar). Within the next hour, nuclear condensation in the sinusoidal lining cells and congestion/extravasation of erythrocytes becomes obvious (21Wanner G.A. Mica L. Wanner-Schmid E. Kolb S.A. Hentze H. Trentz O. Ertel W. FASEB J. 1999; 13: 1239-1248Crossref PubMed Scopus (48) Google Scholar) (Figs. 2 and 3). Death of sinusoidal lining cells could damage vascular integrity, resulting in focal hemorrhage. Our data suggest that focal hemorrhage induces secondary damage to hepatic parenchymal cells and further loss of sinusoid integrity. As the process progresses, the liver becomes severely hemorrhagic. Massive bleeding is always observed in the livers of moribund mice and is coincident with death. Together with the FcγR-dependent induction of apoptosis of targetsin vitro, our in vivo study provides strong evidence that hemorrhage in the liver is responsible for the lethality induced by Jo2. Transgenic mice in which the bcl-2 transgene is controlled by a pyruvate kinase regulatory sequence are resistant to Jo2 (23Lacronique V. Mignon A. Fabre M. Viollet B. Rouquet N. Molina T. Porteu A. Henrion A. Bouscary D. Varlet P. Joulin V. Kahn A. Nat. Med. 1996; 2: 80-86Crossref PubMed Scopus (348) Google Scholar). In contrast, transgenic mice in which the bcl-2 transgene is linked with the α1-antitrypsin promoter (pAAT-bcl-2) are susceptible (9Huang D.C.S. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (274) Google Scholar, 24Rodriguez I. Matsuura K. Ody C. Nagata S. Vassalli P. J. Exp. Med. 1996; 183: 1031-1036Crossref PubMed Scopus (175) Google Scholar). Our model can explain this conundrum. The resistance is likely due to the expression of thebcl-2 transgene in sinusoidal lining cells because the pyruvate kinase regulatory sequence used is not cell type-specific. In contrast, the α1-antitrypsin promoter is hepatocyte-specific. Therefore, the sinusoidal lining cells in thepAAT-bcl-2 transgenic mice should remain susceptible to Jo2. Also of great relevance to the present study is the observation that anti-FLAG-aggregated FasL is lethal to the pATT-bcl-2 mice (9Huang D.C.S. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (274) Google Scholar), whereas recombinant sFasL alone is not even toward normal mice. According to our model, the anti-FLAG-aggregated FasL are focused onto sinusoidal lining cells by the co-engagement of Fas and FcγRII. Interestingly, lethality occurs with severe hemorrhage in liver but little apoptosis of hepatocytes in the pATT-bcl-2 mice (9Huang D.C.S. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (274) Google Scholar,24Rodriguez I. Matsuura K. Ody C. Nagata S. Vassalli P. J. Exp. Med. 1996; 183: 1031-1036Crossref PubMed Scopus (175) Google Scholar), providing further support that hepatocyte death is not necessary for lethality. In addition to the study using anti-FLAG-aggregated rsFasL, several other observations also support the critical role of FcγR in Jo2-induced lethality. At present, Jo2 is the only anti-Fas mAb capable of inducing lethal fulminant hepatitis. The acute FcγR-facilitated cytotoxicity was not reported for other anti-Fas mAbs that do not induce lethal fulminant hepatitis (7Nishimura Y. Kirabayashi Y. Matsuzaki Y. Musette P. Ishii A. Nakauchi H. Inoue T. Yonehara S. Int. Immunol. 1996; 9: 307-316Crossref Scopus (64) Google Scholar). It appears that Jo2-induced lethality is unique to this mAb. Thus, FcγRII-nonreactive anti-Fas mAb and FasL and their derivatives may not induce hepatic toxicity, raising the possibility that they may be used as therapeutic agents. It should be noted, however, that lethality has been induced with an excessive amount of rsFasL (25Schneider P. Holler N. Bodmer J.-L. Hahne M. Frei K. Fontana A. Tschopp J. J. Exp. Med. 1998; 187: 1205-1213Crossref PubMed Scopus (694) Google Scholar, 26Tanaka M. Suda T. Yatomi Y. Nakamura N. Nagata S. J. Immunol. 1997; 158: 2303-2309PubMed Google Scholar), suggesting that the relative sensitivity of target tissues versus liver to FcγRII-nonreactive, Fas-binding agents is an important factor to consider for therapeutic purposes. Because sinusoidal lining cells form the barrier between blood and hepatocytes, their death leads to focal hemorrhage. As the welfare of hepatocytes depends on a functional barrier, hemorrhage-induced biochemical events and the loss of sinusoidal integrity may induce secondary damage to hepatocytes. This hypothesis could reconcile the strong resistance of hepatocytes to Jo2 in in vitro study and their rapid death upon Jo2 administration in vivo(3Ogasawara J. Fukunaga-Watanabe R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. Nagata S. Nature. 1993; 364: 806-809Crossref PubMed Scopus (1800) Google Scholar, 9Huang D.C.S. Hahne M. Schroeter M. Frei K. Fontana A. Villunger A. Newton K. Tschopp J. Strasser A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14871-14876Crossref PubMed Scopus (274) Google Scholar, 21Wanner G.A. Mica L. Wanner-Schmid E. Kolb S.A. Hentze H. Trentz O. Ertel W. FASEB J. 1999; 13: 1239-1248Crossref PubMed Scopus (48) Google Scholar). This hypothesis predicts that circulating Jo2 would attack sinusoidal lining cells first. Indeed, injected fluorescein isothiocyanate-Jo2 (100 μg intravenously) strongly stained sinusoidal lining cells but not hepatocytes, whereas fluorescein isothiocyanate-conjugated hamster Ig did not stain sinusoidal lining cells (data not shown). TUNEL assays indicated focal hemorrhage and apoptotic sinusoidal lining cells around the damaged sinusoids (Fig.3). Jo2-induced toxicity of hepatocytes apparently emanates from damaged sinusoids, and regions where sinusoids are intact the hepatocytes remained viable. Our study suggests that sinusoidal lining cells are the primary targets, and hepatocyte death is secondary to the destruction of sinusoids. This is reasonable because the welfare of hepatocytes depends on functional and intact sinusoids. The mechanism by which hepatocyte death is induced requires further study. In addition to Jo2-induced lethality, severe hemorrhage in the liver is observed in several other models of lethal hepatic failure. The acute liver toxicity of acetaminophen has been shown to be the result of a preferential toxicity toward sinusoidal endothelial cells (27DeLeve L.D. Wang X. Kaplowitz N. Shulman H.M. Bart J.A. van der Hoek A. Biochem. Pharmacol. 1997; 53: 133-1345Crossref Scopus (82) Google Scholar). Consistent with the present study, severe hemorrhage in liver was observed in moribund mice (27DeLeve L.D. Wang X. Kaplowitz N. Shulman H.M. Bart J.A. van der Hoek A. Biochem. Pharmacol. 1997; 53: 133-1345Crossref Scopus (82) Google Scholar). In the lethal fulminant hepatitis in which the apoptosis-inducing factor implicated is TNF-α or FasL, the lethality has been attributed to hepatocyte death (19Tiegs G. Wolter M. Wendel A. Biochem. Pharmacol. 1989; 38: 627-631Crossref PubMed Scopus (301) Google Scholar, 26Tanaka M. Suda T. Yatomi Y. Nakamura N. Nagata S. J. Immunol. 1997; 158: 2303-2309PubMed Google Scholar, 28Onda M. Willingham M. Wang Q.-C. Kreitman R.J. Tsutsumi Y. Nagata S. Pastan I. J. Immunol. 2000; 165: 7150-7156Crossref PubMed Scopus (59) Google Scholar). Our hypothesis suggests that hemorrhage is the underlying cause for lethality in these models. As with the FcγRII-mediated focusing of Jo2 on the sinusoidal lining cells, TNF-α produced by the LPS-activated Kupffer cells could preferentially attack the neighboring sinusoidal endothelial cells (20Bahjat F.R. Dharnidharka V.R. Fukuzuka K. Morel L. Crawford J.M. Clare-Salzler M.J. Moldawer L.L. J. Immunol. 2000; 165: 6559-6567Crossref PubMed Scopus (28) Google Scholar,28Onda M. Willingham M. Wang Q.-C. Kreitman R.J. Tsutsumi Y. Nagata S. Pastan I. J. Immunol. 2000; 165: 7150-7156Crossref PubMed Scopus (59) Google Scholar). Although the Jo2-induced lethality is an acute model for fulminant hepatitis, sinusoidal lining cells can be critical targets in chronic hepatic injury as well. Sinusoidal endothelial cells have been implicated as early targets in veno-occlusive disease observed in the setting of hematopoietic cell transplantation (29DeLeve L.D. Shulman H.M. McDonald G.B. Semin. Liver Dis. 2002; 22: 27-42Crossref PubMed Scopus (526) Google Scholar). In this setting, following injury of the sinusoidal endothelial cells, a series of biochemical processes lead to circulatory compromise of centrilobular hepatocytes, fibrosis, and obstruction of liver blood flow. Other clinical settings include the inflammation around sinusoidal lining cells in lpr bone marrow chimera in irradiatedMRL hosts (30Bobe P. Bonardelle D. Reynes M. Godeau F. Mahiou J. Joulin V. Kiger N. J. Immunol. 1997; 159: 4197-4202PubMed Google Scholar), the reperfusion injury of sinusoidal lining cells associated with liver transplantation (31Natori S. Selzner M. Valentino K.L. Fritz L.C. Srinivasan A. Clavien P.A. Gores G.J. Transplantation. 1999; 68: 89-96Crossref PubMed Scopus (212) Google Scholar), and liver hemorrhage observed after a successful post-operative period in patients of liver transplantation (32Hubscher S.G. Adams D.H. Buckels J.A. McMaster P. Neuberger J. Elias E. J. Clin. Pathol. 1989; 42: 360-370Crossref PubMed Scopus (41) Google Scholar). Finally, recent studies (6Rodriguez I. Matsuura K. Ody C. Nagata S. Vassalli P. J. Exp. Med. 1996; 184: 2067-2072Crossref PubMed Scopus (271) Google Scholar, 21Wanner G.A. Mica L. Wanner-Schmid E. Kolb S.A. Hentze H. Trentz O. Ertel W. FASEB J. 1999; 13: 1239-1248Crossref PubMed Scopus (48) Google Scholar) have shown that caspase inhibitors can block the lethality and hepatocyte death induced by Jo2 and TNF-α. Our study indicates that these inhibitors could potentially be used to treat certain hepatic injuries such as peritonitis or perfusion-induced liver damage by protecting the sinusoidal lining cells and hepatocytes from apoptosis.
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