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Apo-1)-mediated liver apoptosis in Kupffer cell-depleted mice
Hepatology Research 24 (2002) 192– 204
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The regulation of Fas (CD95/Apo-1)-mediated liver apoptosis in Kupffer cell-depleted mice Ion V. Deaciuc a,*, Nympha B. D’Souza b, Mariana Nikolova-Karakashian c, Willem J.S. de Villiers a, Theodore G. Sarphie d, Daniell B. Hill e, Craig J. McClain e,f a
Di6ision of Digesti6e Diseases and Nutrition, Department of Iinternal Medicine, 800 Rose Street, room N649 -0084, Uni6ersity of Kentucky College of Medicine, Lexington, KY 40536, USA b Di6ision of Pulmonary/Critical Care, Department of Internal Medicine, Uni6ersity of Kentucky College of Medicine, Lexington, KY 40536, USA c Department of Physiology, Uni6ersity of Kentucky College of Medicine, Lexington, KY 40536, USA d Department of Anatomy and Cell Biology, Louisiana State Uni6ersity Health Sciences Center, New Orleans, LA 70112, USA e Di6ision of Gastroenterology/Hepatology, Department of Internal Medicine, Uni6ersity of Louis6ille, Louis6ille, KY 40292, USA f Veterans Administration Medical Center, Louis6ille, KY 40292, USA Received 11 October 2001; received in revised form 15 January 2002; accepted 6 February 2002
Abstract The purpose of this study was to further characterize Fas-mediated liver apoptosis. We investigated whether Fas-mediated apoptosis in the liver requires induction of apoptosis-related regulators and whether Kupffer cells play a role in this process. Mice were injected with GdCl3 to deplete/suppress Kupffer cells, followed by treatment with an anti-Fas agonistic antibody, Jo2. Hepatic mRNA levels of several pro- and anti-apoptotic regulators were determined 0.5, 1.5 and 4.0 h after Jo2 injection. Liver histology, TUNEL response, the activity of caspases-3, -8, and -9, and reduced and oxidized glutathione, were also evaluated. Jo2 dramatically increased the number of apoptotic nuclei in the liver, up-regulated mRNA for Bcl-w, Bfl-1, and Bcl-XL, but did not affect pro-apoptotic regulator mRNA expression. Caspase-3, -8 and -9 activity increased at 1.5 h after Jo2-injection. GdCl3 treatment was associated with an increase in the apoptotic effect of Jo2. No effect of Jo2 was recorded on redox state of the free cellular thiol system. These data suggest that: (1) the prompt apoptotic response to Fas-mediated signaling in the liver does not require induction of pro-apoptotic factors; (2) Kupffer cells may play a major role in the liver apoptotic response to Fas ligation by clearing apoptotic cells by phagocytosis; (3) oxidative stress does not seem to play an important role in Fas-mediated liver apoptosis. © 2002 Published by Elsevier Science B.V. Keywords: Anti-apoptotic regulators; Pro-apoptotic regulators; Gadolinium chloride; Glutathione
* Tel.: + 1-859-233-4511x5240; fax: + 1-859-281-4989 E-mail address: [email protected] (I.V. Deaciuc). 1386-6346/02/$ - see front matter © 2002 Published by Elsevier Science B.V. PII: S 1 3 8 6 - 6 3 4 6 ( 0 2 ) 0 0 0 2 1 - 9
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1. Introduction The question whether cells signaled to undergo apoptosis require induction of apoptosisassociated factors/regulators or whether they rely on preexisting proteins is currently under investigation [1]. Recent experimental data indicate that two groups of cells can be distinguished: one group comprises cells that fully rely on preexisting, immediately recruitable apoptotic proteins while another group includes cells that need to induce at least some of the apoptotic machinery proteins in order to undergo apoptosis [1]. It is generally accepted that aberrations from an apoptosis basal rate, characteristic for a given tissue/organ, may lead to both structural and functional alterations and eventually disease. The identification of factors causing changes in the basal apoptotic rate would therefore be crucial. Important information would also include the mechanisms of action and requirement for induction of apoptotic factors. This will determine the end result of an external intervention to reestablish the ‘apoptotic homeostasis’ of a tissue/organ. The above considerations along with our interest in the apoptotic response of the liver to various injurious factors, including alcohol [2– 4], prompted us to determine if Fas-mediated liver apoptosis in the mouse involves induction of apoptotic proteins. Apoptosis was induced by an agonistic Fas antibody, Jo2, one of the most potent liver apoptosis-inducing agents. We furthermore investigated other aspects of Fas-mediated liver apoptosis. Intercellular communication within the liver has recently been demonstrated to be important in apoptosis [5,6]. We explored the contribution of Kupffer cells to Fas-mediated liver apoptosis. We therefore eliminated/suppressed Kupffer cells by in vivo administration of GdCl3 prior to Fas antibody injection and assessed gene expression of a number of apoptosis-associated factors in the liver. In view of the role currently ascribed to oxidative stress as a factor involved in apoptosis [7], we also explored whether Fas-mediated liver
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apoptosis is associated with changes in the redox state of the intracellular free thiol system. For this we determined the content of GSH and GSSG in the liver at different time points after Fas antibody administration.
2. Materials and methods
2.1. Animals and treatment protocols All experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD, Publication no. 86-23, 1985). Male, BALB/c mice, (Harlan Laboratories, Indianapolis, IN), weighing 25–30 g, were acclimated in our animal facility for 8–10 days before experimentation. Housing conditions consisted of constant light–dark cycles (12 h each), mouse chow, water ad libitum and constant temperature and humidity. Forty-eight and 24 h before Jo2 or IgG injection, animals were injected intravenously (penile vein) with either GdCl3 (Aldrich, Milwaukee, WI; 10 mg/kg body weight; corrected for water content) or sterile saline, while under light metofane anesthesia. Twenty-four h after the second GdCl3 injection, the animals were injected intravenously (penile vein) with either IgG or Fas antibody (Jo2), both at 40 mg/100 g body weight (BD Pharmingen, San Diego, CA), and sacrificed 0.5, 1.5 and 4.0 h later.
2.2. Tissue sampling and processing Animals were anesthetized with Nembutal® (Na pentobarbital, 60 mg/kg body weight, intraperitoneally), the abdominal cavity opened and blood (0.5–0.7 ml) withdrawn from the inferior vena cava with citrate-containing syringes. The liver was flushed with phosphate-buffered saline via the portal vein, following infrarenal transection of the inferior vena cava. The liver was quickly blotted on filter paper and a resected lobe placed in formalin for histology. The rest of the liver was placed in liquid nitrogen for the assays described below. Similar lobes were used for similar types of assay.
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2.3. Li6er histology Hematoxylin-eosin staining was used for routine histologic examination of the liver while immunohistochemistry was used to evaluate the number of Kupffer cells using a macrophage marker (F4/80) and the corresponding secondary, horse-radish peroxidase-conjugated antibody (both from Serotec Inc., Raleigh, NC). TUNEL assay was performed using a kit supplied by Intergen (Purchase, NY). Light microscopy images of liver sections for TUNEL were captured with the aid of a Nikon microscope (Nikon Eclipse E600, Nikon, Huntley, IL) equipped with a digital camera (Spot Diagnostic Instruments, Inc., Sterling Heights, MI), processed using a computer program (Adobe Photoshop 5.5, Adobe Systems, Inc., San Jose, CA) and printed with a HewlettPackard DeskJet color printer.
2.4. Biochemical assays Plasma alanine: and aspartate:2-oxoglutarate aminotransferases were assayed using commercially available kits (Sigma Chem. Co., St. Louis, MO). Total RNA extraction and liver processing for caspase activity measurements were described in a previous publication [2]. mRNA for apoptosis-associated factors/regulators was determined with the aid of RNAse protection assay using kits and templates purchased from BD Pharmingen, according to manufacturers’ instructions. Twenty microgram of total RNA were loaded on gels for all mRNAs assays. Caspase-3, -8 and -9 activity was assayed fluorometrically using kits supplied by BD Pharmingen and a luminescence spectrometer with microplate reading capability (LS50B; Perkin-Elmer, Ltd, Beaconsfield, UK). The measurements were performed under the conditions of linearity of the reaction velocity as a function of time (0–90 min) and the amount of protein in sample (0.3–1.0 mg protein) with an r 2 =0.985 – 0.995 for both measurements. GSH and GSSG were assayed in 5% metaphosphoric acid-extracts of the liver with the aid of an enzymatic procedure (Oxis International, Inc., Portland, OR). Protein assay was performed with Bradford’s procedure using a BioRad kit (Hercules, CA) and
bovine serum albumin as standard. Gels were imaged on Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and quantified with ImageQuant computer program (Molecular Dynamics).
2.5. Statistics The results were statistically evaluated according to Student-Neuman-Keul’s t-test with P0 0.05 as the limit of significance.
3. Results
3.1. Li6er histology The liver of GdCl3-treated mice displayed significantly decreased number of Kupffer cells (19.69 4.5 in GdCl3-treated versus 52.090.8 cells/microscopic field in saline-treated mice; 10 fields were examined for each of 3 livers in both groups; PB0.05; an example is shown in Fig. 1A and B). In control animals the sinusoids appeared to be almost completely filled with normal Kupffer cells. In contrast, Kupffer cells were sparse in GdCl3-treated mice and apparently smaller in size. Hematoxylin-eosin staining displayed normal appearance of the liver in IgG-treated groups at all time points (Fig. 2A; only 4 h time point is shown). In Jo2-treated group, with or without GdCl3, the liver preserved its normal appearance at both 0.5 and 1.5 h after the treatment (not shown). In the 4 h group, however, Jo2 induced marked necrosis with neutrophil infiltration (Fig. 2B). A somewhat more accentuated necrosis, expressed as larger necrotic areas, was seen in GdCl3 + Jo2-treated mice 4 h after Jo2 injection (Fig. 2C). TUNEL reaction analysis of the livers showed that in Saline+IgG injected mice, 0.5 h after injection, the number of apoptotic nuclei was very low (0.390.02%; Fig. 3A). One and a half h after Jo2 injection, a marked increase in the number of apoptotic nuclei was observed (13.391.2%; Fig. 3B). GdCl3 treatment had a dual effect on apoptosis: it both increased the number of apoptotic nuclei and changed their morphology. Thus, in the liver of Jo2-treated mice the apoptotic nuclei
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Fig. 1. Section through livers of a GdCl3 (A)- or saline (B)-treated mouse showing Kupffer cell depletion by GdCl3 treatment. Kupffer cells are stained brown and a few of them are indicated by arrowheads. Note that Kupffer cells in GdCl3-treated mice are fewer and appear smaller compared with more numerous and larger Kupffer cells filling the sinusoid in saline-treated mice. Isotype-matched negative controls are not shown. Original magnification: ×400.
were small and mostly located in the sinusoidal space (or in its immediate vicinity; Fig. 3B) while in GdCl3 +Jo2-treated mice most of the apoptotic nuclei were large and predominantly located in the extra-sinusoidal space (Fig. 3C). Four h after Jo2 injection, the number of apoptotic nuclei was slightly larger than at 1.5 h (19.891.5%; Fig. 3D). GdCl3 further increased this number (38.29 4.5%) and also reduced the occurrence of smaller apoptotic nuclei (Fig. 3E). Numerical data and statistical significance on the frequency of apoptotic nuclei are given in Fig. 4.
tested, i.e. Bcl-w, Bfl-1, and Bcl-XL and they consisted of up-regulation at the 1.5 and 4 h time points (Fig. 6). Bcl-2 was not detected. No changes were recorded at 0.5 h after either Jo2 or IgG injection, nor were there any effects of GdCl3 on mRNA expression of Bcl-w, Bfl-1 and Bcl-XL. mRNA expression for a number of pro-apoptotic regulators, including Bak, Bad, Bax, Fas, FasL, RIP and FADD, did not undergo significant changes (Fig. 7; shown for Bak, Bad and Bax only). A representative gel obtained with RNAse protection assay is given in Fig. 8.
3.2. Plasma li6er enzyme acti6ity
3.4. Caspase-3, -8, and -9 acti6ity
The activity of both alanine: and aspartate:2oxoglutarate aminotransferases was markedly increased 4 h after Jo2 administration (Fig. 5). No changes were observed at 0.5 and 1.5 h after Jo2 administration nor were there any effects of GdCl3 on either of the enzymes.
Caspase-3 activity was significantly increased by Jo2 in 1.5 h group and, to a lesser extent, in the 4 h group (Fig. 9). Interestingly, GdCl3 enhanced the effect of Jo2 in the 1.5 h group. A similar picture was observed for caspase-8 and caspase-9 activity (Fig. 9). All three caspases displayed lower activity in the 4 h group. To test possible spillover of the enzyme from the markedly necrotic liver seen 4 h after Jo2 injection, we measured caspase-3 activity in plasma (Fig. 10). This demonstrated a dramatic (107-fold) increase in enzyme activity in the 4 h group as compared to the 1.5 h group (Fig. 10). In this latter group the enzyme activity in plasma was close to the limits of detection. No changes were observed in either of the caspases’ activity 0.5 h after Jo2 or IgG injection.
3.3. Gene expression of apoptosis-associated factors/regulators The following apoptosis-associated factors’ mRNA expression was determined in this study: Bcl-w, Bfl-1, Bcl-XL, Bcl-XS, Bcl-2, Bak, Bad, Bax, Fas, FasL, RIP, FADD, and caspases-3, -8 and -9. Significant changes in the mRNA expression were observed for anti-apoptotic regulators
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Fig. 2. Liver histology as revealed by hematoxylin-eosin staining. (A) Section of a liver from an IgG-treated mouse 4 h after treatment displaying normal appearance. (B) Section of a liver from a Jo2-treated mouse 4 h after antibody injection. Marked necrosis (large arrowheads indicating a few necrotic areas) and neutrophil infiltration (small arrowheads) are present in this liver. (C) Section of a liver from GdCl3 + Jo2-treated mouse showing more pronounced necrosis. Labeling as in (C). Original magnification: × 400.
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3.5. Li6er glutathione content GSH content remained unchanged in the 0.5 h group under all experimental conditions tested, but was decreased by Jo2 in the 1.5 h group only in GdCl3-treated animals (Fig. 11). In the 4 h group, Jo2, with or without GdCl3, markedly decreased the GSH content. GSSG content was significantly increased by GdCl3 in the 1.5 h group but decreased in the 4 h group by both Jo2 and GdCl3. A decrease in the GSH/GSSG ratio was recorded both in the 1.5 and 4 h groups but only in the GdCl3-treated mice, with or without Jo2 injection (Fig. 11). Of note, in Sal+Jo2-treated animals no marked
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changes in the GSH/GSSG ratio were observed at any of the time points tested.
4. Discussion
4.1. Fas-mediated apoptosis in the mouse li6er does not require induction of pro-apoptotic proteins The major finding of this study pertains to the gene expression of apoptosis-associated factors during the course of Fas-mediated apoptosis in the mouse liver in vivo. To our knowledge, this is
Fig. 3. Liver TUNEL response in various treatment groups. (A) Section of a liver from IgG-treated mouse at 1.5 h after IgG injection. Note the presence of very few apoptotic nuclei (arrowheads). Eight high magnification fields ( × 400) from 4 livers were examined here and in subsequent TUNEL pictures and the numbers were averaged. (B) Section of a liver from Saline + Jo2-treated mouse, 1.5 h after Jo2 injection. Note the higher frequency of apoptotic nuclei (13.3 9 1.2% of total nuclei; arrowheads). The small brown bodies (small arrowheads) represent apoptotic bodies during the final stage of apoptosis. Very few large apoptotic nuclei are present (large arrowheads). (C) Section of a liver from GdCl3 +Jo2-treated mouse 1.5 h after Jo2 injection. When compared to the corresponding control (i.e. Saline + Jo2 group, see (C) above), the number of apoptotic nuclei (arrowheads) increased significantly and there was a higher proportion of large apoptotic nuclei denoting less phagocytosis and/or higher apoptosis. (D) Section of a liver of Saline+ Jo2-treated mouse 4 h after Jo2 injection. The number of apoptotic nuclei (arrowheads) was similarly increased compared to the corresponding group at 1.5 h after treatment (C) and the number of large apoptotic nuclei appeared higher. (E) Section of a liver of a GdCl3 + Jo2-treated mouse 4 h after Jo2 injection. The number of apoptotic nuclei (arrowheads) markedly increased as compared to (D). This change was associated with an even higher number of ‘large’ apoptotic nuclei indicating less phagocytosis and/or higher apoptotic rate. Original magnification: ×400. Numerical data and statistical significance on apoptotic nuclei/bodies are given in Fig. 4.
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the first study to undertake such an approach of the Fas-mediated liver apoptosis. Assessment of gene expression in this study covered a wide spectrum of apoptosis genes including Fas-associated signaling molecules (e.g. Fas receptor, FasL, death-domain bearing proteins, such as FADD and RIP), apoptosis adaptors (e.g. Bcl-w, Bfl-1, Bcl-XL, Bcl-XS, Bcl-2, Bak, Bad, and Bax) and
executioners of apoptosis (e.g. caspase-3, -8, and -9). Our data show that only a few anti-apoptotic factors, i.e. Bcl-w, Bcl-XL and Bfl-1, underwent changes consisting of up-regulation of mRNA expression. No changes in gene expression of tested pro-apoptotic factors were observed. We therefore infer that Fas-dependent apoptotic sig-
Fig. 3. (Continued)
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the liver against massive apoptosis induced by Fas antibody [9]. Our data suggest that de novo protein synthesis may be implicated in the modulation of anti-apoptotic proteins.
4.2. Kupffer cells play an important role in Fas-mediated li6er apoptosis
Fig. 4. Time course of the number of apoptotic nuclei in the liver. Plotted are means 9SEM (vertical bars) for 4 individuals in a group. Here and in subsequent graphs, the following abbreviations are used: GdCl, gadolinium chloride (GdCl3); Sal, saline. (a) Significantly different (P B0.05) from Sal + Jo2 group; (b) significantly different (PB 0.05) from Sal + IgG and Gd+ IgG groups.
nal finds the mouse liver replete with all the necessary equipment to promptly execute apoptotic death. Thus, de novo protein synthesis is not required in order for the cells to undergo Fas-mediated apoptosis. A similar conclusion was reached in an earlier study by Leist et al. [8] in which a different experimental approach was used: administration of protein synthesis inhibitors in vivo to mice subsequently treated with anti-Fas antibody. However, in the cited study no proof of complete inhibition of protein synthesis was given. In addition, Leist et al.’s study does not allow any conclusion on a possible involvement of transcriptional/translational steps in the regulation of anti-apoptotic factors. Our study provides information on this issue showing that the regulation of anti-apoptotic factors in response to Fas receptor ligation may be effected at the level of transcription. The up-regulation of anti-apoptotic factors observed in this study may be regarded as an attempt of cells to counteract the apoptotic signal action and to avoid death. This contention is consistent with earlier studies on Fas-mediated liver apoptosis in the mouse showing that Bcl-XL was up-regulated shortly (2 h) after Fas antibody injection and even more so after HGF treatment of Fas antibody-injected mice [9]. HGF protects
In order to assess a role for Kupffer cells in the apoptotic response of the liver to Fas antibody, we treated the mice with GdCl3 prior to antibody injection. This treatment has previously been shown to have two main effects: a partial depletion of the Kupffer cell population and a marked reduction of the phagocytic capacity of the remaining Kupffer cells. We confirmed that, under our experimental conditions, GdCl3 treatment reduces the Kupffer cell population significantly in
Fig. 5. Plasma alanine: and aspartate:2-oxoglutarate aminotransferase activity (ALT and AST, respectively). Plotted are means 9SEM (vertical Ý bars) for 4 – 6 animals in a group. (a) PB 0.05 versus IgG-treated groups.
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One and, perhaps, the most important role of Kupffer cell in the apoptotic response of the liver consists of phagocytosis of apoptotic cells. It is currently accepted that Kupffer cells are the primary phagocytes of the liver involved in apoptotic cell clearance [13,14]. Our experimental results are consistent with this observation. Under the experimental conditions presented in this study, a
Fig. 6. Hepatic mRNA expression of Bcl-w, Bfl-1 and Bcl-XL. Plotted are means 9 SEM (vertical Ý bars) for 4 –6 individuals in a group. (a) PB 0.05 versus IgG-treated groups; (b) PB 0.05 versus GdCl + Jo2-treated group; L32, a house keeping gene.
the mouse liver. It is important to note that GdCl3 treatment, as applied here and in other studies on rats and mice [10– 12], does not completely deplete the Kupffer cell population. The remaining Kupffer cells, however, have a markedly impaired phagocytosing capacity, which is an important issue for the interpreation of our results because it allows a fair estimate of Kupffer cell contribution to phagocytosis of apoptotic cells.
Fig. 7. Hepatic mRNA expression for three of the tested pro-apoptotic adaptors/regulators. Plotted are means 9SEM (vertical Ý bars) for 4 – 6 individuals in a group. No significant differences were observed as a function of either treatment or time after injection. Note that in this graph the Y scale is expanded to allow a better examination of the plotted parameters.
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Fig. 8. Representative RNAse protection assay gel showing changes in Bcl-w, Bfl-1 and Bcl-XL 4 h after Jo2 (lanes 1 – 6) or IgG (lanes 7 – 12) injection. L32, a housekeeping gene.
marked increase in the number of apoptotic nuclei was seen both at 1.5 and, particularly, 4 h after Jo2 injection. Such an increase could be ascribed to two factors: a potentiation by GdCl3 of Jo2 apoptotic effects (Figs. 4 and 9) and the decrease in phagocytosis of apoptotic cells due to a diminution of both the number of Kupffer cells and their phagocytic capacity. The mechanism by which GdCl3 enhances Jo2-induced apoptosis has not been approached in this study and remains to be elucidated. The partial elimination/suppression of Kupffer cells in the livers of GdCl3 +Jo2-treated mice was associated with a marked increase in the number of ‘full size’ apoptotic nuclei along with a significant reduction in the number of smaller apoptotic nuclei when compared to the livers of Jo2-treated controls. The ‘small’ apoptotic nuclei (or apoptotic bodies) may represent nuclei during the last phase of apoptosis. In our experiments the exacerbated apoptosis, well expressed at 1.5 h after Jo2 injection, was not associated with necrosis as clearly demonstrated by histologic examination of the liver and by the lack of an increase in plasma ALT activity. Necrosis ensued later, i.e. at 4 h after Jo2 injection
and, for the following reasons, we believe that necrosis is caused by an exacerbated apoptosis. First, apoptosis preceded necrosis. Therefore, the positively stained nuclei in the TUNEL reaction that occurred before the 3 h time point can be a considered as resulting exclusively from apoptosis; however, at a later time point as necrosis settled, the number of apoptotic nuclei increased as a result of both apoptosis and necrosis. Second, prevention of Jo2 in vivo apoptotic effects on the liver by various agents markedly reduces liver necrosis as assessed with histologic and biochemical procedures [15–18]. Third, Jo2 does not produce solely necrosis, hence the latter cannot be dissociated from Jo2 apoptotic effects. It is not known why enhanced apoptosis such as occurs in this model of Fas antibody administration is followed by necrosis. We agree with an earlier suggestion that the transition from apoptosis to necrosis is caused by the abrupt increase of the number of apoptotic cells surpassing the phagocytic capacity of the tissue, thus leading to necrosis [19,20]. How much apoptosis is needed to overwhelm the liver’s capacity to phagocytose apoptotic cells is not known. Our data show that in the saline-in-
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jected, Jo2-treated mice the number of apoptotic nuclei increased with the time reaching 13.3% at 1.5 h (Fig. 3C) and 19.8% at 4 h (Fig. 3D). However, necrosis was seen only at 4 h after Jo2 injection suggesting that when the number of apoptotic cells lies between 13 and 20%, the phagocytosing capacity of the liver is surpassed by the increase in the number of apoptotic cells
Fig. 10. Caspase-3 activity in plasma. Plotted are means 9 SEM (vertical Ý bars) for 4 – 6 individuals in a group. (a) PB 0.05 versus IgG-treated animals in the 1.5 h group; (b) PB 0.05 versus Sal + Jo2 and GdCl +Jo2-treated mice.
thus leading to necrosis. This range may change when Kupffer cells are partially depleted by GdCl3 treatment.
4.3. Fas-mediated li6er apoptosis is not associated with changes in the intracellular free thiol redox system
Fig. 9. Caspase-3, -8 and -9 (abbreviated as Casp-) activity in the liver of mice. Plotted are means 9SEM (vertical Ý bars) for 4– 6 individuals in a group. (a) PB 0.05 versus GdCl + IgG-treated animals; (b) PB 0.05 versus GdCl + Jo2-treated mice.
It has been documented that cytokine-induced apoptosis in a hepatocyte cell line (Hep 3B) was preceded and paralleled by a decrease in GSH which reflected elevated oxidative stress [21]. Jo2induced liver apoptosis, as seen at 1.5 h after Fas antibody injection, was neither preceded by, nor associated with, any alteration in the redox state of the glutathione system in the liver. This was shown by a lack of change in the GSH to GSSG ratio at the 0.5, 1.5 and 4 h time points after Jo2 injection. While our results do not strongly implicate oxidative stress in Jo2-induced liver apoptosis, they nevertheless are consistent with the idea that an intact GSH–GSSG system is required for liver apoptosis to proceed [22]. At 4 h after Jo2 injection a dramatic decrease in the content of both GSH and GSSG could be seen which did not result in a change in GSH/GSSG ratio. These decreases should be regarded as a consequence rather than as a cause of apoptosis/necrosis. In the 4 h Jo2-treatment group some of the changes in the liver
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glutathione content might also have been caused by massive organ cytolysis as demonstrated by marked spillover into the circulation of aminotransferases and caspase-3. It is intriguing to note that Kupffer cell depletion associated with GdCl3 treatment with or without Jo2, shifted the glutathione system to a more oxidized state as shown by decreased GSH/ GSSG ratio. Although the exact cause of such changes needs to be determined, this may indicate an important role for Kupffer cells in the maintenance of a physiological redox state of the liver GSH –GSSG system. In summary our study allows the following conclusions: (1) Fas-mediated liver apoptosis does not require up-regulation of Fas-associated death domain proteins (i.e. FADD and RIP), pro-apoptotic adaptors (i.e. Bcl-XS, Bak, Bad, Bax,) or executioners of apoptosis (caspases -3, -8 and -9); rather, preexisting apoptotic equipment is sufficient for a prompt apoptotic response. In addition, Fas-mediated liver apoptosis is associated with up-regulation of anti-apoptotic factors in an attempt to avoid death; (2) Kupffer cells seemingly by and, therefore, does not involve as a causative factor, changes in the intracellular free thiol system redox state, as reflected by GSH/GSSG ratio.
Acknowledgements
Fig. 11. Content of reduced (GSH) and oxidized (GSSG) glutathione and GSH to GSSG ratio in the liver of mice. Plotted are means 9 SEM (vertical Ý bars) for 4 –6 individuals in a group. For GSH: (a) PB 0.05 versus Sal + Jo2-treated group; (b) P B 0.05 versus Sal – IgG-treated group; (c) PB 0.05 versus Gd – IgG and Sal –IgG-treated groups. For GSSG: (a) P B 0.05 versus Sal – IgG; (b) PB0.05 versus Sal –Jo2-treated group; (c) P B 0.05 versus corresponding subgroup in the 1.5 h group. For GSH/GSSG ratio: (a) PB0.05 versus Sal –IgGtreated group; (b) P B 0.05 versus Sal –Jo2-treated group; (c) and (d) P B 0.05 versus Sal –Jo2-treated group.
This study was supported by the National Institute on Alcohol Abuse an Alcoholism Grants AA10762 (to CJM), AA12314 (to IVD), and AA 12774 (to WdV), and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to CJM). This material is also the result of work supported with resources and the use of facilities at the Lexington Veterans Administration Medical Center, KY 40506.
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[2] Deaciuc IV, Fortunato F, D’Souza NB, Hill DB, Schmidt J, Lee EY, et al. Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide. Alcohol Clin Exp Res 1999;23:349 –56. [3] Deaciuc IV, D’Souza NB, Fortunato F, Hill DB, Sarphie TG, McClain CJ. Alcohol-induced sinusoidal endothelial cell dysfunction in the mouse is associated with exacerbated apoptosis and can be reversed by caspase inhibition. Hepatol Res 2001;19:85 –97. [4] Deaciuc IV, Fortunato F, D’Souza NB, Hill DB, McClain CJ. Chronic alcohol exposure of rats exacerbates apoptosis in hepatocytes and sinusoidal endothelial cells. Hepatol Res 2001;19:306 –24. [5] Lawson JA, Fisher MA, Simmons CA, Farhood A, Jaeschke H. Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration of neutrophils in murine models of endotoxin and Fas-antibody-induced liver injury. Hepatology 1998;28:761 –7. [6] Hamada E, Nishida T, Uchiyama Y, Nakamura J, Isahara K, Kazuo H, et al. Activation of Kupffer cells and caspase-3 involved in rat hepatocyte apoptosis induced by endotoxin. J Hepatol 1999;30:807 –18. [7] Jabs T. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol 1999;57:231 –45. [8] Leist M, Gantner F, Kunstle G, Bohlinger I, Tiegs G, Bluethmann H, et al. The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure. Mol Med 1996;2:109 – 24. [9] Kosai K, Matsumoto K, Nagata S, Tsujimoto Y, Nakamura T. Abrogation of Fas-induced fulminant hepatic failure in mice by hepatocyte growth factor. Biochem Biophys Res Commun 1998;244:683 – 90. [10] Callery MP, Kamei T, Flye MW. Kupffer cell blockade increases mortality during intra-abdominal sepsis despite improving systemic immunity. Arch Surg 1990;125:36 –41. [11] Ru¨ ttinger D, Vollmar B, Wanner GA, Messmer K. In vivo assessment of hepatic alterations following gadolinium chloride-induced Kupffer cell blockade. J Hepatol
1996;25:960 – 7. [12] Hardonk MJ, Dijkhuis FWJ, Hulstaert CE, Koudstaal J. Heterogeneity of rat liver and spleen macrophages in gadolinium chloride-induced elimination and repopulation. J Leuk Biol 1992;52:296 – 302. [13] Savill J. Recognition and phagocytosis of cells undergoing apoptosis. Br Med Bul 1997;53:491 – 508. [14] Olynyk JK, Britton RS, Stephenson AH, Leicester KL, O’Neill R, Bacon BR. An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells. Liver 1999;19:418 – 22. [15] Okamoto T. Pentoxifylline inhibits anti-Fas antibody-induced hepatitis by affecting downstream of CPP32-like activity in mice. Intl J Mol Med 1999;4:601 – 3. [16] Wanner GA, Mica L, Wanner-Schmidt E, Kolb SA, Hentze H, Trentz O, et al. Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity. FASEB J 1999;13:1239 – 48. [17] Yamamoto M, Miura N, Ohtake N, Amagaya S, Ishige A, Sasaki H, et al. Genipin, a metabolite derived from herbal medicine inchin-ko-to, and suppression of Fas-induced lethal liver apoptosis in mice. Gastroenterology 2000;118:380 – 9. [18] Li XK, Fujino M, Guo L, Okuyama T, Funeshima N, Hashimoto M, Okabe K, et al. Inhibition of Fas-mediated fulminant hepatitis in CrmA gene-transfected mice. Biochem Biophys Res Commun 2000;273:101 – 9. [19] Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasuga T, Kitamura Y, et al. Lethal effect of the anti-Fas antibody in mice. Nature 1993;364:806 – 9. [20] Maher J, Gores GJ. Apoptosis: silent killer or neutron bomb? Hepatology 1998;28:865 – 7. [21] Ribeiro A, Bronk SF, Roberts PJ, Urrutia R, Gores GJ. The transforming growth factor b1-inducible transcription factor, TIEG1, mediates apoptosis through oxidative stress. Hepatology 1999;30:1490 – 7. [22] Hentze H, Ku¨ nstle G, Volbracht C, Ertel W, Wendel A. CD95-mediated murine hepatic apoptosis requires an intact glutathione status. Hepatology 1999;30:177 – 85.
www.elsevier.com/locate/ihepcom
The regulation of Fas (CD95/Apo-1)-mediated liver apoptosis in Kupffer cell-depleted mice Ion V. Deaciuc a,*, Nympha B. D’Souza b, Mariana Nikolova-Karakashian c, Willem J.S. de Villiers a, Theodore G. Sarphie d, Daniell B. Hill e, Craig J. McClain e,f a
Di6ision of Digesti6e Diseases and Nutrition, Department of Iinternal Medicine, 800 Rose Street, room N649 -0084, Uni6ersity of Kentucky College of Medicine, Lexington, KY 40536, USA b Di6ision of Pulmonary/Critical Care, Department of Internal Medicine, Uni6ersity of Kentucky College of Medicine, Lexington, KY 40536, USA c Department of Physiology, Uni6ersity of Kentucky College of Medicine, Lexington, KY 40536, USA d Department of Anatomy and Cell Biology, Louisiana State Uni6ersity Health Sciences Center, New Orleans, LA 70112, USA e Di6ision of Gastroenterology/Hepatology, Department of Internal Medicine, Uni6ersity of Louis6ille, Louis6ille, KY 40292, USA f Veterans Administration Medical Center, Louis6ille, KY 40292, USA Received 11 October 2001; received in revised form 15 January 2002; accepted 6 February 2002
Abstract The purpose of this study was to further characterize Fas-mediated liver apoptosis. We investigated whether Fas-mediated apoptosis in the liver requires induction of apoptosis-related regulators and whether Kupffer cells play a role in this process. Mice were injected with GdCl3 to deplete/suppress Kupffer cells, followed by treatment with an anti-Fas agonistic antibody, Jo2. Hepatic mRNA levels of several pro- and anti-apoptotic regulators were determined 0.5, 1.5 and 4.0 h after Jo2 injection. Liver histology, TUNEL response, the activity of caspases-3, -8, and -9, and reduced and oxidized glutathione, were also evaluated. Jo2 dramatically increased the number of apoptotic nuclei in the liver, up-regulated mRNA for Bcl-w, Bfl-1, and Bcl-XL, but did not affect pro-apoptotic regulator mRNA expression. Caspase-3, -8 and -9 activity increased at 1.5 h after Jo2-injection. GdCl3 treatment was associated with an increase in the apoptotic effect of Jo2. No effect of Jo2 was recorded on redox state of the free cellular thiol system. These data suggest that: (1) the prompt apoptotic response to Fas-mediated signaling in the liver does not require induction of pro-apoptotic factors; (2) Kupffer cells may play a major role in the liver apoptotic response to Fas ligation by clearing apoptotic cells by phagocytosis; (3) oxidative stress does not seem to play an important role in Fas-mediated liver apoptosis. © 2002 Published by Elsevier Science B.V. Keywords: Anti-apoptotic regulators; Pro-apoptotic regulators; Gadolinium chloride; Glutathione
* Tel.: + 1-859-233-4511x5240; fax: + 1-859-281-4989 E-mail address: [email protected] (I.V. Deaciuc). 1386-6346/02/$ - see front matter © 2002 Published by Elsevier Science B.V. PII: S 1 3 8 6 - 6 3 4 6 ( 0 2 ) 0 0 0 2 1 - 9
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1. Introduction The question whether cells signaled to undergo apoptosis require induction of apoptosisassociated factors/regulators or whether they rely on preexisting proteins is currently under investigation [1]. Recent experimental data indicate that two groups of cells can be distinguished: one group comprises cells that fully rely on preexisting, immediately recruitable apoptotic proteins while another group includes cells that need to induce at least some of the apoptotic machinery proteins in order to undergo apoptosis [1]. It is generally accepted that aberrations from an apoptosis basal rate, characteristic for a given tissue/organ, may lead to both structural and functional alterations and eventually disease. The identification of factors causing changes in the basal apoptotic rate would therefore be crucial. Important information would also include the mechanisms of action and requirement for induction of apoptotic factors. This will determine the end result of an external intervention to reestablish the ‘apoptotic homeostasis’ of a tissue/organ. The above considerations along with our interest in the apoptotic response of the liver to various injurious factors, including alcohol [2– 4], prompted us to determine if Fas-mediated liver apoptosis in the mouse involves induction of apoptotic proteins. Apoptosis was induced by an agonistic Fas antibody, Jo2, one of the most potent liver apoptosis-inducing agents. We furthermore investigated other aspects of Fas-mediated liver apoptosis. Intercellular communication within the liver has recently been demonstrated to be important in apoptosis [5,6]. We explored the contribution of Kupffer cells to Fas-mediated liver apoptosis. We therefore eliminated/suppressed Kupffer cells by in vivo administration of GdCl3 prior to Fas antibody injection and assessed gene expression of a number of apoptosis-associated factors in the liver. In view of the role currently ascribed to oxidative stress as a factor involved in apoptosis [7], we also explored whether Fas-mediated liver
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apoptosis is associated with changes in the redox state of the intracellular free thiol system. For this we determined the content of GSH and GSSG in the liver at different time points after Fas antibody administration.
2. Materials and methods
2.1. Animals and treatment protocols All experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD, Publication no. 86-23, 1985). Male, BALB/c mice, (Harlan Laboratories, Indianapolis, IN), weighing 25–30 g, were acclimated in our animal facility for 8–10 days before experimentation. Housing conditions consisted of constant light–dark cycles (12 h each), mouse chow, water ad libitum and constant temperature and humidity. Forty-eight and 24 h before Jo2 or IgG injection, animals were injected intravenously (penile vein) with either GdCl3 (Aldrich, Milwaukee, WI; 10 mg/kg body weight; corrected for water content) or sterile saline, while under light metofane anesthesia. Twenty-four h after the second GdCl3 injection, the animals were injected intravenously (penile vein) with either IgG or Fas antibody (Jo2), both at 40 mg/100 g body weight (BD Pharmingen, San Diego, CA), and sacrificed 0.5, 1.5 and 4.0 h later.
2.2. Tissue sampling and processing Animals were anesthetized with Nembutal® (Na pentobarbital, 60 mg/kg body weight, intraperitoneally), the abdominal cavity opened and blood (0.5–0.7 ml) withdrawn from the inferior vena cava with citrate-containing syringes. The liver was flushed with phosphate-buffered saline via the portal vein, following infrarenal transection of the inferior vena cava. The liver was quickly blotted on filter paper and a resected lobe placed in formalin for histology. The rest of the liver was placed in liquid nitrogen for the assays described below. Similar lobes were used for similar types of assay.
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2.3. Li6er histology Hematoxylin-eosin staining was used for routine histologic examination of the liver while immunohistochemistry was used to evaluate the number of Kupffer cells using a macrophage marker (F4/80) and the corresponding secondary, horse-radish peroxidase-conjugated antibody (both from Serotec Inc., Raleigh, NC). TUNEL assay was performed using a kit supplied by Intergen (Purchase, NY). Light microscopy images of liver sections for TUNEL were captured with the aid of a Nikon microscope (Nikon Eclipse E600, Nikon, Huntley, IL) equipped with a digital camera (Spot Diagnostic Instruments, Inc., Sterling Heights, MI), processed using a computer program (Adobe Photoshop 5.5, Adobe Systems, Inc., San Jose, CA) and printed with a HewlettPackard DeskJet color printer.
2.4. Biochemical assays Plasma alanine: and aspartate:2-oxoglutarate aminotransferases were assayed using commercially available kits (Sigma Chem. Co., St. Louis, MO). Total RNA extraction and liver processing for caspase activity measurements were described in a previous publication [2]. mRNA for apoptosis-associated factors/regulators was determined with the aid of RNAse protection assay using kits and templates purchased from BD Pharmingen, according to manufacturers’ instructions. Twenty microgram of total RNA were loaded on gels for all mRNAs assays. Caspase-3, -8 and -9 activity was assayed fluorometrically using kits supplied by BD Pharmingen and a luminescence spectrometer with microplate reading capability (LS50B; Perkin-Elmer, Ltd, Beaconsfield, UK). The measurements were performed under the conditions of linearity of the reaction velocity as a function of time (0–90 min) and the amount of protein in sample (0.3–1.0 mg protein) with an r 2 =0.985 – 0.995 for both measurements. GSH and GSSG were assayed in 5% metaphosphoric acid-extracts of the liver with the aid of an enzymatic procedure (Oxis International, Inc., Portland, OR). Protein assay was performed with Bradford’s procedure using a BioRad kit (Hercules, CA) and
bovine serum albumin as standard. Gels were imaged on Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and quantified with ImageQuant computer program (Molecular Dynamics).
2.5. Statistics The results were statistically evaluated according to Student-Neuman-Keul’s t-test with P0 0.05 as the limit of significance.
3. Results
3.1. Li6er histology The liver of GdCl3-treated mice displayed significantly decreased number of Kupffer cells (19.69 4.5 in GdCl3-treated versus 52.090.8 cells/microscopic field in saline-treated mice; 10 fields were examined for each of 3 livers in both groups; PB0.05; an example is shown in Fig. 1A and B). In control animals the sinusoids appeared to be almost completely filled with normal Kupffer cells. In contrast, Kupffer cells were sparse in GdCl3-treated mice and apparently smaller in size. Hematoxylin-eosin staining displayed normal appearance of the liver in IgG-treated groups at all time points (Fig. 2A; only 4 h time point is shown). In Jo2-treated group, with or without GdCl3, the liver preserved its normal appearance at both 0.5 and 1.5 h after the treatment (not shown). In the 4 h group, however, Jo2 induced marked necrosis with neutrophil infiltration (Fig. 2B). A somewhat more accentuated necrosis, expressed as larger necrotic areas, was seen in GdCl3 + Jo2-treated mice 4 h after Jo2 injection (Fig. 2C). TUNEL reaction analysis of the livers showed that in Saline+IgG injected mice, 0.5 h after injection, the number of apoptotic nuclei was very low (0.390.02%; Fig. 3A). One and a half h after Jo2 injection, a marked increase in the number of apoptotic nuclei was observed (13.391.2%; Fig. 3B). GdCl3 treatment had a dual effect on apoptosis: it both increased the number of apoptotic nuclei and changed their morphology. Thus, in the liver of Jo2-treated mice the apoptotic nuclei
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Fig. 1. Section through livers of a GdCl3 (A)- or saline (B)-treated mouse showing Kupffer cell depletion by GdCl3 treatment. Kupffer cells are stained brown and a few of them are indicated by arrowheads. Note that Kupffer cells in GdCl3-treated mice are fewer and appear smaller compared with more numerous and larger Kupffer cells filling the sinusoid in saline-treated mice. Isotype-matched negative controls are not shown. Original magnification: ×400.
were small and mostly located in the sinusoidal space (or in its immediate vicinity; Fig. 3B) while in GdCl3 +Jo2-treated mice most of the apoptotic nuclei were large and predominantly located in the extra-sinusoidal space (Fig. 3C). Four h after Jo2 injection, the number of apoptotic nuclei was slightly larger than at 1.5 h (19.891.5%; Fig. 3D). GdCl3 further increased this number (38.29 4.5%) and also reduced the occurrence of smaller apoptotic nuclei (Fig. 3E). Numerical data and statistical significance on the frequency of apoptotic nuclei are given in Fig. 4.
tested, i.e. Bcl-w, Bfl-1, and Bcl-XL and they consisted of up-regulation at the 1.5 and 4 h time points (Fig. 6). Bcl-2 was not detected. No changes were recorded at 0.5 h after either Jo2 or IgG injection, nor were there any effects of GdCl3 on mRNA expression of Bcl-w, Bfl-1 and Bcl-XL. mRNA expression for a number of pro-apoptotic regulators, including Bak, Bad, Bax, Fas, FasL, RIP and FADD, did not undergo significant changes (Fig. 7; shown for Bak, Bad and Bax only). A representative gel obtained with RNAse protection assay is given in Fig. 8.
3.2. Plasma li6er enzyme acti6ity
3.4. Caspase-3, -8, and -9 acti6ity
The activity of both alanine: and aspartate:2oxoglutarate aminotransferases was markedly increased 4 h after Jo2 administration (Fig. 5). No changes were observed at 0.5 and 1.5 h after Jo2 administration nor were there any effects of GdCl3 on either of the enzymes.
Caspase-3 activity was significantly increased by Jo2 in 1.5 h group and, to a lesser extent, in the 4 h group (Fig. 9). Interestingly, GdCl3 enhanced the effect of Jo2 in the 1.5 h group. A similar picture was observed for caspase-8 and caspase-9 activity (Fig. 9). All three caspases displayed lower activity in the 4 h group. To test possible spillover of the enzyme from the markedly necrotic liver seen 4 h after Jo2 injection, we measured caspase-3 activity in plasma (Fig. 10). This demonstrated a dramatic (107-fold) increase in enzyme activity in the 4 h group as compared to the 1.5 h group (Fig. 10). In this latter group the enzyme activity in plasma was close to the limits of detection. No changes were observed in either of the caspases’ activity 0.5 h after Jo2 or IgG injection.
3.3. Gene expression of apoptosis-associated factors/regulators The following apoptosis-associated factors’ mRNA expression was determined in this study: Bcl-w, Bfl-1, Bcl-XL, Bcl-XS, Bcl-2, Bak, Bad, Bax, Fas, FasL, RIP, FADD, and caspases-3, -8 and -9. Significant changes in the mRNA expression were observed for anti-apoptotic regulators
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Fig. 2. Liver histology as revealed by hematoxylin-eosin staining. (A) Section of a liver from an IgG-treated mouse 4 h after treatment displaying normal appearance. (B) Section of a liver from a Jo2-treated mouse 4 h after antibody injection. Marked necrosis (large arrowheads indicating a few necrotic areas) and neutrophil infiltration (small arrowheads) are present in this liver. (C) Section of a liver from GdCl3 + Jo2-treated mouse showing more pronounced necrosis. Labeling as in (C). Original magnification: × 400.
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3.5. Li6er glutathione content GSH content remained unchanged in the 0.5 h group under all experimental conditions tested, but was decreased by Jo2 in the 1.5 h group only in GdCl3-treated animals (Fig. 11). In the 4 h group, Jo2, with or without GdCl3, markedly decreased the GSH content. GSSG content was significantly increased by GdCl3 in the 1.5 h group but decreased in the 4 h group by both Jo2 and GdCl3. A decrease in the GSH/GSSG ratio was recorded both in the 1.5 and 4 h groups but only in the GdCl3-treated mice, with or without Jo2 injection (Fig. 11). Of note, in Sal+Jo2-treated animals no marked
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changes in the GSH/GSSG ratio were observed at any of the time points tested.
4. Discussion
4.1. Fas-mediated apoptosis in the mouse li6er does not require induction of pro-apoptotic proteins The major finding of this study pertains to the gene expression of apoptosis-associated factors during the course of Fas-mediated apoptosis in the mouse liver in vivo. To our knowledge, this is
Fig. 3. Liver TUNEL response in various treatment groups. (A) Section of a liver from IgG-treated mouse at 1.5 h after IgG injection. Note the presence of very few apoptotic nuclei (arrowheads). Eight high magnification fields ( × 400) from 4 livers were examined here and in subsequent TUNEL pictures and the numbers were averaged. (B) Section of a liver from Saline + Jo2-treated mouse, 1.5 h after Jo2 injection. Note the higher frequency of apoptotic nuclei (13.3 9 1.2% of total nuclei; arrowheads). The small brown bodies (small arrowheads) represent apoptotic bodies during the final stage of apoptosis. Very few large apoptotic nuclei are present (large arrowheads). (C) Section of a liver from GdCl3 +Jo2-treated mouse 1.5 h after Jo2 injection. When compared to the corresponding control (i.e. Saline + Jo2 group, see (C) above), the number of apoptotic nuclei (arrowheads) increased significantly and there was a higher proportion of large apoptotic nuclei denoting less phagocytosis and/or higher apoptosis. (D) Section of a liver of Saline+ Jo2-treated mouse 4 h after Jo2 injection. The number of apoptotic nuclei (arrowheads) was similarly increased compared to the corresponding group at 1.5 h after treatment (C) and the number of large apoptotic nuclei appeared higher. (E) Section of a liver of a GdCl3 + Jo2-treated mouse 4 h after Jo2 injection. The number of apoptotic nuclei (arrowheads) markedly increased as compared to (D). This change was associated with an even higher number of ‘large’ apoptotic nuclei indicating less phagocytosis and/or higher apoptotic rate. Original magnification: ×400. Numerical data and statistical significance on apoptotic nuclei/bodies are given in Fig. 4.
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the first study to undertake such an approach of the Fas-mediated liver apoptosis. Assessment of gene expression in this study covered a wide spectrum of apoptosis genes including Fas-associated signaling molecules (e.g. Fas receptor, FasL, death-domain bearing proteins, such as FADD and RIP), apoptosis adaptors (e.g. Bcl-w, Bfl-1, Bcl-XL, Bcl-XS, Bcl-2, Bak, Bad, and Bax) and
executioners of apoptosis (e.g. caspase-3, -8, and -9). Our data show that only a few anti-apoptotic factors, i.e. Bcl-w, Bcl-XL and Bfl-1, underwent changes consisting of up-regulation of mRNA expression. No changes in gene expression of tested pro-apoptotic factors were observed. We therefore infer that Fas-dependent apoptotic sig-
Fig. 3. (Continued)
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the liver against massive apoptosis induced by Fas antibody [9]. Our data suggest that de novo protein synthesis may be implicated in the modulation of anti-apoptotic proteins.
4.2. Kupffer cells play an important role in Fas-mediated li6er apoptosis
Fig. 4. Time course of the number of apoptotic nuclei in the liver. Plotted are means 9SEM (vertical bars) for 4 individuals in a group. Here and in subsequent graphs, the following abbreviations are used: GdCl, gadolinium chloride (GdCl3); Sal, saline. (a) Significantly different (P B0.05) from Sal + Jo2 group; (b) significantly different (PB 0.05) from Sal + IgG and Gd+ IgG groups.
nal finds the mouse liver replete with all the necessary equipment to promptly execute apoptotic death. Thus, de novo protein synthesis is not required in order for the cells to undergo Fas-mediated apoptosis. A similar conclusion was reached in an earlier study by Leist et al. [8] in which a different experimental approach was used: administration of protein synthesis inhibitors in vivo to mice subsequently treated with anti-Fas antibody. However, in the cited study no proof of complete inhibition of protein synthesis was given. In addition, Leist et al.’s study does not allow any conclusion on a possible involvement of transcriptional/translational steps in the regulation of anti-apoptotic factors. Our study provides information on this issue showing that the regulation of anti-apoptotic factors in response to Fas receptor ligation may be effected at the level of transcription. The up-regulation of anti-apoptotic factors observed in this study may be regarded as an attempt of cells to counteract the apoptotic signal action and to avoid death. This contention is consistent with earlier studies on Fas-mediated liver apoptosis in the mouse showing that Bcl-XL was up-regulated shortly (2 h) after Fas antibody injection and even more so after HGF treatment of Fas antibody-injected mice [9]. HGF protects
In order to assess a role for Kupffer cells in the apoptotic response of the liver to Fas antibody, we treated the mice with GdCl3 prior to antibody injection. This treatment has previously been shown to have two main effects: a partial depletion of the Kupffer cell population and a marked reduction of the phagocytic capacity of the remaining Kupffer cells. We confirmed that, under our experimental conditions, GdCl3 treatment reduces the Kupffer cell population significantly in
Fig. 5. Plasma alanine: and aspartate:2-oxoglutarate aminotransferase activity (ALT and AST, respectively). Plotted are means 9SEM (vertical Ý bars) for 4 – 6 animals in a group. (a) PB 0.05 versus IgG-treated groups.
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One and, perhaps, the most important role of Kupffer cell in the apoptotic response of the liver consists of phagocytosis of apoptotic cells. It is currently accepted that Kupffer cells are the primary phagocytes of the liver involved in apoptotic cell clearance [13,14]. Our experimental results are consistent with this observation. Under the experimental conditions presented in this study, a
Fig. 6. Hepatic mRNA expression of Bcl-w, Bfl-1 and Bcl-XL. Plotted are means 9 SEM (vertical Ý bars) for 4 –6 individuals in a group. (a) PB 0.05 versus IgG-treated groups; (b) PB 0.05 versus GdCl + Jo2-treated group; L32, a house keeping gene.
the mouse liver. It is important to note that GdCl3 treatment, as applied here and in other studies on rats and mice [10– 12], does not completely deplete the Kupffer cell population. The remaining Kupffer cells, however, have a markedly impaired phagocytosing capacity, which is an important issue for the interpreation of our results because it allows a fair estimate of Kupffer cell contribution to phagocytosis of apoptotic cells.
Fig. 7. Hepatic mRNA expression for three of the tested pro-apoptotic adaptors/regulators. Plotted are means 9SEM (vertical Ý bars) for 4 – 6 individuals in a group. No significant differences were observed as a function of either treatment or time after injection. Note that in this graph the Y scale is expanded to allow a better examination of the plotted parameters.
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Fig. 8. Representative RNAse protection assay gel showing changes in Bcl-w, Bfl-1 and Bcl-XL 4 h after Jo2 (lanes 1 – 6) or IgG (lanes 7 – 12) injection. L32, a housekeeping gene.
marked increase in the number of apoptotic nuclei was seen both at 1.5 and, particularly, 4 h after Jo2 injection. Such an increase could be ascribed to two factors: a potentiation by GdCl3 of Jo2 apoptotic effects (Figs. 4 and 9) and the decrease in phagocytosis of apoptotic cells due to a diminution of both the number of Kupffer cells and their phagocytic capacity. The mechanism by which GdCl3 enhances Jo2-induced apoptosis has not been approached in this study and remains to be elucidated. The partial elimination/suppression of Kupffer cells in the livers of GdCl3 +Jo2-treated mice was associated with a marked increase in the number of ‘full size’ apoptotic nuclei along with a significant reduction in the number of smaller apoptotic nuclei when compared to the livers of Jo2-treated controls. The ‘small’ apoptotic nuclei (or apoptotic bodies) may represent nuclei during the last phase of apoptosis. In our experiments the exacerbated apoptosis, well expressed at 1.5 h after Jo2 injection, was not associated with necrosis as clearly demonstrated by histologic examination of the liver and by the lack of an increase in plasma ALT activity. Necrosis ensued later, i.e. at 4 h after Jo2 injection
and, for the following reasons, we believe that necrosis is caused by an exacerbated apoptosis. First, apoptosis preceded necrosis. Therefore, the positively stained nuclei in the TUNEL reaction that occurred before the 3 h time point can be a considered as resulting exclusively from apoptosis; however, at a later time point as necrosis settled, the number of apoptotic nuclei increased as a result of both apoptosis and necrosis. Second, prevention of Jo2 in vivo apoptotic effects on the liver by various agents markedly reduces liver necrosis as assessed with histologic and biochemical procedures [15–18]. Third, Jo2 does not produce solely necrosis, hence the latter cannot be dissociated from Jo2 apoptotic effects. It is not known why enhanced apoptosis such as occurs in this model of Fas antibody administration is followed by necrosis. We agree with an earlier suggestion that the transition from apoptosis to necrosis is caused by the abrupt increase of the number of apoptotic cells surpassing the phagocytic capacity of the tissue, thus leading to necrosis [19,20]. How much apoptosis is needed to overwhelm the liver’s capacity to phagocytose apoptotic cells is not known. Our data show that in the saline-in-
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jected, Jo2-treated mice the number of apoptotic nuclei increased with the time reaching 13.3% at 1.5 h (Fig. 3C) and 19.8% at 4 h (Fig. 3D). However, necrosis was seen only at 4 h after Jo2 injection suggesting that when the number of apoptotic cells lies between 13 and 20%, the phagocytosing capacity of the liver is surpassed by the increase in the number of apoptotic cells
Fig. 10. Caspase-3 activity in plasma. Plotted are means 9 SEM (vertical Ý bars) for 4 – 6 individuals in a group. (a) PB 0.05 versus IgG-treated animals in the 1.5 h group; (b) PB 0.05 versus Sal + Jo2 and GdCl +Jo2-treated mice.
thus leading to necrosis. This range may change when Kupffer cells are partially depleted by GdCl3 treatment.
4.3. Fas-mediated li6er apoptosis is not associated with changes in the intracellular free thiol redox system
Fig. 9. Caspase-3, -8 and -9 (abbreviated as Casp-) activity in the liver of mice. Plotted are means 9SEM (vertical Ý bars) for 4– 6 individuals in a group. (a) PB 0.05 versus GdCl + IgG-treated animals; (b) PB 0.05 versus GdCl + Jo2-treated mice.
It has been documented that cytokine-induced apoptosis in a hepatocyte cell line (Hep 3B) was preceded and paralleled by a decrease in GSH which reflected elevated oxidative stress [21]. Jo2induced liver apoptosis, as seen at 1.5 h after Fas antibody injection, was neither preceded by, nor associated with, any alteration in the redox state of the glutathione system in the liver. This was shown by a lack of change in the GSH to GSSG ratio at the 0.5, 1.5 and 4 h time points after Jo2 injection. While our results do not strongly implicate oxidative stress in Jo2-induced liver apoptosis, they nevertheless are consistent with the idea that an intact GSH–GSSG system is required for liver apoptosis to proceed [22]. At 4 h after Jo2 injection a dramatic decrease in the content of both GSH and GSSG could be seen which did not result in a change in GSH/GSSG ratio. These decreases should be regarded as a consequence rather than as a cause of apoptosis/necrosis. In the 4 h Jo2-treatment group some of the changes in the liver
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glutathione content might also have been caused by massive organ cytolysis as demonstrated by marked spillover into the circulation of aminotransferases and caspase-3. It is intriguing to note that Kupffer cell depletion associated with GdCl3 treatment with or without Jo2, shifted the glutathione system to a more oxidized state as shown by decreased GSH/ GSSG ratio. Although the exact cause of such changes needs to be determined, this may indicate an important role for Kupffer cells in the maintenance of a physiological redox state of the liver GSH –GSSG system. In summary our study allows the following conclusions: (1) Fas-mediated liver apoptosis does not require up-regulation of Fas-associated death domain proteins (i.e. FADD and RIP), pro-apoptotic adaptors (i.e. Bcl-XS, Bak, Bad, Bax,) or executioners of apoptosis (caspases -3, -8 and -9); rather, preexisting apoptotic equipment is sufficient for a prompt apoptotic response. In addition, Fas-mediated liver apoptosis is associated with up-regulation of anti-apoptotic factors in an attempt to avoid death; (2) Kupffer cells seemingly by and, therefore, does not involve as a causative factor, changes in the intracellular free thiol system redox state, as reflected by GSH/GSSG ratio.
Acknowledgements
Fig. 11. Content of reduced (GSH) and oxidized (GSSG) glutathione and GSH to GSSG ratio in the liver of mice. Plotted are means 9 SEM (vertical Ý bars) for 4 –6 individuals in a group. For GSH: (a) PB 0.05 versus Sal + Jo2-treated group; (b) P B 0.05 versus Sal – IgG-treated group; (c) PB 0.05 versus Gd – IgG and Sal –IgG-treated groups. For GSSG: (a) P B 0.05 versus Sal – IgG; (b) PB0.05 versus Sal –Jo2-treated group; (c) P B 0.05 versus corresponding subgroup in the 1.5 h group. For GSH/GSSG ratio: (a) PB0.05 versus Sal –IgGtreated group; (b) P B 0.05 versus Sal –Jo2-treated group; (c) and (d) P B 0.05 versus Sal –Jo2-treated group.
This study was supported by the National Institute on Alcohol Abuse an Alcoholism Grants AA10762 (to CJM), AA12314 (to IVD), and AA 12774 (to WdV), and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to CJM). This material is also the result of work supported with resources and the use of facilities at the Lexington Veterans Administration Medical Center, KY 40506.
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