Caspase 9–dependent killing of hepatic stellate cells by activated Kupffer cells

GASTROENTEROLOGY 2002;123:845– 861

Caspase 9 –Dependent Killing of Hepatic Stellate Cells by Activated Kupffer Cells RICHARD FISCHER, ALEXANDRA CARIERS, ROLAND REINEHR, and DIETER HA¨USSINGER Department of Gastroenterology, Hepatology and Infectiology, Medizinische Einrichtungen der Heinrich–Heine Universita¨t, Du¨sseldorf, Germany

Background & Aims: Hepatic stellate cells play an important role in liver fibrogenesis, and hepatic stellate cell death may be involved in the termination of this response. Methods: Molecular mechanisms of hepatic stellate cell killing were studied in hepatic stellate cell/ Kupffer cell cocultures. Results: Lipopolysaccharide stimulation of hepatic stellate cell/Kupffer cell cocultures, but not of hepatic stellate cell monocultures, induced profound alterations of hepatic stellate cell morphology and hepatic stellate cell death. Kupffer cell– induced hepatic stellate cell killing required hepatic stellate cell/Kupffer cell contacts and was prevented by dexamethasone, prostaglandin E2, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor 2 antagonists, and down-regulation of receptor-interacting protein, but not by antioxidants, tumor necrosis factor receptor, or CD95 antagonists. Hepatic stellate cell death was characterized by activation of caspases 3, 8, and 9, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling negativity, lack of gross calcium overload, and TRAIL trafficking to the plasma membrane. Inhibition of caspase 9, but not of caspases 3, 8, or 10, prevented hepatic stellate cell death. Lipopolysaccharide induced a dexamethasoneand prostaglandin E2–sensitive expression of TRAIL in Kupffer cells. TRAIL receptors 1 and 2, FLIP (caspase 8 –inhibitory protein), and receptor-interacting protein were up-regulated during hepatic stellate cell transformation; however, TRAIL addition did not induce hepatic stellate cell death. Hepatic stellate cell susceptibility toward Kupffer cell–induced death paralleled receptorinteracting protein and TRAIL-receptor expression levels. Conclusions: Activated Kupffer cell can effectively kill hepatic stellate cell by a caspase 9 – and receptorinteracting protein– dependent mechanism, possibly involving TRAIL. The data may suggest a novel form of hepatic stellate cell death.

epatic stellate cells (HSC) play an important role in the pathogenesis of liver disease.1–3 In the injured liver and during culture, quiescent HSC transform to myofibroblast-like cells, which are characterized by increased proliferation and contractility,4,5 altered gene

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expression,6 –12 and increased production of extracellular matrix proteins.2,3,9 Activation and transformation of HSC into myofibroblast-like cells corresponds to a wound-healing response, whose termination is thought to involve HSC apoptosis.3 However, the pathways of HSC death are poorly understood. Activated HSC express both the CD95 receptor and ligand, and CD95mediated apoptosis was reported to occur in parallel with HSC activation.10 However, cultured HSC are fairly resistant against CD95-mediated apoptosis11,12 unless cycloheximide is present, but transformation of HSC to myofibroblast-like cells was paralleled by an increasing sensitivity toward soluble CD95 ligand (CD95L)–mediated apoptosis, which was related to a strong decrease of Bcl-2 and Bcl-XL expression.12 Transforming growth factor ␤ and tumor necrosis factor (TNF)-␣ inhibit both proliferation and apoptosis in activated HSC in vitro.13 More recently, ligands of the peripheral type benzodiazepine receptor were shown to be potent inducers of HSC apoptosis, which involves inactivation of protein kinase B/Akt, dephosphorylation of Bad, and an early collapse of the mitochondrial membrane potential.11 HSC apoptosis is also induced by gliotoxin14 or rapamycin.15 However, nothing is known about the role of liver macrophages (Kupffer cells; KC) and other apoptosis-inducing systems, such as TNF–related apoptosis-inducing ligand (TRAIL), for HSC apoptosis. Abbreviations used in this paper: CD95L, CD95 ligand; DMEM, Dulbecco’s modified Eagle medium; Erk, extracellular regulated kinase; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; FLIP, caspase 8 –inhibitory protein; GFAP, glial fibrillary acidic protein; HSC, hepatic stellate cell; KC, Kupffer cells; KHB, Krebs–Henseleit buffer; LPS, lipopolysaccharide; PGE2 , prostaglandin E2; PI, propidium iodide; RIP, receptor interacting protein; SMA, smooth muscle actin; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; TRAIL-R1, TRAIL receptor 1; TRITC, tetramethylrhodamine isothiocyanate; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; Z-VAD-FMK, benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.35384

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TRAIL was identified on the basis of sequence homology to other members of the TNF family.16 Binding of TRAIL to TRAIL receptors 1 and 2 (TRAIL-R1 and -R2) can induce apoptosis, whereas TRAIL-R3 and R4 act as putative decoy receptors and do not transmit the death signal.17 The TRAIL system has received interest by its selective antitumor activity in mice.18 Here, systemic application of TRAIL did not seem toxic to normal tissues, in contrast to the fulminant hepatotoxicity induced by CD95L administration.17 TRAIL expression was found on the surface of lipopolysaccharide (LPS)and interferon gamma–stimulated monocytes and macrophages,19,20 stimulated T cells,21 and natural killer cells.22 The death domain kinase receptor-interacting protein (RIP) was shown to be essential for TRAIL- or TNF-␣–induced activation of I␬B kinase and c-Jun Nterminal kinase, but it is not involved in TRAIL-induced apoptosis in mouse fibroblasts.23 However, recent data suggest a role for RIP-1 as an effector molecule, which triggers an alternative, caspase 8 –independent, probably necrotic cell death pathway, which is activated by CD95L, TNF-␣, or TRAIL.24 In addition, a novel nonapoptotic form of programmed cell death, called paraptosis, was recently described.25 This pathway requires caspase 9 activity and is characterized by cycloheximide sensitivity, cytoplasmic vacuolation, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) negativity, and a lack of effect of caspase inhibitors. We therefore studied the role of KC, RIP kinase, and the TRAIL system in HSC death. The data show that LPS stimulation of KC induces rapid HSC death in coculture with features neither fitting to typical apoptosis nor necrosis and involving RIP, caspase 9, and possibly TRAIL.

Materials and Methods Materials Dulbecco’s modified Eagle medium (DMEM) and fetal calf serum (FCS) were from PAA Laboratories (Linz, Austria), and penicillin/streptomycin from Biochrom (Berlin, Germany). Nycodenz was from Nycomed (Oslo, Norway). Pronase was from Merck (Darmstadt, Germany). Deoxyribonuclease I and collagenase were from Boehringer (Mannheim, Germany). Antibodies against ␣-smooth muscle actin (␣-SMA), bovine serum albumin, propidium iodide (PI)– and tetramethylrhodamine isothiocyanate (TRITC)/fluorescein isothiocyanate (FITC)– conjugated phalloidin, LPS, prostaglandin E2 (PGE2), superoxiddismutase, 4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxyde 14-hydroxy-TEMPO, cycloheximide, zymosan A, dexamethasone, octanol, N-acetyl-L-cysteine, catalase, actinomycin D, uridine triphosphate, and L-N 5-(1-iminoethyl) ornithine were obtained from Sigma (Deisenhofen, Germany).

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Antibodies against CD95 receptor were from Santa Cruz Biotech (Santa Cruz, CA). Soluble CD95L was from Alexis Corp. (Gru¨nberg, Germany) and was used together with 1 ␮g/mL of enhancer protein. Cyclosporin A, recombinant CD95/Fc-immunoglobulin fusion molecule, TRAIL-R2:Fc-immunoglobulin fusion molecule, c-Jun N-terminal kinase peptide inhibitor 1 (L-JNK-inhibitor), TRAIL monoclonal antibodies, recombinant TRAIL, D2R ([L-Asp]2 rhodamine 110), and trolox were from Alexis Corp. Recombinant TRAIL from R&D (Wiesbaden, Germany) was also examined and gave the same results, as did recombinant TRAIL purchased from TEBU (Frankfurt, Germany). The caspase assays and inhibitors, the TUNEL assay, and the kit for detection of mitochondrial membrane potential disruption, TNF-␣, and anti–TNF-␣ monoclonal antibodies were from R&D. Annexin V FITC was from Bender Med. Systems (Vienna, Austria). All fluorochrome-conjugated secondary antibodies were from Jackson Corp. (West Grove, PA). Horseradish peroxidase– conjugated anti-mouse IgG, antirabbit IgG, and the Bio-Rad protein assay were from Bio-Rad (Hercules, CA); the enhanced chemiluminescence detection kit was from Amersham (Braunschweig, Germany). Interferon gamma was from Biosource (Camarillo, CA). Geldanamycin, the mitogen-activated protein kinase kinase inhibitor PD98059, and NG-monomethyl-L-arginine were from Calbiochem (Bad Soden, Germany). Formaldehyde and uric acid were from Sigma-Aldrich (Steinheim, Germany). Mouse anti-rat ED-1/FITC and mouse anti-rat macrophage MCA343 were from Serotec (Oxford, UK). TNF-␣ receptor antagonists were from Biomol (Hamburg, Germany). Fura-2 AM (acetomethyl ester) was from Molecular Probes (Eugene, OR). Antagonistic antibodies against TRAIL-R1 and -R2 were kindly provided by Prof. Dr. Harald Wajant (Institute of Cell Biology and Immunology, University of Stuttgart, Germany).

Isolation and Culture of HSC HSC from 1- to 3-year-old male Wistar rats were prepared and cultured as previously described4 by collagenase/ pronase perfusion and isolated by Nycodenz gradient. The cells were seeded at a density of 0.15 ⫻ 106/cm2 on glass coverslips in a 24-well culture plate or 60-mm well (Falcon, Heidelberg, Germany) and maintained in DMEM containing 10% (vol/vol) heat-inactivated FCS and 1% (wt/vol) penicillin/streptomycin. The purity of HSC was ⬎95%, as assessed 24 hours after seeding by their typical light-microscopic appearance and vitamin A–specific autofluorescence, their inability to phagocytose fluorescent 1.1-␮m latex particles, and their positive immunofluorescent staining for desmin, glial fibrillary acidic protein (GFAP), and, beyond the seventh day of culture, for ␣-SMA. Experiments were performed in DMEM containing 1% (vol/vol) FCS and 1% (wt/vol) penicillin/streptomycin.

Isolation and Culture of KC: Coculture KC were prepared by collagenase/pronase perfusion and separated by a single Nycodenz gradient and centrifugal elutriation, as described previously.26 The purity of KC was ⬎95%, as assessed 24 hours after seeding by their positive

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immunofluorescent staining for ED-1 and anti-macrophage antibodies. KC culture conditions were identical to those for HSC. For coculture experiments, KC were plated on HSC and maintained in DMEM containing 10% FCS and 1% penicillin/ streptomycin. Experiments were started after 48 hours of coculture by using DMEM medium containing 1% heatinactivated fetal bovine serum. If not stated otherwise, HSC, which had been in monoculture for 14 days, were cocultured with KC at a KC/HSC ratio of 2.

In Vivo Studies Male Wistar rats (200 –250 g) received an initial dose of CCl4 (150 ␮L/100 g body weight in paraffin oil 1:1) intraperitoneally followed by 2 injections per week intraperitoneally of CCl4 (100 ␮L/100 g), as described previously.11 Control was performed by injecting the vehicle (paraffin oil). Groups of 3 CCl4-treated animals and single controls were killed after 3 weeks of treatment. Acute in vivo LPS challenge was performed by intraperitoneal injection of LPS (4 mg/kg body weight) 12 hours before removal of the livers. All rats were fed ad libitum on stock diet and held according to the local ethical guidelines. Livers were removed and used for immunohistochemistry.

Determination of Protein Expression Specific cellular protein expression was determined with Western blotting as described previously.11 Proteincontaining blots were blocked in 5% (wt/vol) bovine serum albumin– containing 20 mmol/L Tris, pH 7.5; 150 mmol/L NaCl; and 0.1% Tween 20 and then incubated at 4°C for 2 hours with the first antibody (dilution 1:20,000; antibodies used: RIP, c–FLIP [caspase 8]–inhibitory protein, phosphoextracellular regulated kinase (Erk)-1/Erk-2, TRAIL-R1 to -R4, TRAIL, and CD95L). After extensive washing with 0.1% Tween in Tris-buffered saline and incubation with horseradish peroxidase– coupled anti-mouse IgG, anti-goat IgG, or antirabbit IgG antibody (dilution 1:20,000) at 4°C for 2 hours, the blot was washed extensively and developed with enhanced chemiluminescent detection (Amersham). Blots were exposed to Kodak X-OMAT AR-5 film (Eastman Kodak Co., Rochester, MN) for 1–30 minutes. Suitably exposed films were then analyzed by densitometry scanning (Kodak digital image station 440CF).

Measurement of Intracellular Calcium Cells were washed with KHB (Krebs–Henseleit buffer) 1 hour before calcium measurements and loaded with 5 ␮mol/L Fura-2 AM for 30 minutes. After the loading period, the coverslips were mounted in a perfusion chamber on an inverted fluorescence microscope (Zeiss, Oberkochen, Germany) and continuously superfused with KHB at a rate of 10 mL/min. The KHB was equilibrated with oxygen and CO2 (95:5; vol/vol). The temperature was kept at 37°C. One single cell per coverslip was investigated; determination of intracellular calcium was performed as previously described.4

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Immunofluorescence Staining of ␣-SMA, ED-1, RIP, TRAIL-R1, and CD95 Cells were fixed with 4% formaldehyde in phosphate buffer for 10 minutes and subsequently incubated with Triton X-100 (0.1% in phosphate-buffered saline [PBS]) for 5 minutes. After washing, the cells were incubated for at least 2 hours with rabbit anti–␣-SMA (1:200), mouse anti–ED-1 antibody (1:200), mouse anti–TRAIL-R1 (1:200), and mouse anti-RIP (1:200). The cells were extensively washed and incubated for 2 hours with FITC-conjugated anti-mouse IgG for labeling of RIP and TRAIL-R1, FITC-conjugated anti-rabbit IgG for detection of ␣-SMA, and Cy3-conjugated anti-mouse IgG for ED-1. Control cells were processed without initial incubation with the specific antibody. For determination of membrane surface targeting of CD95, cells were pretreated for 24 hours with 0.5 ␮mol/L cycloheximide and then exposed to 100 ng/mL CD95L plus 1 ␮g/mL enhancer for 1 hour. CD95 or TRAIL-R1 were stained in unpermeabilized cells for 45 minutes at 4°C with a rabbit anti-CD95 (1:500) and a Cy3-labeled goat anti-rabbit antibody (1:500). Cells were washed in PBS supplemented with 2% FCS and 0.1% NaN3. Microscopy was performed with a confocal laser scanning system (argon/krypton laser; Leica, Heidelberg, Germany). Images were acquired from 1 or 2 channels at 488 nm (for FITC) and 568 nm (for Cy3 and TRITC) wavelengths. Colocalization of the green (FITC) and red (Cy3) fluorescent dyes results in an orange to yellow signal. PI yields a red signal at both excitation wavelengths. Alternatively, immunostained cell samples were analyzed with an Axioskop (Zeiss, Jena, Germany) with a 3CCD Camera (Intas, Go¨ttingen, Germany).

Detection of Apoptosis and Necrosis The in vitro caspase 3, 8, and 9 assays measure the colorimetric reaction of the cleavage of the amino acid motif Ast-Glu-Val-Ast, Ile-Glu-Thr-Asp, and Leu-Glu-His-Asp, respectively, thereby releasing the chromophore p-nitroanilide, which was quantitated spectrophotometrically at a wavelength of 405 nm. Data were corrected for background (no substrate or no cell lysate) and expressed as percentage of activity found in untreated controls. Intracellular caspase 3 activity was measured in situ by fluorescence microscopy at an excitation wavelength of 488 nm and an emission at 550 nm, after loading the cells for 15 minutes with the caspase 3 substrate D2R ([L-Asp]2 rhodamine 110). For detection of DNA fragmentation, the TUNEL method was used according to the manufacturer’s protocol as previously described.11 Before the labeling reaction, cells were incubated with 0.5 ␮g/mL of FITC-conjugated phalloidin for detection of the cell shape. The number of apoptotic cells was determined by counting the percentage of TRITC-positive cells. At least 100 cells were counted for each experiment. Mitochondrial membrane potential was determined by using 5,5⬘,6,6⬘,7 tetrachloro-1,1⬘,4,4⬘tetraethylbenzimidazolyl carbocyaniniodide (JC-1), as described previously.11

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For determination of the loss of symmetry of cell membrane phospholipids as an early event in apoptosis, FITC-conjugated annexin V was used as described previously.11 PI was used as a marker of necrotic cells. Cells were incubated simultaneously with both reagents for 20 minutes and then subjected to fluorescence microscopy. Fluorescein-positive cells without nuclear PI staining were regarded as apoptotic, whereas fluorescein- and PI-positive cells were regarded as necrotic and differentiated from healthy cells by phase-contrast microscopy. KC were differentiated from HSC by their ability to phagocytose Cy-3–labeled microsphere latex particles and by their typical morphology.

Quantification of Cells HSC in monoculture or coculture with KC were fixed with formaldehyde (4% in PBS) and subsequently incubated with Triton X-100 (0.1% in PBS) for 5 minutes. Staining of F actin was performed by incubating the cells with phalloidin/ FITC (1 ␮g/mL) together with PI (4.4 ␮g/mL), which stains nucleic acids (20 minutes in PBS, respectively). HSC and KC were identified by their typical morphologies and quantified under a fluorescence microscope by counting 6 fields at magnification 40⫻. The number of HSC found in control incubations was set to 100%, and the HSC number found in experimental conditions was expressed as the percentage thereof. The difference in cell number counted in the control fields between different preparations was less than 10%. For all experiments, at least 3 independent HSC and KC preparations were used.

Analysis of Results and Statistics Data are expressed as means ⫾ SEM (n ⫽ number of cell preparations). SEM was omitted when smaller than symbol size. Each experiment was performed in at least 3 different cell preparations. Results were analyzed with the Student t test. P ⬍ 0.05 was considered statistically significant.

Results KC-Induced HSC Killing After LPS Stimulation HSC were kept in monoculture for 14 days and thereafter cocultured with KC for another 2 days. Then, experimental conditions were instituted for the time periods indicated. In the coculture system, KC were in close contact with HSC (Figure 1A). Whereas the number of HSC did not significantly change under control conditions, the addition of LPS led to a rapid loss of HSC in the cocultures (Figures 1B, C, and I–M and 2A). The number of HSC decreased by approximately 50%, 65%, and 90% in the cocultures within 12, 24, and 48 hours after LPS stimulation, respectively (Figure 2A). LPSinduced HSC death (14 days culture) in the cocultures was dependent on the LPS concentration used (Figure

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2B) and the relative density of KC in the cocultures (Figure 2C). However, LPS did not induce HSC killing or morphological changes of HSC, which were monocultured for 14 days in the absence of KC (not shown). Killing of HSC by LPS stimulation of KC/HSC cocultures was strongly dependent on the HSC preculture duration. When 1-day– cultured HSC were cocultured with KC for another 48 hours and were thereafter exposed for 24 hours to LPS, virtually no HSC killing was observed (not shown), whereas the same procedure with HSC being cultured for 7 or 14 days resulted in a decrease of HSC numbers to 58% ⫾ 12% and 37% ⫾ 1% (n ⫽ 3), respectively. These findings suggest that LPS-induced HSC killing by KC occurs only after HSC transformation into myofibroblasts. Thus, if not stated otherwise, the experiments described were performed with HSC, which were kept for 14 days in culture, before coculture with KC was started. Characteristics of KC-Induced HSC Killing As shown in Figure 1A, HSC in coculture with KC for 48 –96 hours showed the characteristic myofibroblast-like morphology (Figure 1A), indistinguishable from that found in HSC monocultures (see figure 1 in Peters-Regehr et al.8). On the addition of LPS, the HSC morphology changed remarkably in the cocultures (compare Figure 1C and D). Twenty-four hours after the addition of LPS, the remaining HSC had experienced loss of cytoplasm and showed a spider-like morphology, with long extensions containing cytoskeletal elements (Figure 1B, C, and I–M). Under these conditions ␣-SMA became detectable in the cytoplasm of KC, suggestive of phagocytosis of HSC-derived material (Figure 1B; compare Figure 1A). Some HSC with spider-like morphology were already visible 1–2 hours after LPS addition to the cocultures (Figure 1I ). Even after 24 hours of LPS treatment of the cocultures, some of these spider-like HSC were still viable with preserved mitochondrial membrane potential (Figure 3B) and had the potential to regain, within approximately 7 days, a normal myofibroblast morphology when LPS was removed from the cocultures (not shown). Dexamethasone (Figure 1H ) and PGE2 largely prevented HSC killing by LPS stimulation of the cocultures (Table 1) and the morphological alterations. LPS stimulation of the HSC/KC cocultures led to a loss of the mitochondrial membrane potential (Figure 3A and B) and the appearance of annexin V–positive cells (Figures 3D–F and 4). Approximately 50% of the annexin V–positive cells also stained positive for PI, suggesting that HSC death showed signs of necrosis (Figures 3D–F and 4). TUNEL positivity was found in less than 1% of HSC in the LPS-treated HSC/KC

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Figure 1. Effect of LPS on HSC morphology in cocultures of HSC and KC. HSC were kept in culture for 14 days and subsequently cocultured with KC for 2 days. Thereafter the cocultures were treated for the time periods indicated with LPS (1 ␮g/mL) and/or other effectors. (A–D) Cells in cocultures were fixed and stained with antibodies against ␣-smooth muscle actin (SMA)/FITC (green), an HSC-specific marker, and propidium iodide for visualization of nuclei. White arrows indicate KC and blue arrows, HSC; the bar corresponds to 20 ␮m. (A, D) Morphology of HSC and KC in coculture in the absence of LPS (control). KC were in close contact to HSC, which show typical myofibroblast morphology, indistinguishable from that observed in monocultured HSC (compare Figure 1A8). (B, C) On 24-hour exposure to LPS, the number of HSC has decreased considerably (Table 1; Figure 2), and the residual HSC developed a dendritic, spider-like morphology. Under these conditions KC contained green fluorescence, indicative of phagocytosis of HSC-derived material (B). (E–M) Cells in coculture were fixed and stained with phalloidin-TRITC (red) and FITC labeling (green) of DNA. (E) Unstimulated HSC/KC coculture (control). (F ) The caspase 8 inhibitor Z-IETD-FMK (50 ␮mol/L) does not prevent LPS-induced changes of HSC morphology (compare Figure 1M). (G, H) The caspase 9 inhibitor Z-LEHD-FMK (50 ␮mol/L) and dexamethasone (1␮mol/L) largely abolish the LPS-induced changes in HSC morphology and HSC killing. (I–M) Time course of the LPS-induced alterations of HSC density and morphology. Some spider-like HSC are already found after 1 hour of LPS treatment of the HSC/KC cocultures.

cocultures, whereas more than 90% of these cocultured HSC became TUNEL positive after apoptosis induction with CD95L plus cycloheximide (Figure 5A and B). These findings suggest that DNA fragmenta-

tion (as assessed by the TUNEL reaction) is not a prominent feature of LPS/KC-induced HSC killing but can be induced by CD95 ligation even on top of LPS-induced killing.

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Figure 2. LPS-induced HSC loss in HSC/KC coculture. HSC were kept in culture for 14 days and then cocultured with KC for another 2 days. Thereafter the cocultures were treated with LPS (1 ␮g/mL) for the time periods (A) or LPS concentrations (B) indicated. The KC/HSC ratio was 2:1. In (C), the effect of KC density on LPS-induced HSC loss is shown. Formaldehyde-fixed cells were stained with phalloidin FITC and propidium iodide and quantified under a fluorescence microscope. The mean ⫾ SEM is shown (n ⫽ 3). *Statistical significance of P ⬍ 0.05 with the Student t test compared with control. For further details, see Materials and Methods. (A) Time course of HSC disappearance after stimulation of KC/HSC cocultures with LPS. Cocultures of HSC and KC were incubated without (square symbols; control) or with LPS (1 ␮g/mL) (circles; LPS) for the time periods indicated. (B) Dependence of HSC disappearance on the LPS concentration. LPS was added for 24 hours (light bars) or 48 hours (dark bars). (C) Dependence of HSC disappearance on Kupffer cell density. KC and HSC were cocultured at the KC/HSC ratio indicated for 48 hours and thereafter exposed to LPS (1 ␮g/mL) for 24 hours. LPSinduced HSC disappearance was dependent on KC density.

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HSC disappearance in response to LPS in the KC/HSC cocultures was accompanied by an increase of caspase 3, 8, and 9 activity (Figure 6). Only inhibition of caspase 9, but not of caspases 3 and 8, could significantly prevent HSC killing (Table 1; Figure 1F and G). The pan-caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartic acidfluoromethylketone (Z-VAD-FMK) was similarly effective in preventing HSC death as the caspase 9 inhibitor (Table 1). Inhibition of caspase 3 or 8 did not prevent caspase 9 activation (Figure 6C). However, inhibition of caspase 9, but not of caspase 8, blocked caspase 3 activation under these conditions (Figure 6A), indicating that caspase 3 activation occurred in a caspase 9 – dependent, but caspase 8 –independent, way. Inhibition of caspase 3 or 9 completely blocked caspase 8 activation (Figure 6B). LPS-induced HSC killing in cocultures was not affected after the combined inhibition of caspases 8 and 10 (Table 1), whereas these inhibitors prevented the HSC apoptosis in response to CD95L plus cycloheximide (Table 2). Conversely, caspase 9 inhibition did not prevent CD95L/cycloheximide-induced apoptosis in HSC monoculture, in contrast to HSC killing by LPS stimulation of KC/HSC cocultures. Caspase 3 activation occurred in HSC, as shown in single-cell fluorescence experiments using the fluorescent caspase 3 substrate D2R (Figure 3C). Intracellular caspase 3 activation was completely blocked in the presence of the caspase 3 inhibitor Z-DEVD-FMK (not shown). This, however, did not prevent HSC killing (Table 1), indicating that caspase 3 activation is not essential for execution of HSC death by LPS-activated KC. Dexamethasone prevented the LPS-induced caspase 3 and 9 activation (Figure 6A and C), the decrease in HSC number in the coculture system (Table 1), and the alterations of HSC morphology (Figure 1H ). Likewise, inhibitors of protein synthesis, such as cycloheximide or actinomycin D, largely prevented the HSC killing in LPS-treated KC/HSC cocultures (Table 1). Role of KC-Derived Soluble Factors for HSC Killing by LPS-Stimulated KC LPS addition to HSC/KC coculture systems, in which KC and HSC were placed separately (by use of cell culture inserts) in a way that KC/HSC contacts, but not fluid exchange, were prevented, did not induce HSC death. Also, supernatants obtained from 24-hour LPStreated KC in monoculture were ineffective to trigger HSC death in HSC monocultures (not shown). These data indicate that long-lived soluble factors secreted by LPS-stimulated KC were not the immediate triggers for HSC killing.

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Figure 3. LPS treatment of KC/HSC cocultures leads in HSC to a loss of mitochondrial membrane potential (A, B), intracellular caspase 3 activation (C), and positive annexin V staining (D–F). Fourteen-day cultured HSC were cocultured with KC for another 2 days and thereafter treated with LPS (B, C, E, F) or control media (A, D) for 24 hours. For each fluorescent micrograph, phase contrast images are given. (A, B) Mitochondrial membrane potential was assessed by 5,5⬘,6,6⬘,7 tetrachloro-1,1⬘,4,4⬘-tetraethylbenzimidazolyl carbocyaniniodide (JC-1) staining. In the absence of LPS (A), HSC show a red punctate JC-1 staining, indicative of preserved mitochondrial membrane potential (MMP), whereas LPS exposure (B) led to the loss of MMP (arrow) in some, but not all HSC (B). (C) LPS treatment of the cocultures for 24 hours leads to caspase 3 activation in HSC. Intracellular caspase 3 activation was assessed by the fluorescence generated from the caspase 3 substrate D2R. (D–F ) Cells in coculture were incubated with annexin V FITC and propidium iodide. No annexin V staining is found in control cocultures (LPS absent; D), whereas after LPS treatment annexin V positivity develops (E, F; arrows) with negative (E) or positive (F; arrow) propidium iodide staining of the nuclei, indicative of the occurrence of both apoptotic (E) and necrotic (F ) HSC death.

In another set of experiments, monocultured KC were exposed to LPS for 8 hours and thereafter extensively washed and incubated for another 16 hours in LPS-free medium. When this latter supernatant was added to unstimulated KC/HSC cocultures, HSC killing was observed (Table 3). These findings indicate that LPS primes KC to produce a soluble mediator, which by itself does not induce HSC killing but can stimulate resting KC to do so. These data also suggest that HSC sensitization by

LPS is not required for the induction of KC-induced HSC death and that LPS acts primarily on KC. As further shown in Table 3, dexamethasone, but not geldanamycin, when added during the 8-hour LPS-priming period of monocultured KC, abrogated the killing potency of the 16-hour supernatant in unstimulated KC/HSC cocultures. These data suggest that prevention of HSC killing in LPS-stimulated KC/HSC cocultures by dexamethasone (Table 1) may reside in its effect on KC,

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Table 1. Modulation of LPS-Induced HSC Disappearance in KC/HSC Coculture Effector Control LPS (1 ␮g/mL) Interferon ␥ (100 U/mL) Zymosan A (0.5 mg/mL) LPS ⫹ dexamethasone (1 ␮mol/L) LPS ⫹ indomethacin (5 ␮mol/L) LPS ⫹ prostaglandin E2 (10 ␮mol/L) LPS ⫹ Z-VAD-FMK (pan-caspase inhibitor) LPS ⫹ Z-VDVAD-FMK (caspase 2 inhibitor) LPS ⫹ Z-DEVD-FMK (caspase 3 inhibitor) LPS ⫹ Z-VEID-FMK (caspase 6 inhibitor) LPS ⫹ Z-IETD-FMK (caspase 8 inhibitor) LPS ⫹ Z-LEHD-FMK (caspase 9 inhibitor) LPS ⫹ Z-AEVD-FMK (caspase 10 inhibitor) LPS ⫹ Z-IETD-FMK ⫹ Z-AEVD-FMK (caspase 8 plus caspase 10 inhibitor) LPS ⫹ geldanamycin (5 ␮mol/L) LPS ⫹ cycloheximide (0.5 ␮mol/L) LPS ⫹ actinomycin D (1 ␮mol/L) LPS ⫹ TNF-␣ receptor antagonist (10 ␮g/mL) LPS ⫹ anti–TNF-␣ antibody (50 ␮g/mL) LPS ⫹ anti-CD95 ligand antibody (10 ␮g/mL) LPS ⫹ CD95-Ig:Fc (5 ␮g/mL) LPS ⫹ cyclosporine A (1 ␮mol/L) LPS ⫹ TRAIL receptor-1 antagonistic antibody (10 ␮g/mL) LPS ⫹ TRAIL receptor-2 antagonistic antibody (10 ␮g/mL) LPS ⫹ antagonistic TRAIL receptor-1 and -2 antibody (10 ␮g/mL) LPS ⫹ L-JNK inhibitor (5 ␮mol/L) LPS ⫹ MEK-1 inhibitor PD98059 (10 ␮mol/L) LPS ⫹ superoxiddismutase (6000 U/mL)/catalase (8000 U/mL) LPS ⫹ 4-hydroxy-TEMPO (1 ␮mol/L) LPS ⫹ uric acid (300 ␮mol/L) LPS ⫹ N-acetylcysteine (60 mmol/L) LPS ⫹ ascorbic acid (250 ␮mol/L) LPS ⫹ trolox (20 ␮mol/L) LPS ⫹ L-NMMA (1 mmol/L) LPS ⫹ n-iminoornithine (200 ␮mol/L)

Number of HSC (%) 100 37 ⫾ 1 100 ⫾ 9 98 ⫾ 3a 108 ⫾ 22a 18 ⫾ 2 70 ⫾ 9a 69 ⫾ 7a 39 ⫾ 4 31 ⫾ 2 33 ⫾ 2 31 ⫾ 6 67 ⫾ 6a 33 ⫾ 6 33 ⫾ 2 108 ⫾ 8a 90 ⫾ 12a 71 ⫾ 4a 28 ⫾ 2 31 ⫾ 2 28 ⫾ 1 35 ⫾ 4 41 ⫾ 2 42 ⫾ 2 68 ⫾ 9a 79 ⫾ 10a 41 ⫾ 1 46 ⫾ 4 43 ⫾ 4 26 ⫾ 2 42 ⫾ 6 38 ⫾ 2 42 ⫾ 6 42 ⫾ 7 31 ⫾ 4 31 ⫾ 2

NOTE. HSC were kept in culture for 14 days and thereafter cocultured with KC for another 2 days. Then control medium, interferon ␥, zymosan, LPS (1 ␮g/mL), or LPS (1 ␮g/mL), together with effectors as indicated, were added for 24 hours and the number of HSC determined. To downregulate RIP (Figure 8), geldanamycin incubation was started 16 hours before LPS addition. All caspase inhibitors were used at a concentration of 50 ␮mol/L. For determination of HSC number, cells were fixed with formaldehyde and stained with phalloidin FITC/propidium iodide and quantified under a fluorescence microscope (see Materials and Methods). Data are expressed as means ⫾ SEM and are from at least 3 independent cell preparations. The HSC number found in control experiments (without LPS or other additions) was set to 100%, and the effect of LPS and other effectors is presented as percentage thereof. CD95-Ig:Fc, recombinant CD95:Fcimmunoglobulin fusion molecule; L-NMMA, NG-monomethyl-L-arginine; TRAILR2-Ig:Fc, recombinant TRAIL-receptor 2:Fc-immunoglobulin fusion molecule; 4-hydroxy-TEMPO, 4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy; L-JNK-inhibitor, c-Jun N-terminal kinase peptide inhibitor 1, L-stereoisomer. aStatistically significantly different (P ⬍ 0.05) from the LPS condition.

Figure 4. Time course of LPS-induced caspase 3 activation and annexin V and propidium iodide (PI) staining of HSC in KC/HSC cocultures. HSC/KC cocultures were treated with LPS (1 ␮g/mL; filled symbols) or control medium (open symbols) for the time periods indicated. The percentage of annexin V–positive, but PI-negative, HSC is represented by ‚ and Œ and the percentage of annexin V–positive and PI-positive HSC, by { and }. Annexin V–positive, but PI-negative, cells were regarded as apoptotic and annexin V– and PI-positive HSC as necrotic. The percentage of HSC with positive intracellular caspase 3 activity is indicated by E and ●. Data are given as means ⫾ SEM (at least 3 different experiments).

whereas the protective effect of geldanamycin (Table 1) is probably due to the action of this compound on HSC. Roles of CD95L and TNF-␣ in KC-Induced HSC Killing Stimulation of KC by LPS is known to increase expression of CD95L26,27 and TNF-␣,28 and TNF and Fas receptors are expressed by HSC.10,12,13 However, the addition of neither TNF-␣ nor CD95L was sufficient to trigger HSC death in monocultures (Table 2). In line with previous data,11,12 HSC killing was induced by CD95L only when cycloheximide was present (Table 2). Furthermore, addition of TNF-␣–receptor antagonists, anti–TNF-␣, or inhibitors of the CD95 system to KC/ HSC cocultures did not prevent HSC killing in response to LPS (Table 1). These findings suggest that induction of HSC death in LPS-stimulated cocultures was not mediated by TNF-␣ or CD95L. In line with this, LPS stimulation of KC/HSC cocultures did not lead to membrane surface targeting of CD95 in HSC (Figure 5C). However, CD95 trafficking to the plasma membrane of HSC was induced in the LPS-treated cocultures on treatment with CD95L/cycloheximide (Figure 5D), indicating that apoptotic signals can still be induced on top of

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KC/LPS-induced HSC killing. Furthermore, interferon gamma, which was shown to induce CD95L in KC monocultures,26,27 did not induce HSC killing in KC/ HSC cocultures (Table 1), and cyclosporin A, known to downregulate CD95L in KC,26 had no effect on LPSinduced HSC killing (Table 1). These findings suggest that neither soluble nor membrane-bound forms of TNF-␣ or CD95L can explain the HSC killing induced by LPS-stimulated KC. LPS Induces TRAIL Expression in KC As shown in Figure 7A, LPS led not only to a strong induction of CD95L, as reported previously,27 but

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also to induction of TRAIL in KC monocultures. TRAIL induction was inhibited by dexamethasone (1 ␮mol/L) or PGE2 (10 ␮mol/L). Increased TRAIL expression was also found in KC in the rat liver in vivo 12 hours after intraperitoneal injection of LPS (Figure 8D–F) compared with control (Figure 8A–C), suggestive of an in vivo relevance of the finding. Role of TRAIL in the HSC Killing by Activated KC TRAIL-R1 and -R2 were hardly detectable in HSC that were monocultured for 1 day, but TRAIL-R1 and -R2 expression strongly increased from the seventh day of culture onward, whereas expression of the decoy receptors tended to decrease (Figure 7C). An increased expression of TRAIL-R1 and -R2 after HSC activation and transformation was also found in vivo. As shown in Figure 8G–L, in livers from rats which were treated for 3 weeks with CCl4 and have experienced activation of HSC,6 TRAIL-R1 colocalized with ␣-SMA– expressing HSC. Similar findings were obtained for TRAIL R-2 (not shown). The addition of commercially available recombinant TRAIL to HSC monocultures was ineffective to induce HSC killing (Table 2) and did not induce TRAIL-receptor trafficking to the plasma membrane (not shown). However, TRAIL addition caused an activation of extracellular regulated matrix (Erks) (Figure 7B) and significantly increased the intracellular calcium concentration Š Figure 5. Effect of LPS addition to KC/HSC cocultures on TUNEL reactivity, CD95 receptor, TRAIL-R1, and RIP kinase immunostaining in HSC. Cocultures of HSC and KC were incubated with LPS (1 ␮g/mL) (A–E, G, I) or control medium (F, H, J) for 24 hours. (A, B) F-actin was stained with phalloidin FITC. LPS treatment of cocultures does not lead to the appearance of TUNEL-positive HSC (A), but TUNEL positivity develops on additional stimulation of the LPS-treated cocultures with CD95L (5 ng/mL) plus cycloheximide (0.5 ␮mol/L) (B; arrows indicate TUNEL-positive nuclei in HSC). (C, D) LPS treatment of KC/ HSC cocultures did not induce positive staining for CD95 in nonpermeabilized cells, suggesting that no trafficking of CD95 to the plasma membrane of HSC occurred (C). However, additional treatment with CD95L (1 hour; 100 ng/mL) and cycloheximide (0.5 ␮mol/L) induced strong CD95 cell-surface staining (D). (E, F) LPS stimulation of KC/ HSC cocultures induces strong staining for TRAIL-R1 in nonpermeabilized HSC, suggestive of TRAIL receptor trafficking to the plasma membrane (E), but not in unstimulated controls, in which HSC show only weak punctate staining (F ). (G, H) In the LPS-treated cocultures, immunostaining of TRAIL-R1 is strongly increased in permeabilized spider-like HSC (G; arrow), whereas TRAIL-R1 in untreated controls (LPS absent) shows weak, punctate immunoreactivity for TRAIL-R1 in HSC (H ). A similarly faint punctate staining for TRAIL-R1 is also found in those HSC in LPS-treated cocultures that have not experienced morphological alterations (G). (I, J) Immunostaining for RIP kinase shows a strong staining in spider-like cells (arrow) after LPS treatment of cocultures (I ), whereas in control cocultures (LPS absent) only a faint punctate staining is observed ( J).

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([Ca2⫹]i) by 103 ⫾ 16 nmol/L (n ⫽ 16). These findings indicate that TRAIL signaling occurred but apparently was modified in a way that cell killing was prevented. However, the addition of antagonistic antibodies against TRAIL R-2, but not TRAIL-R1, which were characterized recently,29,30 largely abolished LPS-induced HSC killing in the coculture system (Table 1). In nonpermeabilized HSC in coculture with unstimulated KC, only faint, punctate immunostaining for TRAIL-R1 was found (Figure 5F ); however, on stimulation with LPS, the HSC strongly immunostained for

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Table 2. Effect of Cytokines and Other Effectors on HSC Number in Monoculture Additions Control TRAIL (100 ng/mL) TRAIL ⫹ cycloheximide (0.5 ␮mol/L) TNF-␣ (1 ␮g/mL) CD95L (50 ng/mL) CD95L (50 ng/mL) ⫹ cycloheximide (0.5 ␮mol/L) CD95L/Chx ⫹ Z-IETD-FMK (caspase 8 inhibitor) CD95L/Chx ⫹ Z-IETD-FMK ⫹ Z-AEVD-FMK (caspase 8 plus caspase 10 inhibitor) CD95L/Chx ⫹ Z-LEHD-FMK (caspase 9 inhibitor) CD95L/Chx ⫹ Z-DEVD-FMK (caspase 3 inhibitor) CD95L/Chx ⫹ Z-VAD-FMK (pan-caspase inhibitor) CD95L/Chx ⫹ geldanamycin (1 ␮g/mL) CD95L/Chx ⫹ CD95-Ig:Fc (5 ␮g/mL)

Number of HSC (%) 100 80 ⫾ 25 95 ⫾ 20 103 ⫾ 10 118 ⫾ 8a 15 ⫾ 3 41 ⫾ 18 69 ⫾ 18a 14 ⫾ 6 23 ⫾ 5 91 ⫾ 7a 10 ⫾ 6 105 ⫾ 14a

NOTE. HSC were monocultured for 14 days and then exposed to cytokines and inhibitors for 24 hours. Formaldehyde-fixed cells were stained with phalloidin FITC/propidium iodide and quantified under a fluorescence microscope. Cell numbers are expressed as percentage of the HSC numbers found in control experiments. Data are given as means ⫾ SEM (n ⫽ 3). All caspase inhibitors were used at a concentration of 50 ␮mol/L. The concentration of CD95 ligand (CD95L) was 50 ng/mL, and that of cycloheximide (Chx) was 0.5 ␮mol/L. CD95Ig:Fc, recombinant CD95:Fc-immunoglobulin fusion molecule. aStatistically significantly different (P ⬍ 0.05) from the condition with CD95 ligand plus cycloheximide.

Figure 6. LPS-induced activation of caspase 3 (A), caspase 8 (B), and caspase 9 (C) in HSC/KC cocultures. HSC/KC cocultures were treated with LPS (1 ␮g/mL) for the time periods indicated (A) or for 24 hours (B, C), if not otherwise indicated. Caspase 3, 8, and 9 activities are given as percentage of activity found in controls (100%) and were determined as described in Materials and Methods. Caspase inhibitors, dexamethasone, and cycloheximide were added together with LPS, and geldanamycin was added 16 hours before LPS addition. (A) Time course of LPS-induced caspase 3 activation and its sensitivity to dexamethasone (1 ␮mol/L) and the caspase 9 inhibitor Z-LEHD-FMK (50 ␮mol/L), but not to the caspase 8 inhibitor Z-IETD-FMK (50 ␮mol/L). (B) LPS-induced caspase 8 activation is sensitive to inhibition of caspases 3, 6, and 9 by Z-DEVD-FM (50 ␮mol/L), Z-VEID-FMK (50 ␮mol/L), or Z-LEHD-FMK (50 ␮mol/L), respectively. (C) LPSinduced caspase 9 activation is insensitive to inhibition of caspase 3 or 8 by Z-DEVD-FM (50 ␮mol/L) or Z-IETD-FMK (50 ␮mol/L), respectively, but is sensitive to geldanamycin (5 ␮mol/L), cycloheximide (0.5 ␮mol/L), and dexamethasone (1 ␮mol/L).

TRAIL receptors (Figure 5E). These data indicate that in contrast to CD95 (Figure 5C and D), TRAIL-R1 is targeted to the plasma membrane of HSC after LPS stimulation of the KC/HSC coculture system. No TRAIL trafficking to the plasma membrane was observed in HSC monocultures in response to LPS or TRAIL treatment (not shown). When permeabilized cells were studied, TRAIL-R1 expression was found in both KC and HSC (Figure 5G and H ). Whereas only a faint punctate TRAIL-R1 staining in HSC in unstimulated KC/HSC cocultures was observed (Figure 5H ), immunoreactivity for TRAIL-R1 in permeabilized HSC was enhanced in cocultures on LPS exposure for 24 hours (Figure 5G). Here, TRAIL receptor immunoreactivity was most pronounced in the morphologically altered, spider-like HSC, whereas those HSC that maintained near-normal myofibroblast morphology continued to show the faint punctate staining (Figure 5G and H ). These findings may suggest that LPS stimulation of KC/HSC cocultures leads not only to TRAILreceptor membrane targeting, but also to overexpression in those HSC undergoing KC-induced morphological changes. Role of RIP for KC-Induced HSC Killing RIP was shown to associate with the death-inducing signaling complex of TNF-␣, CD95, and TRAIL

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Table 3. Effect of Supernatants of LPS-Stimulated Kupffer Cells on HSC Number in Coculture Supernatant of KC stimulated for 8 hours with

Number of HSC [%] in coculture

Control LPS (1 ␮g/mL) LPS (1 ␮g/mL) ⫹ dexamethasone (1 ␮mol/L) LPS (1 ␮g/mL) ⫹ geldanamycin (5 ␮mol/L)

100 ⫾ 13a 45 ⫾ 5 98 ⫾ 19a 36 ⫾ 3

NOTE. Monocultures of KC were stimulated with LPS and different inhibitors for 8 hours. Cells were consecutively washed 8 times and incubated with culture medium for 16 hours. Cocultures of HSC and KC were incubated with this supernatant for 24 hours. Formaldehydefixed cells were stained with phalloidin FITC/propidium iodide and quantified under a fluorescence microscope. Cell numbers are expressed as percentage of the HSC numbers found in control experiments (supernatant of unstimulated KC). Data are given as means ⫾ SEM (n ⫽ 3). aStatistically significantly different (P ⬍ 0.05) from the condition with supernatants derived from LPS-treated KC.

receptors, and its activation was reported to be antiapoptotic through activation of c-Jun-N-terminal kinase ( JNK) and the nuclear factor-␬B system in some cell types.23,31 However, RIP-mediated pathways toward cell killing were described in other cell types.24 RIP expression was not detectable in HSC during the first days of culture but strongly increased after 1–3 weeks (Figure 9A). Thus, RIP expression roughly paralleled the susceptibility of HSC toward LPS-induced killing in KC/ HSC cocultures (see previously). An activation-dependent RIP expression in HSC was also found in vivo. Whereas no RIP was detectable in the GFAP-positive HSC from control livers, strong RIP immunoreactivity, which colocalized with GFAP, was found in livers from rats that underwent CCl4 treatment for 3 weeks (Figure 8M–R). A transient expression of antiapoptotic FLIP was also found from days 7–14 of culture (Figure 9A), which may explain the resistance of HSC against CD95L-induced apoptosis, unless FLIP is down-regulated by cycloheximide (not shown). As reported previously,31 the addition of geldanamycin for 16 hours to HSC monocultures led to a strong down-regulation of RIP (Figure 9B). After geldanamycin-induced RIP down-regulation, HSC killing in response to LPS in the cocultures was fully abolished (Table 1), and activation of caspase 9 was no longer observed (Figure 6C). Also, cycloheximide resulted in a marked down-regulation of RIP in HSC (Figure 9B) and prevented both HSC killing (Table 1) and caspase 9 activation in response to LPS (Figure 6C). HSC in unstimulated cocultures showed a faint punctate RIP immunostaining (Figure 5J), which was strongly enhanced in the morphologically altered, spider-like HSC after LPS treatment (Figure 5I ).

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Role of Oxidative Stress KC are known to produce high amounts of superoxide anions and other reactive oxygen species (socalled oxidative burst) in response to zymosan,32,33 and oxidative stress is known to induce apoptotic or necrotic death in a variety of cell types.34 Although LPS does not stimulate superoxide formation in KC,32 a potential role of oxidative stress induced by LPS stimulation of KC was studied by the use of antioxidants. As shown in Table 1,

Figure 7. Induction of TRAIL and CD95L in monocultured KC (A), Erk phosphorylation by TRAIL addition to monocultured HSC (B), and culture-dependent TRAIL-receptor expression in monocultured HSC (C). Respective proteins were assessed by Western blot analysis with specific antibodies. Data are representative for at least 3 different experiments for each series. (A) TRAIL expression by monocultured KC is strongly increased by LPS (1 ␮g/mL; 24 hours) in a prostaglandin E2 (PGE2; 10 ␮mol/L)– and dexamethasone (1 ␮mol/L)–sensitive manner. (B) Addition of TRAIL (100 ng/mL) to monocultured HSC (14 days of culture) induces activation of Erks. For detection of the phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2, protein lysates were subjected to Western blot analysis with a phosphospecific antibody against Erk-1/Erk-2. (C) Culture time– dependent expression of TRAIL-R1–R4 in monocultured HSC. Whereas TRAIL-R1 and -R2 are not detectable during the first week of HSC culture, their expression increases thereafter. However, expression of the decoy receptors TRAIL-R3 and -R4 is observed in early culture and declines thereafter.

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Figure 8. In vivo expression of TRAIL in LPS-activated Kupffer cells in rat liver and in vivo expression of TRAIL-R1 and RIP in hepatic stellate cells from fibrotic rat liver. Rats were treated with 4 mg/kg of LPS (D–F) or control medium (A–C) intraperitoneally and killed after 12 hours. Staining of liver cryo sections was performed with antibodies against macrophage-specific ED-1 (green; A, C, D, F) and anti-TRAIL (red; A, B, D, E). Colocalization of ED-1 with TRAIL is seen in LPS-activated KC (D; arrow), but not controls (A). To activate hepatic stellate cells, rats were treated with CCl4 ( J–L, P–R) or vehicle (G–I, M–O) as described in Materials and Methods for 21 days before death. Liver cryo sections were stained with antibodies against TRAIL-R1 (green; G, H, J, K) and ␣-SMA (specific for activated HSC, red; G, I, J, L) or with RIP (green; M, N, P, Q) and HSCspecific GFAP (red; M, N, O, R). TRAIL-R1 colocalizes with activated SMA-expressing HSC (yellow; J ). RIP colocalizes with GFAP-expressing HSC (yellow; P). Stainings are representative for at least 3 independent experiments performed with different animals.

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HSC and KC in monoculture were 82 ⫾ 5 nmol/L (n ⫽ 24) and 68 ⫾ 13 nmol/L (n ⫽ 24), respectively. The addition of LPS to monocultured HSC had no effect on [Ca2⫹]i (Figure 10A) but increased [Ca2⫹]i in KC monocultures by 45 ⫾ 15 nmol/L (n ⫽ 24). In the KC/HSC coculture system (without LPS), [Ca2⫹]i in HSC was increased to 141 ⫾ 13 nmol/L (n ⫽ 24), whereas [Ca2⫹]i in KC was 74 ⫾ 8 nmol/L (n ⫽ 24) and not significantly different from that in KC monoculture. When LPS was added to KC/HSC cocultures, the [Ca2⫹]i response of KC was similar to that obtained in KC monocultures; i.e., there was an increase of [Ca2⫹]i by 44 ⫾ 15 nmol/L (n ⫽ 24). However, under these conditions, HSC also responded with an increase of [Ca2⫹]i by 30 ⫾ 8 nmol/L

Figure 9. Culture time– dependent RIP and FLIP expression in monocultured HSC (A) and modulation of RIP expression by geldanamycin and cycloheximide (B). (A) HSC were kept in monoculture for the time periods indicated, and RIP and FLIP protein expression were assessed by Western blot analysis with specific antibodies. (B) Downregulation of RIP expression by geldanamycin and cycloheximide. HSC were monocultured for 16 days and incubated for 16 hours with geldanamycin (5 ␮mol/L), 24 hours with cycloheximide (0.5 ␮mol/L), or with control medium (B).

neither 4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxyde, N-acetylcysteine, uric acid, the water-soluble vitamin E analogue trolox, nor incubations with superoxiddismutase/catalase were able to prevent KC-induced HSC killing after stimulation of the cocultures with LPS. Thus, these findings strongly argue against a major role of reactive oxygen species in the LPS-induced killing of HSC by KC. In line with this, stimulation of KC in KC/HSC cocultures with zymosan, which strongly stimulates superoxide production by KC,32 did not induce HSC killing in KC/HSC cocultures (Table 1). LPS is known to induce inducible nitric oxide synthase in KC,35 however, the nitric oxide synthase inhibitors NG-monomethyl-L-arginine and iminoornithine did not prevent HSC death in cocultures (Table 1), suggestive of a nitric oxide–independent mechanism of HSC killing by activated KC. Role of Intracellular Calcium Single-cell fluorescence analyses were performed to study the effect of LPS on [Ca2⫹]i in KC and HSC in mono- and coculture (Figure 10). The resting [Ca2⫹]i of

Figure 10. Effect of LPS, TRAIL, and CD95 ligand on intracellular calcium concentration in HSC in monoculture and coculture with KC. Intracellular calcium concentrations were determined in single-cell measurements of fura-2–loaded HSC as described in Materials and Methods. HSC in monoculture show no calcium response to LPS (A), whereas HSC in coculture with KC show an increase of intracellular calcium concentration in response to LPS (B). When cocultures were treated with LPS for 24 hours, the resting intracellular calcium was increased, and additional LPS evoked no further calcium response (C). The recording given in (C) comes from a spider-like HSC (compare Figure 1C). HSC in monoculture show a calcium response to TRAIL (D), but not to CD95L (E). For statistical analysis and calcium responses of KC, see Results.

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(n ⫽ 24) (Figure 10B), resulting in a [Ca2⫹]i of 169 ⫾ 17 nmol/L (n ⫽ 12). It is interesting to note that this [Ca2⫹]i response was found only in those HSC that were in close contact with KC (not shown). The LPS-induced [Ca2⫹]i increase in cocultured HSC was sustained; after a 24-hour treatment with LPS, the [Ca2⫹]i was increased in spider-like HSC to 176 ⫾ 10 nmol/L (n ⫽ 24), and further LPS addition was ineffective (Figure 10C). These findings suggest that HSC killing in LPS-stimulated cocultures is not accompanied by a gross calcium overload of HSC. When HSC in monoculture were exposed to TRAIL, an increase of the [Ca2⫹]i was observed (Figure 10D), resembling that observed in KC/HSC cocultures in response to LPS (compare Figure 10B and D). No calcium signal was elicited on the addition of CD95L (Figure 10E).

Discussion Characteristics of HSC Killing by LPS Activation of KC/HSC Cocultures As shown in this study, LPS stimulation of KC/ HSC cocultures results in HSC death, whereas LPS is ineffective in HSC monocultures. KC/HSC contacts are apparently required for this effect, which was accompanied by remarkable morphological alterations of HSC, which to our knowledge have not been reported yet. Spider-like cells with long dendrites began to develop as soon as 1–2 hours after LPS addition, and within 4 hours, activation of caspase 3 and annexin V positivity was observed. Approximately half of the annexin V–positive HSC showed nuclear staining with PI, suggestive of necrosis; however, a gross calcium overload of the cells was not detectable. Not only the morphological features of HSC death induced by LPS treatment of KC/HSC cocultures make it difficult to classify HSC death as purely necrotic or purely apoptotic. Although HSC killing was accompanied by caspase activation and was sensitive to caspase 9 or pan-caspase inhibition, TUNELpositive cells were scarcely detectable, but typical DNA fragmentation (TUNEL positivity) was rapidly inducible even in the spider-like cells on addition of CD95L plus cycloheximide. These findings argue against typical apoptosis underlying the HSC death induced by LPSstimulated KC. Antioxidants were ineffective in preventing KC-induced HSC killing, in line with the previous demonstration that LPS does not increase superoxide formation in KC.32 Furthermore, stimulation of superoxide formation by KC in response to zymosan32 did not induce HSC killing in the coculture system. From these data we concluded that oxidative stress induced by LPS

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stimulation of KC probably does not explain the killing of HSC. Clearly, protein expression is required for the induction of HSC death in LPS-treated cocultures in view of the protective effects of cycloheximide, actinomycin, geldanamycin, and dexamethasone, suggestive of some kind of programmed cell death. LPS increases the expression of CD95L and TNF-␣ in KC, and the corresponding receptors are present on transformed HSC. Shedded death receptor ligands derived from LPS-stimulated KC are clearly not responsible for HSC death, because supernatants derived from LPStreated KC were ineffective to induce HSC killing, as was the addition of commercially available ligands. However, supernatants derived from LPS-treated KC were potent inducers of parenchymal cell apoptosis and thymic lymphocytes.26,27 It is interesting to note that LPS-free supernatants from LPS-activated KC are highly potent in triggering HSC death in coculture. This points to the existence of other factors mediating KC-induced HSC killing that may be of pathophysiological importance. Therefore, although the LPS concentrations used in this study are rather high, it represents a good model for triggering activated KC-induced HSC death. Soluble and membrane-bound death receptor ligands may differ in their death-inducing potential.30,36 However, several lines of evidence argue against a role of KC-bound CD95L in mediating HSC killing. (1) Maneuvers to antagonize the action of membrane-bound TNF-␣ or CD95L in the cocultures by adding neutralizing antibodies or receptor antagonists did not prevent HSC killing. (2) Interferon gamma, which is known to induce hepatocyte killing via CD95L expression of KC,26 did not induce HSC killing in KC/HSC cocultures. (3) Cyclosporin A, which down-regulates CD95L expression by KC,26 did not prevent LPS-induced HSC killing. (4) CD95 trafficking to the HSC plasma membrane was not observed in KC/HSC cocultures on LPS stimulation, but this occurred on apoptosis induction by the addition of CD95L plus cycloheximide. (5) Combined inhibition of caspases 8 and 10 prevented HSC apoptosis induced by CD95L plus cycloheximide but was ineffective with respect to LPS-induced HSC killing in the cocultures. (6) No spider-like HSC are generated when apoptosis is induced by CD95L/cycloheximide in the absence of LPS. (7) TUNEL-positive cells are a hallmark of CD95/cycloheximide-induced apoptosis, but not of the LPS-induced HSC killing in KC/HSC cocultures. Role of the TRAIL System in HSC Killing by LPS-Activated KC Several observations may point to an involvement of the TRAIL system in KC-induced HSC killing.

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(1) TRAIL-R1 and -R2 were not detectable in HSC during the first 3 days of culture, and these cells were resistant toward killing by LPS-activated KC. (2) The increase of TRAIL-R1 and -R2 expression from day 7 to 14 of HSC culture was paralleled by an increased sensitivity of HSC toward killing by LPS-stimulated KC. (3) LPS up-regulates expression of TRAIL in KC in a dexamethasone- and PGE2-sensitive way, and dexamethasone and PGE2 also inhibited KC-induced HSC killing. (4) LPS stimulation of KC/HSC cocultures leads to TRAIL receptor trafficking to the HSC plasma membrane and increased TRAIL receptor immunoreactivity. (5) Antagonistic antibodies against TRAIL R-2,30 but not against TRAIL-R1, largely abolished HSC killing by LPS-stimulated KC. However, the addition of commercially available TRAIL to HSC monocultures induced neither TRAIL receptor trafficking to the plasma membrane nor HSC killing, but it activated Erks and increased [Ca2⫹]i. Although this last finding may at first glance argue against a role of TRAIL in HSC killing, it could be misleading because of the known complexity of TRAIL pharmacology and signaling (see reviews17,18). For example, the failure of TRAIL contained in the supernatants from LPS-treated KC to induce HSC killing could be explained by the requirement of membrane-bound TRAIL to induce HSC death via TRAIL-R230 or of other costimulatory or signal-modulating factors, which are expressed on the surface of activated KC and mediate both TRAIL receptor trafficking to the plasma membrane and sensitivity against TRAIL-induced cell death. In this respect it is interesting to note that cell death can be induced in Jurkat cell lines by membrane-bound TRAIL, but not soluble TRAIL.30 Role of RIP and Caspases KC-induced HSC killing led to the activation of caspases 3, 8, and 9; however, only inhibition of caspase 9 largely prevented HSC death, whereas caspase 3, 8, and 10 inhibitors were ineffective. Also, caspase 3 and 8 activation were blocked by the caspase 9 inhibitor, indicating that caspase 3 and 8 activation might have occurred in response to caspase 9 activation but are apparently not required for induction of HSC death. The sensitivity pattern of caspase activation toward the different caspase inhibitors found in this study resembles the hierarchy of cytochrome c–inducible caspase activation events in Jurkat cell-free extracts (see Figure 1D37). One important feature of KC-induced HSC killing is its caspase 9 dependence. Recent studies showed that caspase 9 can be activated without proteolytic cleavage by yet-unknown cytosolic factors.25,38 Studies on the dependence receptor DCC (deleted in colorectal cancer)

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showed that cell death can be driven independently of both the mitochondrial and the death receptor/caspase 8 pathway, through a cytochrome c– or apoptotic protease-activating factor-1–independent caspase 9 activation.39 Also, an alternative form of programmed cell death (paraptosis) was shown to be driven by an alternative apoptotic protease-activating factor-1–independent caspase 9 activity.40 However, paraptosis was insensitive to pan-caspase inhibition,40 in contrast to the KC-induced HSC killing in response to LPS in this study. Recent studies indicated that recruitment of RIP to TRAIL receptors can cause a caspase 8 –independent form of cell death resembling necrosis.24 A potential link between RIP and caspase 9 may consist of the caspase recruitment domain of RIP that can bind caspase 9.41 Furthermore, the proapoptotic RIP-3 isoform was recently shown to coimmunoprecipitate with caspase 942 and to translocate to mitochondria.43 In this study, down-regulation of RIP by geldanamycin31 abolished caspase 9 activation and HSC killing by LPS activation of KC/HSC cocultures. Likewise, cycloheximide down-regulated RIP and prevented HSC killing. In line with an involvement of RIP in mediating KC-induced HSC killing is also the finding that the culture time– dependent RIP expression in HSC roughly paralleled their susceptibility toward killing by LPS-activated KC; however, a cause/effect relationship remains to be proven. Nonetheless, one may speculate that HSC killing by activated KC is the result of a RIP/caspase 9 interaction, which triggers both cell death and activation of other caspases. Taken together, this study shows potent HSC killing by LPS-activated KC in a caspase 9 – and RIP-dependent way, with properties suggestive of a novel form of cell death. TRAIL receptor activation may play a role in this type of cell killing. Further insight into the molecular mechanisms underlying KC-induced HSC killing is required.

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Received November 13, 2001. Accepted May 31, 2002.

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Address requests for reprints to: Prof. Dr. Dieter Ha ¨ussinger, Medizinische Einrichtungen der Heinrich–Heine Universita ¨t, Klinik fu ¨r Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-40225 Du ¨sseldorf, Germany. Fax: (49) 211-811-8838. This study was supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 575, “Experimentelle Hepatologie.” The authors thank Priv. Doz. Dr. F. Schliess, Dr. U. Warskulat, Dr. R. Kubitz, and Prof. Dr. G. Paumgartner for their fruitful discussions, Prof. Dr. Harald Wajant for kindly providing antagonistic antibodies against TRAIL receptors 1 and 2, and Claudia Rupprecht for excellent technical assistance.