1. Caspase 9–dependent killing of hepatic stellate cells by activated Kupffer cells 
Richard Fischer, Alexandra Cariers, Roland Reinehr, Dieter Häussinger 
Gastroenterology 
Volume 123, Issue 3, Pages (September 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 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 μg/mL) and/or other effectors. (A–D) Cells in cocultures were fixed and stained with antibodies against α-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 μm. (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 μmol/L) does not prevent LPS-induced changes of HSC morphology (compare Figure 1M). (G, H) The caspase 9 inhibitor Z-LEHD-FMK (50 μmol/L) and dexamethasone (1μmol/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.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 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 μg/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 μg/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 μg/mL) for 24 hours. LPS-induced HSC disappearance was dependent on KC density.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 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.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 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 μg/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 ▴ 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 ○ and ●. Data are given as means ± SEM (at least 3 different experiments).
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6. Fig. 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 μg/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 μmol/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 μmol/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).
Gastroenterology  , DOI: ( /gast )
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7. Fig. 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 μg/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 μmol/L) and the caspase 9 inhibitor Z-LEHD-FMK (50 μmol/L), but not to the caspase 8 inhibitor Z-IETD-FMK (50 μmol/L). (B) LPS-induced caspase 8 activation is sensitive to inhibition of caspases 3, 6, and 9 by Z-DEVD-FM (50 μmol/L), Z-VEID-FMK (50 μmol/L), or Z-LEHD-FMK (50 μmol/L), respectively. (C) LPS-induced caspase 9 activation is insensitive to inhibition of caspase 3 or 8 by Z-DEVD-FM (50 μmol/L) or Z-IETD-FMK (50 μmol/L), respectively, but is sensitive to geldanamycin (5 μmol/L), cycloheximide (0.5 μmol/L), and dexamethasone (1 μmol/L).
Gastroenterology  , DOI: ( /gast )
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8. Fig. 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 μg/mL; 24 hours) in a prostaglandin E2 (PGE2; 10 μmol/L)– and dexamethasone (1 μmol/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.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 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 α-SMA (specific for activated HSC, red; G, I, J, L) or with RIP (green; M, N, P, Q) and HSC-specific 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.
Gastroenterology  , DOI: ( /gast )
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10. Fig. 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) Down-regulation of RIP expression by geldanamycin and cycloheximide. HSC were monocultured for 16 days and incubated for 16 hours with geldanamycin (5 μmol/L), 24 hours with cycloheximide (0.5 μmol/L), or with control medium (B).
Gastroenterology  , DOI: ( /gast )
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11. Fig. 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.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions