Anti-CD95-induced Lethality Requires Radioresistant FcγRII+ Cells

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.

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 (7)(8)(9). 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 (3,(7)(8)(9). 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 (3). 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 (7). 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 Fc␥R expression. Both hepatocytes and sinusoidal lining cells express Fas, but only the latter express Fc␥R (10 -12). Because a co-engagement of Fas and Fc␥R can facilitate binding and strengthen the subsequent signaling process, we asked the question of whether the Jo2induced lethality requires co-engagement of Fas and Fc␥R. We found that Jo2-induced lethality is critically dependent on host Fc␥RII 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 Fc␥RII binding activity as useful therapeutic agents.

MATERIALS AND METHODS
Cytotoxicity Assays-Cytotoxicity assays against the Fc␥R ϩ A20 and the Fc␥R Ϫ variant IIA1.6 target cells (obtained from Dr. C. J. Janeway, Yale University, New Haven, CT) were conducted as described previously (13). Target cells (2 ϫ 10 4 ), labeled with Na 2 51 CrO 4 (PerkinElmer Life Sciences), were cultured with various doses of Jo2 (BD Biosciences) or FasL vesicle preparation (FasL VP) (13,14) 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 51 Cr release, which is determined by the formula: 100% ϫ (experimental release Ϫ background release)/(total cpm released by 0.5% Nonidet P-40 Ϫ 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) (14).
Jo2-induced Lethality-B6, B6;129SF2/J, B6;129S-Fcgr2 tm1Rav (Fc␥RII KO), C57BL/6-Fcgr3 tm1Sjv (Fc␥RIII 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-treated B6 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).
Radioresistance of the Target of Jo2-To determine the role of Fc␥R and target radiosensitivity in the Jo2-induced lethal fulminant hepatitis, B6 and Fc␥RII 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 with B6 splenic cells (10 8 /mouse) by intravenous injection of single cell suspension prepared from B6 spleen cells. In other experiments, mice were injected intravenously with purified, Fc␥R-specific 2.4G2 or control rat Ig (Jackson ImmunoResearch, Atlanta, GA) 2 h before the intraperitoneally injection of Jo2.
Treatment with D-Gal Plus Lipopolysaccharide (LPS)-Both B6 and Fc␥RII KO mice, 3 mice per group, were injected with D-Gal (0.5 g/kg body weight) plus bacterial LPS (50 g/kg body weight) intraperitoneally as described (15,16). 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.

Fc␥R Expression on Target Cells
Regulates the Cytotoxicity of Jo2-We tested Jo2 anti-Fas mAb for Fc␥R-dependent cytotoxicity using 51 Cr-labeled, Fc␥R ϩ A20 and Fc␥RII Ϫ variant IIA1.6 cells (derived from A20) as targets in a 5-h cytotoxicity assay (Fig. 1). Jo2 effectively killed Fc␥R ϩ A20 cells but not IIA1.6 cells (Fig. 1a). Both targets were equally sensitive to FasL VP, the apoptosis-inducing membrane vesicles that induce cell death independent of target Fc␥R expression (Fig. 1b). In addition, the killing of Fc␥R ϩ A20 targets by Jo2 was inhibited by 2.4G2 mAb that blocks Fc binding to Fc␥RII and Fc␥RIII (17,18). The control rat IgG2b did not inhibit the killing (Fig. 1c). In addition, 2.4G2 did not inhibit FasL VPmediated killing (Fig. 1d). These results indicate that the acute cytotoxicity of Jo2 depends on Fc␥R binding.
Fc␥RII KO Mice Are Resistant to Jo2-Because 2.4G2 blocked both Fc␥RII and Fc␥RIII, we determined whether Jo2 induces lethality in Fc␥RII KO mice (B6;129S-Fcgr2 tm1Rav ) or Fc␥RIII KO mice (C57BL/6-Fcgr2 tm1Sjv ). Control groups included B6129SF2/J, B6, and B6.MRL-Fas lpr mice. Fc␥RII 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/J mice died even when treated with a lower dose of Jo2 (1.5 mg/kg body weight). In addition, all of the Fc␥RIII 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 Fc␥RII ϩ and Fas ϩ cells and correlates with Fc␥R-dependent cytotoxicity in vitro.
Fc␥RII KO Mice Are Sensitive to D-Gal/LPS-induced Lethality-To exclude the possibility that Fc␥RII 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 (15,16). The molecule responsible for the lethality is TNF-␣ (19), which, like Jo2, induces caspase-dependent fulminant hepatic failure (20). In contrast to Jo2 treatment, however, Fc␥RII KO, B6;129SF2/J, and B6 mice were equally sensitive to this regimen and died within 7 h after treatment (Table II). Thus, Fc␥RII KO mice are specifically resistant to Jo2-induced lethality.
Serum Transaminase and Liver Hemorrhage Are Not Induced in Jo2-treated Fc␥RII KO Mice-The serum levels of ALT and AST increased dramatically in Jo2-susceptible mice but remained normal in Jo2-treated Fc␥RII KO mice (Table II). However, the Fc␥RII 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 and D-Gal/LPS-treated Fc␥RII KO mice but not in Jo2-treated Fc␥RII KO mice (Fig. 2). Hemorrhage was selec-FIG. 1. Jo2 mediates Fc␥R-dependent cytotoxicity. Varying concentrations of Jo2 mAb (a) or FasL-bearing vesicles (FasL VP) (b) were used to kill Fc␥R ϩ target A20 and the Fc␥R Ϫ target IIA1.6 in a 5-h cytotoxicity assay. The anti-Fc␥R mAb 2.4G2 (300 ng/ml) but not control rat IgG2b inhibited the killing of A20 by Jo2 (c). 2.4G2 did not inhibit the killing of A20 by FasL VP (d). tively induced in liver but not in kidney (Fig. 2a), 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 Fc␥RII KO mice displayed normal structure, similar to those observed in untreated mice (Fig. 2, b and c). These observations indicate that an Fc␥RII-dependent event that mediates lethality by Jo2 must occur before the death of hepatocytes and the hemorrhage in the liver.
The Target Cells of Jo2 Responsible for Lethality Are Radioresistant-In the hematopoietic system, Fc␥RII is abun-dantly expressed on macrophages, B cells, neutrophils, eosinophils, and platelets (10,17,18). In the liver, Fc␥RII is found on the sinusoidal lining cells but not on hepatocytes, and Fc␥RIII expression on the sinusoidal lining cells is controversial (11,12). Because many Fc␥RII ϩ hematopoietic cells are radiosensitive, mice were treated with 600 rads of ␥-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 Fc␥RII KO mice remained resistant to Jo2 (Table III). Irradiated Fc␥RII KO mice that had been reconstituted with B6 splenic cells 24 h earlier remained resistant to Jo2. In addition, we blocked Fc␥R in irradiated B6 mice with 2.4G2 and 2 h later injected Jo2. Fc␥R 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 Fc␥RII ϩ cells.
Coincidental Display of Sinusoid Destruction, Hemorrhage, and Hepatocyte Death-Because Fc␥R express in sinusoidal lining cells but not hepatocytes, the critical requirement of Fc␥RII 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 Fc␥RII-dependent destruction of sinusoids is the primary mechanism for Jo2-induced lethality by causing severe hemorrhage in liver and hepatocyte apoptosis.

A Novel Mechanism for Jo2-induced Lethal Fulminant
Hepatitis-Our study supports a model in which the critical targets of Jo2 are the sinusoidal lining cells that co-express Fc␥RII 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 Fc␥RII expression. Under in vitro conditions, we showed that the co-engagement of Fas and Fc␥RII provided a strong apoptotic signal, and presumably the Fc␥R-engaged Jo2 could enhance binding and resist endocytosis. Under in vivo conditions and within 1 h of application, Jo2 induces the de-tachment of sinusoidal lining cells and inhibits their microvascular perfusion function (21). Within the next hour, nuclear condensation in the sinusoidal lining cells and congestion/extravasation of erythrocytes becomes obvious (21) (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 Fc␥R-dependent induction of apoptosis of targets in 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 (23). In contrast, transgenic mice in which the bcl-2 transgene is linked with the ␣ 1 -antitrypsin promoter (pAAT-bcl-2) are susceptible (9,24). Our model can explain this conundrum. The resistance is likely due to the expression of the bcl-2 transgene in sinusoidal lining cells because the pyruvate kinase regulatory sequence used is not cell type-specific. In contrast, the ␣ 1 -antitrypsin promoter is hepatocyte-specific. Therefore, the sinusoidal lining cells in the pAAT-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 (9), 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 Fc␥RII. Interestingly, lethality occurs with severe hemorrhage in liver but little apoptosis of hepatocytes in the pATT-bcl-2 mice (9,24), providing further support that hepatocyte death is not necessary for lethality.
In addition to the study using anti-FLAG-aggregated rs-FasL, several other observations also support the critical role of Fc␥R in Jo2-induced lethality. At present, Jo2 is the only anti-Fas mAb capable of inducing lethal fulminant hepatitis. The acute Fc␥R-facilitated cytotoxicity was not reported for other anti-Fas mAbs that do not induce lethal fulminant hepatitis (7). It appears that Jo2-induced lethality is unique to this mAb. Thus, Fc␥RII-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 (25,26), suggesting that the relative sensitivity of target tissues versus liver to Fc␥RII-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.  3. Sinusoid destruction proceeds with apoptosis of sinusoidal lining cells and coincident with hemorrhage and hepatocyte apoptosis. B6 mice were injected intraperitoneally with Jo2 (5 mg/kg body weight). Untreated B6 mice were used as control as 0 h of treatment (top panel). At 1 h after treatment (middle panel), TUNEL ϩ nuclei of sinusoidal lining cells (arrows) appear at sinusoidal margins, whereas the large nuclei of intact hepatocytes remain TUNEL Ϫ . At 2 h after treatment (lower panel), severe sinusoid destruction was observed. Apoptotic hepatocytes (arrows) with large and strongly stained nuclei were abundant around damaged sinusoids. Note the TUNEL ϩ nuclei of sinusoidal lining cells are smaller than the TUNEL ϩ hepatocytes. Hemorrhage, clusters of erythrocytes, and breakdown of hepatic architecture were evident around the damaged sinusoids. Hepatocyte apoptosis and focal hemorrhage occurred mainly around the damaged sinusoids. Magnification, ϫ200. This hypothesis could reconcile the strong resistance of hepatocytes to Jo2 in in vitro study and their rapid death upon Jo2 administration in vivo (3,9,21). This hypothesis predicts that circulating Jo2 would attack sinusoidal lining cells first. Indeed, injected fluorescein isothiocyanate-Jo2 (100 g 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.
Implication for Sinusoidal Lining Cells as Critical Targets for Hepatic Damage-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 (27). Consistent with the present study, severe hemorrhage in liver was observed in moribund mice (27). In the lethal fulminant hepatitis in which the apoptosis-inducing factor implicated is TNF-␣ or FasL, the lethality has been attributed to hepatocyte death (19,26,28). Our hypothesis suggests that hemorrhage is the underlying cause for lethality in these models. As with the Fc␥RII-mediated focusing of Jo2 on the sinusoidal lining cells, TNF-␣ produced by the LPS-activated Kupffer cells could preferentially attack the neighboring sinusoidal endothelial cells (20,28). 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 (29). 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 irradiated MRL hosts (30), the reperfusion injury of sinusoidal lining cells associated with liver transplantation (31), and liver hemorrhage observed after a successful post-operative period in patients of liver transplantation (32). Finally, recent studies (6,21) have shown that caspase inhibitors can block the lethality and hepatocyte death induced by Jo2 and TNF-␣. 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.