Inhibition of p53 protects liver tissue against endotoxininduced

Transcription

1 Inhibition of p53 protects liver tissue against endotoxininduced apoptotic and necrotic cell death T. SCHÄFER, C. SCHEUER, K. ROEMER,* M. D. MENGER, AND B. VOLLMAR,1 Institute for Clinical and Experimental Surgery, *Department of Virology, University of Saarland, D Homburg/Saar; and Department of Experimental Surgery, University of Rostock, Rostock, Germany ABSTRACT There is increasing evidence that apoptotic and necrotic hepatocyte death following endotoxin-induced liver injury act as signals for leukocyte sequestration in the liver vasculature. p53 has been implicated to promote apoptosis through trans-activation and down-regulation of specific pro- and antiapoptotic genes. Here, we report that inhibition of p53 decreases apoptotic and necrotic tissue injury as well as inflammatory cell response. Sprague-Dawley rats were injected intraperitoneally with 2.2 mg/kg pifithrin- (PFT), a p53-inactivating agent, or the vehicle DMSO 30 min before intravenous exposure to lipopolysaccharide (LPS). In vehicle-pretreated animals, LPS induced significant apoptosis and necrosis of hepatocytes, which was associated with intrahepatic leukocyte recruitment, microvascular dysfunction, and enzyme release. Inhibition of p53 effectively attenuated (P<0.05) hepatocellular apoptosis and necrosis, but also reduced leukocyte recruitment and microvascular dysfunction. Western blot analysis revealed that PFT lowered the nuclear-to-cytoplasmic p53 ratio and reduced both activation of NF- B and cleavage of procaspase 3 (P<0.05). In parallel, immunohistochemistry of PFT-pretreated, but not vehicle-pretreated, endotoxic animals exhibited nuclear p53 exclusion and reduced NF- B p65 staining. This indicates that p53 mediates, at least in part, LPSassociated apoptosis and contributes to inflammatory endotoxic tissue injury through leukocyte activation and intraorgan sequestration. Schäfer, T., Scheuer, C., Roemer, K., Menger, M. D., Vollmar, B. Inhibition of p53 protects liver tissue against endotoxin-induced apoptotic and necrotic cell death. FASEB J. 17, (2003) Key Words: apoptosis necrosis pifithrin-alpha LPS microcirculation caspase 3 NF- B ICAM-1 intravital fluorescence microscopy bisbenzimide A significant cause of morbidity and mortality in patients with gram-negative sepsis is the development of liver dysfunction and liver failure (1, 2). Neutrophils have an important role as mediators of host defense against infection by prompt activation, migration, and elimination of the infectious source. However, these leukocytes are also known to cause parenchymal cell damage by releasing a variety of toxic mediators at the site of inflammation (3). Even though the mechanisms of leukocyte-dependent injury have been subject of intensive investigation during the last two decades, there is still a need to better understand the complex scenario of septic organ injury and to find specific targets for potential therapeutic interventions. Apoptotic cell death has been reported in a number of sepsis-relevant models (4 8). Though the importance of apoptosis for overall injury and organ dysfunction is not conclusively defined, it has been shown that hepatocellular apoptosis may represent an early, general, and possibly causal event for inflammatory liver failure (4). Because apoptotic hepatocytes might also function as a signal that triggers neutrophil transmigration and subsequent parenchymal cell attack (9), apoptotic cell death, even if affecting less than 10% of hepatocytes, has to be considered a relevant issue in overall liver injury. Experimental studies have indicated that TNF- plays a major role in mediating hepatocellular apoptosis because interventions directed against TNF- e.g., neutralizing antibodies, TNF receptor knockout animals, and inhibition of TNF- gene transcription protect against hepatocellular apoptosis and liver injury (6 8, 10). Engagement of the tumor necrosis factor receptor p55 (TNFRp55) seems to make a major contribution to the proapoptotic activities of TNF- (7). On the other hand, there is evidence for TNF- independent mechanisms of induction of hepatocellular apoptosis, including the Fas-FasL-system (7, 11, 12) and TRAIL (13). Because the genes for these membrane death receptors (TNFRp55, Fas/APO1, and TRAIL) are, at least in part, under the transcriptional control of the p53 tumor suppressor and have been shown to be up-regulated under various conditions of stress in a p53-dependent manner (14, 15), it is tempting to speculate that p53 suppression may protect endotoxin-exposed hepatic tissue. We therefore asked whether p53 is involved in endotoxin-associated hepatocellular apoptosis and whether transient inhibition of p53 is effective to reduce endotoxemic liver injury. 1 Correspondence: Department of Experimental Surgery, University of Rostock, D Rostock, Germany. brigitte.vollmar@med.uni-rostock.de /03/ FASEB

2 MATERIALS AND METHODS Animal model and surgical preparation Sprague-Dawley rats of either sex (body weight g) were used for the experiments. Animals were kept on water and standard laboratory chow ad libitum. The experiments were conducted in accordance with the German legislation on protection of animals and the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council). Animals were injected intraperitoneally with 2.2 mg/kg pifithrin- (PFT; Alexis, Grünberg, Germany), which has been shown to reversibly block p53 function (16). PFT- was dissolved at a concentration of 2.2 mg/ml in 99.5% DMSO (DMSO; Sigma, Deisenhofen, Germany). Control animals received equal volumes of the vehicle DMSO (1 L/g body weight). After an additional 30 min, all animals (n 26) were exposed to Escherichia coli lipopolysaccharide (serotype 0128:B12; 10 mg/kg i.v.; Sigma). Intravital fluorescence microscopic analysis of the hepatic microcirculation was performed 16 h after LPS exposure, including the in vivo assessment of hepatocellular apoptosis, sinusoidal perfusion failure, and leukocyte endothelial cell interaction. Liver tissue was sampled for subsequent Western blot analysis, histology and immunohistochemistry (n 7 animals per group). In an additional series of animals liver tissue was sampled 4 h after LPS exposure for subsequent Western blot analysis, histology, and immunohistochemistry (n 4 animals per group). For in vivo microscopy of the liver, pentobarbital anesthetized animals (50 mg/kg i.p.) were tracheotomized to facilitate spontaneous respiration (room air) and placed in supine position on a heating pad for maintenance of body temperature at C. Polyethylene catheters (PtdEtn 50, ID 0.58 mm, Fa. Portex, Hythe, UK) in the right carotid artery and jugular vein allowed for the assessment of systemic hemodynamics, injection of fluorescent dyes (intravital microscopy) and permanent infusion of isotonic saline solution at a rate of 2 ml/kg h. After transverse laparotomy and cannulation of the common bile duct (PE-50) for continuous collection of bile, the animals were positioned on their left side. The left liver lobe was then exteriorized and covered with a glass slide for intravital fluorescence microscopy (17). Intravital fluorescence microscopy By use of a modified fluorescence microscope with a 100-W HBO mercury lamp (Axiotech, Zeiss, Jena, Germany) attached to an UV, a blue and a green filter system, the hepatic microcirculation was analyzed in epi-illumination technique. The microscopic images were recorded by a CCD video camera (FK 6990, COHU, Prospective Measurements Inc., San Diego, CA, USA) and transferred to a video system (S-VHS Panasonic AG 7350, Matsushita, Tokyo, Japan). Using water immersion objectives (W 20 /0.5; W 40 /0.75; Zeiss, Jena, Germany), magnifications of 430 and 860 were achieved on the video screen (PVM-2130 QM, Sony, Munich, Germany). Sinusoidal perfusion failure was quantitatively analyzed after tissue contrast enhancement by sodium fluorescein (2 mol/kg i.v.; Merck, Darmstadt, Germany) using blue light epi-illumination ( nm/ 520 nm, excitation/emission wavelength) (17). Leukocyte endothelial cell interaction was assessed after in vivo white blood cell staining by rhodamine-6g (2 mol/kg i.v.; Merck) using the green filter system ( / 580 nm) (18). Apoptotic cell death was determined in vivo after staining the nuclei of hepatocytes by bisbenzimide (H33342; 2 mol/kg i.v.; Sigma) using the UV filter system ( / 415 nm) (19). Quantitative video analysis Assessment of hepatic microcirculatory parameters was performed off-line by frame-to-frame analysis of the videotaped images using a computer-assisted image analysis system (CapImage; Zeintl, Heidelberg, FRG). Within 10 lobules per animal, sinusoidal perfusion failure was determined by the number of nonperfused sinusoids (given in percent of all sinusoids crossing a 200 m raster line) (20). Leukocyte endothelial cell interaction was analyzed within 10 hepatic lobules and 10 postsinusoidal venules per animal, including 1) the number of stagnant leukocytes, located within sinusoids (given as cells/lobule) and not moving during an observation period of 20 s, and 2) the number of adherent leukocytes, located within postsinusoidal venules (given as cells/mm 2 endothelial surface, calculated from diameter and length of the vessel segment studied, assuming cylindrical geometry) and not moving or detaching from the endothelial lining during an observation period of 20 s (18). Apoptotic cell death was analyzed within 10 lobules per animal by counting the number of cells which showed apoptosis-associated condensation, fragmentation and crescent-shaped formation of chromatin (given in percent of all cells visible) (19). Sampling and assays Bile flow was measured continuously via the catheter in the common bile duct and standardized per gram liver wet weight ( L/min g). At the end of in vivo microscopy of the liver, arterial blood samples were taken for the spectrophotometric determination of aspartate aminotransferase and alanine aminotransferase serum activities, which served as an indicator for hepatocellular disintegration. Liver tissue was sampled for Western blot analysis, histology, and immunohistochemistry. Histology and immunohistochemistry At the end of each experiment, liver tissue was fixed in 4% phosphate-buffered formalin for 2 3 days and embedded in paraffin. From the paraffin-embedded tissue blocks, 5 m sections were cut and stained with hematoxylin-eosin (HE) for histological analysis of apoptosis, necrosis, and endothelial detachment. Apoptotic cells (i.e., cells characterized by cell shrinkage, nuclear and cytoplasmic condensation, nuclear fragmentation, and cellular budding) and necrotic cells were counted and given in percentage of all cells within 25 consecutive high-power fields (HPF; 40 objective) using an Olympus microscope (Model BX60F; Olympus Optical Co., Tokyo, Japan). Endothelial detachment was assessed as the number of venules showing detachment in percent of the total number of venules per 25 HPF: none 0; minimal ( 10% venules) 1; mild (10 40% venules) 2; moderate (40 70% venules) 3; severe ( 70% venules) 4. Neutrophils were stained by the AS-D chloroacetate esterase (CAE) technique. Cells were identified by positive staining and morphology and were counted in 50 HPF. In addition, liver tissue was excised as rapidly as possible, embedded in O.C.T. compound (Miles Inc., Elkhart, IN, USA), and quickly frozen in liquid nitrogen. Thin sections (5 8 m) were cut and collected on poly-l-lysine-coated glass slides, air dried, acetone fixed, and processed immunohistochemically for demonstration of p53 and NF- B p65 using indirect immunofluorescence techniques. p53 IN HEPATIC ENDOTOXEMIA 661

3 Sections were rehydrated with PBS and permeabilized by 0.2% Triton-X 100 for 10 min, followed by 30 min incubation with 10% normal goat serum to block unspecific binding sites. Mouse monoclonal anti-p53 (clone PAb421; Calbiochem, Cambridge, MA, USA) and mouse monoclonal anti- NF- B p65 (Chemicon Int., Temecula, CA, USA) diluted 1:50 and 1:30 were used as primary antibodies and incubated for min at room temperature. Indocarbocyanine (Cy3) -conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:600 was incubated for 1 h as secondary antibody. The sections were counterstained with bisbenzimide (2 g/ml; Sigma, Saint Louis, MO, USA) and examined by fluorescence microscopy (Model BX60F; Olympus) using green and UV epi-illumination. For ICAM-1 immunohistochemistry of cryostat sections, a mouse monoclonal anti-rat ICAM-1 antibody (AMS Biotechnology, Wiesbaden, Germany) was used as primary antibody (1:100; 2 h at 37 C), followed by a biotinylated goat antimouse antibody (1:200) for streptavidin-biotin complex peroxidase staining (Vectastain ABC-peroxidase kits, Camona, Wiesbaden, Germany). 3,3 Diaminobenzidine was used as chromogen. The sections were counterstained with hematoxylin and examined by light microscopy (Model BX60F; Olympus). Western blot analysis The procedure used to prepare nuclear and cytoplasmic protein extracts for assessment of p53 levels is a modification of the method described by Schreiber et al. (21). After manual homogenization of liver in buffer A (10 mm Tris ph 7.3, 10 mm KCl, 1.5 mm MgCl 2, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 0.5 mm -mercaptoethanol) and centrifugation for 25 s at 16,000 g and 4 C, the pellet was resuspended in lysis buffer (buffer A containing 0.4% Nonidet P-40) and kept on ice for 10 min. By centrifugation for 5 min at 16,000 g and 4 C, the supernatant was saved as the cytoplasmic fraction. The nuclei were then resuspended in buffer C (20 mm HEPES ph 7.9, 400 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol (DTT), 1 mm PMSF) and incubated on ice for 15 min with occasional shaking. The sample was then centrifuged for 5 min at 16,000 g and 4 C to save the supernatant as nuclear soluble fraction. For whole protein extracts and Western blot analysis of NF- B, caspase 3, and ICAM-1, liver was homogenized in lysis buffer (10 mm Tris ph 7.5, 10 mm NaCl, 0.1 mm EDTA, 0.5% Triton-X 100, 0.02% NaN 3, 0.2 mm PMSF), incubated for 30 min on ice, and centrifuged for 30 min at 16,000 g. The supernatant was saved as whole protein fraction. Before use, all buffers received protease inhibitor cocktail (1:100 v/v; Sigma). Protein concentrations were determined using the Lowry assay with bovine serum albumin as standard (22). Sixty micrograms protein/lane was separated discontinuously on sodium dodecyl sulfate polyacrylamide gels (10% SDS-PAGE for p53 and NF- B; 12%SDS-PAGE for caspase 3; 8% SDS-PAGE for ICAM-1) and transferred to a polyvinyldifluoride membrane (Westran; Schleicher and Schüll, Dassel, Germany). After blockade of nonspecific binding sites, membranes were incubated for 2 h at room temperature with mouse monoclonal anti-p53 (1:100; clone PAb421; Calbiochem, Cambridge, MA, USA), mouse monoclonal anti-nf- B p65 subunit (1:150; Chemicon Int.), rabbit anti-caspase-3 polyclonal antibody (1:600; StressGen Biotechnologies Corp., Victoria, BC, Canada), and rabbit anti-icam-1 polyclonal antibody (1:200; Santa Cruz, Biotechnology, CA), followed by peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit Ig antibodies (Amersham Pharmacia Biotech, Freiburg, Germany) (dilution of 1:2500 for p53, caspase-3, and ICAM-1; dilution of 1:3750 for NF- B p65) as secondary antibodies. The nuclear and cytoplasmic samples were examined for the presence of a known nuclear (histone H1) and cytoplasmic (I B ) protein (12% SDS-PAGE). Antibodies were diluted 1:200 for anti-histone and anti-i B (Santa Cruz Biotechnology, Heidelberg, Germany). Secondary antibody was peroxidase-conjugated sheep anti-mouse Ig antibody (1:5000; Amersham Pharmacia Biotech). Protein expression was visualized by means of luminolenhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and exposure of the membrane to a blue lightsensitive autoradiography film (Hyperfilm ECL, Amersham, Braunschweig, Germany). Signals were densitometrically assessed (Bio-Rad, Gel-Dokumentations system, Munich, Germany) and, except for p53, normalized to the -actin signals (mouse monoclonal anti- -actin antibody; 1:8000; Sigma) to correct unequal loading. Statistical analysis All data are expressed as mean se. After proving the assumption of normality and equal variance across groups, differences between groups were assessed using the Student s t test. Overall statistical significance was set at P Statistics were performed using the software package Sigma- Stat (Jandel Corporation, San Rafael, CA, USA). RESULTS Animals of the DMSO- and PFT-pretreated group behaved no differently during the initial period after LPS exposure. During the entire 16 h observation period, however, 4 of 11 animals of the DMSO-pretreated group died whereas all of the 7 PFT-pretreated animals survived (P 0.07). Animals that survived the 16 h LPS exposure did not differ in terms of systemic hemodynamics and peripheral blood cell counts (data not shown). Intravital fluorescence microscopy of endotoxemic livers Exposure of DMSO-pretreated animals with LPS resulted in a severe deterioration of sinusoidal perfusion characterized by 40% of nonperfused sinusoids (Fig. 1A). Leukocytes were found stagnant within sinusoids (26 4 cells/acinus) and adherent to the endothelium of postsinusoidal venules ( cells/mm 2 endothelial surface; Fig. 1B). The individual staining of hepatocytes by bisbenzimide H33342 allowed us to assess nuclear morphology with condensation, fragmentation and crescent-shaped formation of chromatin. The overall fraction of hepatocytes exhibiting these apoptotic features averaged % in the DMSO-pretreated animals at 16 h LPS exposure (Fig. 1C). Animals pretreated with PFT showed significant reduction of the number of apoptotic hepatocytes in endotoxemic livers with a mean value of % (P 0.001; Fig. 1C). Concomitantly, inflammatory cell response within postsinusoidal venules was effectively attenuated together with a significant improvement of nutritive sinusoidal perfusion (Fig. 1A, B). Leukocyte 662 Vol. 17 April 2003 The FASEB Journal T. SCHÄFER ET AL.

4 Immunohistochemistry and Western blot analysis of endotoxemic livers PFT significantly affected the nuclear-to-cytoplasmic ratio of the p53 protein inasmuch as PFT lowered nuclear but not cytoplasmic p53 protein (Fig. 3A C). In line with this, immunohistochemistry of liver tissue of PFT-pretreated animals revealed a distinct exclusion of p53 staining within hepatocellular nuclei but strong p53 staining within hepatocellular cytoplasm (Fig. 3E), whereas in DMSO-pretreated endotoxemic animals p53 staining was found more uniformly distributed within these individual hepatocellular compartments (Fig. 3D). Protein levels of NF- B p65, i.e., the active subunit of NF- B, were significantly reduced by 40 and 50% at 4 and 16 h after endotoxin exposure in case of PFT pretreatment (Fig. 4A, B). This finding was confirmed by the only faint immunohistochemical NF- B p65 staining of hepatocytes in PFT-pretreated animals (Fig. 4D), whereas livers of DMSO-pretreated controls exhibited strong immunoreactivity for NF- B p65 (Fig. 4C). Moreover, densitometric analysis of cleaved products of Figure 1. Prevention of LPS-induced perfusion failure (A), venular leukocyte adherence (B), and hepatocellular apoptosis (C) by pretreatment of animals with the p53-inhibiting substance pifithrin- (PFT). DMSO-pretreated animals served as controls. Microcirculatory and cellular parameters were assessed using intravital fluorescence microscopy at 16 h after LPS exposure. Values are means se. *P 0.05 vs. DMSO, unpaired Student s t test. accumulation within the sinusoids, however, remained unaffected by PFT (25 6 cells/acinus). Histological analysis of endotoxemic livers PFT-mediated protection against endotoxemic liver injury was substantiated by analysis of HE-stained tissue sections, which revealed necrotic hepatocytes of only % compared with % in DMSOpretreated controls (P 0.002; Fig. 2A). In line with in vivo analysis of hepatocellular apoptosis, the percentage of hepatocytes exhibiting characteristic morphological features of cell apoptosis was % in the p53-blocked animals vs % in the DMSOpretreated animals (Fig. 2B). In PFT-pretreated animals, venular endothelial detachment ( % vs. DMSO: %;P 0.056) and leukocytic transmigration were markedly reduced (Fig. 2C), implying limited inflammatory cell response and thus tissue injury upon blockade of p53-dependent apoptosis. Figure 2. Prevention of LPS-induced hepatic tissue necrosis (A), hepatocellular apoptosis (B), and leukocyte infiltration (C) by pretreatment of animals with the p53-inhibiting substance pifithrin- (PFT). DMSO-pretreated animals served as controls. Morphological parameters were assessed in HE- (A, B) and CAE-stained tissue sections (C) of livers at 16 h after LPS exposure. Values are means se. *P 0.05 vs. DMSO, unpaired Student s t test. p53 IN HEPATIC ENDOTOXEMIA 663

5 administration of LPS. PFT pretreatment was effective to significantly reduce this elevation of enzyme release, achieving values which corresponded to almost normal conditions (Table 1). DISCUSSION We report here a protection against LPS-induced liver injury documented by attenuation of hepatocellular apoptosis and necrosis, sinusoidal perfusion failure, and inflammatory leukocytic response. PFT pretreatment resulted in a reduction of nuclear p53 levels and Figure 3. Modulation of cytoplasmic (cyt) and nuclear (nuc) p53 distribution, as densitometrically assessed by protein Western blot analysis of p53 in the individual hepatocellular compartments (A) of endotoxemic animals pretreated with the p53-inhibiting substance pifithrin- (PFT). DMSO-pretreated animals served as controls. B) Nuclear-to-cytoplasmic ratio of p53 protein in DMSO- and PFT-pretreated animals. Liver tissue was sampled at 4 h after LPS exposure. Values are means se.*p 0.05 vs. DMSO, unpaired Student s t test. C) Representative Western blot of p53 in cytoplasmic and nuclear extracts of endotoxemic liver tissue in DMSO- and PFT-pretreated animals. D, E) Representative immunofluorescent images of p53 staining in endotoxemic liver tissue 4 h after LPS exposure. Note the distinct exclusion of p53 staining within the hepatocellular nuclei in the PFT-pretreated animal (E), whereas in the DMSO-pretreated animal p53 staining was found more uniformly distributed within these individual hepatocellular compartments (D). Bars represent 20 m. caspase 3 revealed a marked inhibition of caspase activation in liver tissues of PFT-pretreated animals (Fig. 5) when compared with those of DMSO-pretreated ones. Neither Western blot analysis nor immunohistochemistry indicated a significant difference in ICAM-1 expression between livers of PFT- and DMSOpretreated animals (data not shown). Liver enzymes activities and bile flow In DMSO-pretreated animals, plasma activities of liver enzymes were found markedly elevated 4 and 16 h after Figure 4. Reduced activation of NF- B, as densitometrically assessed by Western blot analysis of the active NF- B p65 subunit (A) in livers of endotoxemic animals pretreated with the p53-inhibiting substance pifithrin- (PFT). DMSO-pretreated animals served as controls. Liver tissue was sampled 4 and 16 h after LPS exposure. Values are means se. *P 0.05 vs. DMSO, unpaired Student s t test. B) Representative Western blot of NF- B p65 (arrowhead) in four experiments per group. Signals were normalized to -actin (arrow) to correct unequal loading. C, D) Representative immunofluorescent images of NF- B p65 staining in endotoxemic liver tissue 16 h after LPS exposure. Note the pronounced staining in the DMSO-pretreated animal (C) vs. faint or even absent staining in the PFT-pretreated animal (D). Bars represent 20 m. 664 Vol. 17 April 2003 The FASEB Journal T. SCHÄFER ET AL.

6 Figure 5. Inhibition of activation of caspase 3, as densitometrically assessed by Western blot analysis of the cleaved products of caspase 3 (A) in livers of endotoxemic animals pretreated with the p53-inhibiting substance pifithrin- (PFT). DMSO-pretreated animals served as controls. Liver tissue was sampled 4 and 16 h after LPS exposure. Values are means se. *P 0.05 vs. DMSO, unpaired Student s t test. B) Representative Western blot of caspase 3 in two experiments per group, displaying the precursor form of caspase 3 (arrow) together with the active cleaved products (arrowheads). caspase 3 activity, and in the attenuation of apoptotic cell death upon LPS exposure. Finally, PFT pretreatment of endotoxemic animals attenuated NF- B activation, prevented inflammatory leukocyte recruitment, and reduced sepsis-associated hepatic tissue injury and excretory dysfunction. As a consequence, PFT pretreatment reduced the mortality within the first 16 h upon LPS-challenge. p53 is known to induce the expression of multiple genes whose products are implicated in cell cycle arrest or apoptosis. Most of the p53-induced apoptosis-related genes are activated soon after toxin exposure. Among them are sequences encoding proteins that affect the integrity of mitochondria, such as Bax, and others producing membrane death and decoy receptors of the tumor necrosis factor receptor (TNFR) family (14). Upon binding their respective ligands, the death receptors form trimers and recruit adaptor molecules and initiator caspases (caspase 8, caspase 10) to their intracellular/cytoplasmic death domains. Autocatalytic cleavage and activation of these initiator caspases in turn result in the cleavage of downstream effector caspases (caspase 3, caspase 7) and cell death (15, 23). In the liver, activation of the death receptors has been recognized as a hallmark of the acute inflammatory response in endotoxemia and sepsis with TNF- as the most important early mediator in this scenario. With experimental studies addressing the respective ligands (12), receptors (7), and intracellular caspases (12, 24), the mechanistic factors that underlie the control of apoptotic cell death have meanwhile been broadly integrated in the complex biology of sepsis-related liver injury. To our knowledge, however, the present study is the first to address the role of p53 and the potential to manipulate endotoxic liver injury by targeting p53- dependent apoptosis. In the present study, we have chosen the 16 h time point for analysis of apoptotic cell death. It has been well established that expression of p53-regulated proapoptotic genes upon toxin exposure usually requires 6 to 12 h. Thus, the execution phase of apoptosis cannot be expected to take place during the initial few hours. We abstained from analysis at later time points because the liver represents the organ with the largest pool of sessile macrophages (i.e., Kupffer cells) that rapidly phagocytize and clear apoptotic cells. In 1999, pifithrin- was shown to selectively inhibit p53 transcriptional activity in various mouse cell lines, and to prevent DNA damage-induced apoptosis in TABLE 1. Transaminase activities 4 and 16 h after LPS-exposure in animals pretreated with either the p53 inhibitor pifithrin- (PFT) or the vehicle DMSO a 4 h 16 h DMSO PFT DMSO PFT Aspartate aminotransferase (U/L) * Alanine aminotransferase (U/L) * * a Values are given as means se; *P 0.05 vs. DMSO. p53 IN HEPATIC ENDOTOXEMIA 665

7 those cells (16). This small molecule has further been demonstrated to protect normal cells, but not cells from p53 null mice, in vivo against death induced by anticancer treatment (16). Dose and route of application of PFT (2.2 mg/kg i.p.) were chosen in accordance to the above referenced report (16). The ideal log P value of PFT at physiological ph allows us to predict a high permeability not only at the blood brain barrier (25), but also at the gastrointestinal tract. In addition, the effectiveness of PFT in reducing LPS-induced hepatocellular apoptosis suggests accessibility of this drug to the liver. Immunofluorescent staining of p53 in PFT-pretreated endotoxemic livers implies that cytoplasmic retention of p53 and a slight overall reduction of p53 levels mediate the anti-apoptotic effects of PFT. PFT lowered the nuclear but not the cytoplasmic p53 protein in endotoxemic livers, as has been described for the distribution of p53 in PFT-treated ConA cells upon UV irradiation (16). Given the complex and multifaceted nature of the mechanisms by which p53 induces apoptosis (15), modulation of the nuclear to cytoplasmic p53 ratio by PFT is most likely not the only mechanism of anti-apoptotic PFT action. Protection against apoptotic cell death by PFT may also be attributed to reduced expression of the active NF- B p65 subunit, since NF- B can be induced by p53 and NF- B induction seems to be crucial for p53-mediated apoptosis in at least some cell systems (26). NF- B, known to be activated in all liver cell types after LPS exposure (27), is a key protein modulating not only the apoptotic response but numerous other relevant aspects within the inflammation-associated cascade of tissue injury (28). The promotors of several cytokine genes, such as TNF- and interferon, have NF- B binding sites and represent candidate inducible effector molecules involved in the initiation and progression of inflammatory processes. In addition, NF- B-inducible adhesion molecules like ICAM-1, E-selectin, and VCAM may potentially mediate intrahepatic leukocyte sequestration and further promote leukocyte-dependent injury (29). We observed reduced leukocyte endothelial cell interaction and leukocytic tissue infiltration in PFT-pretreated endotoxemic animals without a concomitant change in ICAM-1 expression. However, previous studies have documented that sequestration of leukocytes in mesenteric and hepatic vasculature can be independent of ICAM-1 during endotoxemia (30 32). Parenchymal cells undergoing apoptosis have been accused of stimulating the sequestration of primed leukocytes (9). This intriguing observation is in accord with the present study, which demonstrates that the inhibition of parenchymal cell apoptosis by chemical p53 inhibition causes a concomitant reduction of intrahepatic leukocyte accumulation compared with vehiclepretreated animals. Though not addressed in the present study, there is evidence that the apoptotic hepatocyte itself and/or generation of a leukocyte chemotactic factor are necessary for inflammatory cell attraction (33). With the possibility in mind that apoptotic cells can aggravate inflammatory responses, targeting the apoptotic pathway may constitute a valid strategy against sepsis-related organ injury. This work is supported by a grant of the Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg, Germany (Vo 450/7 1). B.V. is recipient of a Heisenberg Stipendium (Vo 450/6 1 and 6 2) of the Deutsche Forschungsgemeinschaft. REFERENCES 1. Baue, A. E. (1994) Multiple organ failure, multiple organ dysfunction syndrome, and the systemic inflammatory response syndrome where do we stand? Shock 2, Livingston, D. H., and Deitch, E. A. (1995) Multiple organ failure: a common problem in surgical intensive care unit patients. Ann. Med. 27, Jaeschke, H., and Smith, C. W. (1997) Mechanisms of neutrophil-induced parenchymal cell injury. J. Leukoc. Biol. 61, Leist, M., Gantner, F., Bohlinger, I., Tiegs, G., Germann, P. G., and Wendel, A. (1995) Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am. J. Pathol. 146, Haendeler, J., Messmer, U. K., Brune, B., Neugebauer, E., and Dimmeler, S. (1996) Endotoxin shock leads to apoptosis in vivo and reduces Bcl-2. Shock 6, Morikawa, A., Sugiyama, T., Kato, Y., Koide, N., Jiang, G. Z., Takahashi, K., Tamada, Y., and Yokochi, T. (1996) Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxin shock model. Infect. Immun. 64, Tsuji, H., Harada, A., Mukaida, N., Nakanuma, Y., Bluethmann, H., Kaneko, S., Yamakawa, K., Nakamura, S. I., Kobayashi, K. I., and Matsushima, K. (1997) Tumor necrosis factor receptor p55 is essential for intrahepatic granuloma formation and hepatocellular apoptosis in a murine model of bacterium-induced fulminant hepatitis. Infect. Immun. 65, Leist, M., Gantner, F., Jilg, S., and Wendel, A. (1995) Activation of the 55kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis and nitric oxide release. J. Immunol. 154, Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A., and Jaeschke, H. (1998) Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration of neutrophils in murine models of endotoxin and Fas-antibodyinduced liver injury. Hepatology 28, Gantner, F., Uhlig, S., and Wendel, A. (1995) Quinine inhibits release of tumor necrosis factor, apoptosis, necrosis and mortality in a murine model of septic liver failure. Eur. J. Pharmacol. 294, Yin, X. M., Wang, K., Gross, A., Zhao, Y., Zinkel, S., Klocke, B., Roth, K. A., and Korsmeyer, S. J. (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature (London) 400, Wanner, G. A., Mica, L., Wanner-Schmid, E., Kolb, S. A., Hentze, H., Trentz, O., and Ertel, W. (1999) Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity. FASEB J. 13, Jo, M., Kim, T. H., Seol, D. W., Esplen, J. E., Dorko, K., Billiar, T. R., and Strom, S. C. (2000) Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosisinducing ligand. Nature (London) 6, Sheikh, M. S., and Fornace, A. J. (2000) Role of p53 family members in apoptosis. J. Cell. Physiol. 182, Bates, S., and Vousden, K. H. (1999) Mechanisms of p53- mediated apoptosis. Cell. Mol. Life Sci. 55, Komarov, P. G., Komarova, E. A., Kondratov, R. V., Christov- Tselkov, K., Coon, J. S., Chernov, M. V., and Gugkov, A. V. (1999) A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285, Vol. 17 April 2003 The FASEB Journal T. SCHÄFER ET AL.

8 17. Vollmar, B., Glasz, J., Leiderer, R., Post, S., and Menger, M. D. (1994) Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm ischemia-reperfusion. Am. J. Pathol. 145, Vollmar, B., Menger, M. D., Glasz, J., Leiderer, R., and Messmer, K. (1994) Impact of leukocyte endothelial cell interaction in hepatic ischemia-reperfusion injury. Am. J. Physiol. 267, G786 G Menger, M. D., and Vollmar, B. (2000) Role of microcirculation in transplantation. Microcirculation 7, Clemens, M. G., McDonagh, P. F., Chaudry, I. H., and Baue, A. E. (1985) Hepatic microcirculatory failure after ischemia and reperfusion: improvement with ATP-MgCl 2 treatment. Am. J. Physiol. 248, H804 H Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Rapid detection of octamer binding proteins with miniextracts, prepared from a small number of cells. Nucleic Acids Res. 17, Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, Burns, T. F., and El-Deiry, W. S. (1999) The p53 pathway and apoptosis. J. Cell. Physiol. 181, Jaeschke, H., Fisher, M. A., Lawson, J. A., Simmons, C. A., Farhood, A., and Jones, D. A. (1998) Activation of caspase 3 (CPP-32)-like proteases is essential for TNF- -induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model. J. Immunol. 160, Culmsee, C., Zhu, X., Yu, Q. S., Chan, S. L., Camandola, S., Guo, Z., Greig, N. H., and Mattson, M. P. (2001) A synthetic inhibitor of p53 protects neurons against death induced by ischemia and excitotoxic insults, and amyloid -peptide. J. Neurochem. 77, Ryan, K. M., Ernst, M. K., Rice, N. R., and Vousden, K. H. (2000) Role of NF-kappa B in p53-mediated programmed cell death. Nature (London) 404, Essani, N. A., McGuire, G. M., Manning, A. M., and Jaeschke, H. (1996) Endotoxin-induced activation of the nuclear transcription factor B in hepatocytes, Kupffer cells and endothelial cells in vivo. J. Immunol. 156, Baeurele, P. A., and Henkel, T. (1994) Function and activation of NF-kappa B in the immune system. Annu. Rev. Immunol. 12, Jaesche, H. (1997) Cellular adhesion molecules: regulation and functional significance in the pathogenesis of liver diseases. Am. J. Physiol. 273, G602 G Harris, N. R., Russell, J. M., and Granger, D. N. (1994) Mediators of endotoxin-induced leukocyte adhesion in mesenteric postcapillary venules. Circ. Shock 43, Jaeschke, H., Farhood, A., Fisher, M. A., and Smith, C. W. (1996) Sequestration of neutrophils in the hepatic vasculature during endotoxemia is independent of beta 2 integrins and intercellular adhesion molecule-1. Shock 6, Vollmar, B., Senkel, A., and Menger, M. D. (1995) In vivo evidence that intercellular adhesion-molecule-1 does not mediate endotoxin-induced hepatic leukocyte endothelial interaction. J. Hepatol. 23, Rogers, H. W., Callery, M. P., Deck, B., and Unanue, E. R. (1996) Listeria monocytogenes induces apoptosis of infected hepatocytes. J. Immunol. 156, Received for publication August 21, Accepted for publication December 12, p53 IN HEPATIC ENDOTOXEMIA 667