Shedding of TNFR1 in regenerative liver can be induced with TNFα and PMA

AIM: Liver regeneration is associated with apoptosis of hepatocytes, which is mediated via tumor necrosis factor receptor 1(TNFR1). The shedding of TNFR1 in liver regeneration and its mechanism to regulate this shedding were investigated.METHODS: The shedding of TNFR1 in liver regeneration and changes of TNF-α, PMA and plasma membrane purified from hepatocytes on this shedding process were measured with Western blot. Then, the relationship between TNFR1 shedding and apoptosis of hepatocytes induced by TNFα was studied by detecting apoptotic index.RESULTS: The shedding of TNFR1 began at 4 hours and terminated before 2 months after partial hepatectomy. In culture system, serum from rats at 36 h after partial hepatectomy could also promote this shedding process. With the stimulation of TNF α, PMA or purified plasma membrane from hepatocytes at 36 h after partial hepatectomy or from hepatocytes treated with TNF α for 2 h, membranous TNFR1 was also shed. With the stimulation of both TNF α and plasma membrane from hepatocytes affected with TNF α for 2 hor from hepatocytes at 36 h after partial hepatectomy, apoptotic index of hepatocytes decreased from 21% to 7.52 % and 8.45 %, respectively. PMA could also reduce apoptotic index to 13.67 %. This descent occurred in hepatocytes cultured in serum from rats at 36 h after partial hepatectomy too,but not in serum from rats at 2 months after partial hepatectomy and sham-operated rats.CONCLUSION: Shedding of TNFR1 may help reduce apoptosis of hepatocytes induced by TNFα. Membraneanchored metalloprotases could play a role in shedding membranous TNFR1. At the same time, PKC may take part in regulation of this shedding process.     

Sensitization by interleukin-6 of rat hepatocytes to tumor necrosis factor alpha-induced apoptosis

BACKGROUND/AIMS: Tumor necrosis factor (TNF) elicits hepatocyte apoptosis in toxic liver injury and is also central in hepatocyte proliferation after partial hepatectomy. In both circumstances interleukin (IL)-6 levels are also elevated. In mouse liver IL-6 attenuated Fas receptor-mediated apoptosis indicating its interference with pro-apoptotic signal chains. It was, therefore, the aim to examine the modulation by IL-6 of TNFalpha-induced apoptosis in rat hepatocytes. METHODS: Primary rat hepatocytes were treated with IL-6 prior to induction of apoptosis with TNFalpha/actinomycin D or anti-Fas antibody M-20. Apoptosis was detected by determination of caspase-3 activation and bisbenzimide staining of condensed nuclei. Expression of TNFalpha receptors was analyzed by semi-quantitative polymerase chain reaction and ligand binding studies with [125I]-TNFalpha. RESULTS: IL-6 treatment doubled TNFalpha/actinomycin D-induced caspase-3 activity and significantly enhanced chromatin condensation. By contrast IL-6 inhibited Fas-induced increase in caspase-3 activity by 45% and significantly reduced chromatin condensation. IL-6 increased the mRNA level of TNF-R1 1.35-fold and augmented cell surface binding of [125I]-TNFalpha 3-fold. The latter and TNFalpha-mediated caspase activation was attenuated by prostaglandin E(2). CONCLUSIONS: IL-6 - in contrast to its anti-apoptotic modulation of the Fas-induced pathway - exerted a pro-apoptotic effect on the TNFalpha/actinomycin D-induced apoptosis by increasing the number of TNF-R on hepatocytes.     

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice

Tissue transglutaminase (TG2) is a protein cross-linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-out mice within 20 hours. Although TG2-/- thymocytes exposed to anti-Fas antibodies die at the same rate as wild-type mice, TG2-/- hepatocytes show increased sensitivity toward anti-Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIP(L) (FLICE-inhibitory protein), or the rate of I-kappaBalpha degradation, but a decrease in the Bcl-xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl-xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand-induced hepatocyte proliferation with a simultaneous inhibition of the Fas-death pathway plays a determinant role.     

Mouse islet cell lysis mediated by interleukin-1-induced Fas

Summary This study was conducted to investigate the possible involvement of Fas in β-cell death in insulitis of Type 1 (insulin-dependent) diabetes mellitus. Although primary cultured Balb/c mouse islet cells did not express Fas mRNA, 4–12 hours of treatment with 10²-10⁵ U/l of mouse interleukin-1 α (IL-1α) induced the expression of Fas mRNA. Surface Fas expression was detected by immunofluorescence flow cytometry using a non-cytolytic anti-Fas monoclonal antibody after 6 or 12 h of incubation with 10³ U/l of IL-1α. Primary islet cells were resistant to an agonistic anti-Fas monoclonal antibody. However, 12 h pretreatment with IL-1α sensitized islet cells to its cytolytic effect. Significant cell death was observed 24 h after the addition of anti-Fas, and progressively increased until 72 h, when specific ⁵¹Cr release was 72 ± 6 %. Agarose gel electrophoresis of DNA extracted from cells exposed to IL-1α and agonistic anti-Fas showed internucleosomal DNA fragmentation, a hallmark of apoptotic cell death. Since the Fas antibody showed no cross-reactive activity of tumour necrosis factor (TNF), the cytotoxic effect was not mediated by TNF receptors. A protein synthesis inhibitor cycloheximide augmented Fas-mediated islet cell death. The Fas-mediated killing of islet cells was not l-arginine-dependent, or blocked by N^G-monomethyl-l-arginine. β-TC1 cells also expressed Fas mRNA when exposed to IL-1α or IL-1α plus interferon-γ. These observations suggest that Fas-mediated apoptosis may be a mechanism of islet cell death in autoimmune insulitis. [Diabetologia (1996) 39: 1306–1312] Received: 5 April 1996 and in revised form: 7 August 1996     

Sensitization by interleukin-6 of rat hepatocytes to tumor necrosis factor α-induced apoptosis

Background/Aims: Tumor necrosis factor (TNF) elicits hepatocyte apoptosis in toxic liver injury and is also central in hepatocyte proliferation after partial hepatectomy. In both circumstances interleukin (IL)-6 levels are also elevated. In mouse liver IL-6 attenuated Fas receptor-mediated apoptosis indicating its interference with pro-apoptotic signal chains. It was, therefore, the aim to examine the modulation by IL-6 of TNFα-induced apoptosis in rat hepatocytes.Methods: Primary rat hepatocytes were treated with IL-6 prior to induction of apoptosis with TNFα/actinomycin D or anti-Fas antibody M-20. Apoptosis was detected by determination of caspase-3 activation and bisbenzimide staining of condensed nuclei. Expression of TNFα receptors was analyzed by semi-quantitative polymerase chain reaction and ligand binding studies with [¹²⁵I]-TNFα.Results: IL-6 treatment doubled TNFα/actinomycin D-induced caspase-3 activity and significantly enhanced chromatin condensation. By contrast IL-6 inhibited Fas-induced increase in caspase-3 activity by 45% and significantly reduced chromatin condensation. IL-6 increased the mRNA level of TNF-R1 1.35-fold and augmented cell surface binding of [¹²⁵I]-TNFα 3-fold. The latter and TNFα-mediated caspase activation was attenuated by prostaglandin E₂.Conclusions: IL-6 – in contrast to its anti-apoptotic modulation of the Fas-induced pathway – exerted a pro-apoptotic effect on the TNFα/actinomycin D-induced apoptosis by increasing the number of TNF-R on hepatocytes.     

Fas (APO-1/CD95) mRNA is down-regulated in liver regeneration after hepatectomy in rats

The interaction of Fas (APO-1/CD95) and the Fas ligand system induces apoptosis. However, the role of the Fas/Fas ligand system in normal physiological liver cell death remains to be determined. Using northern blotting analysis, we investigated the role of Fas and Fas ligand mRNA expression in liver regeneration in rats until 14 days after partial hepatectomy. Partial hepatectomy led to a significant decrease in Fas mRNA levels at 2 h, with a further gradual reduction until 18 h. The minimum Fas mRNA levels persisted until 5 days after the surgery. Fas mRNA levels then increased markedly at 6 days, and gradually increased to the initial levels by 14 days after the surgery. However, partial hepatectomy did not affect the expression of Fas ligand mRNA levels. These findings suggest that Fas mRNA is down-regulated in liver regeneration after partial hepatectomy, and that when the original liver mass is gradually regenerating, Fas mRNA is again up-regulated to the initial levels. Received: March 4, 1999 / Accepted: July 23, 1999     

Enhanced Sensitivity to CD95-Induced Apoptosis in ob/ob Mice

Hepatocyte apoptosis was recently described for NASH patients. The pathomechanisms are incompletely understood, but upregulation of the death receptor Fas was detectable on hepatocytes of NASH patients. We analyzed the sensitivity of fatty liver against CD95/Fas-mediated apoptotic cell death by injection of agonistic anti-Fas antibody (Jo2) in obese ob/ob mice and lean control animals. Ob/ob mice died within 12 hrs, whereas control animals survived. Liver enzymes were significantly increased compared to those in control mice (P < 0.001). Histological analysis and also TUNEL assay of liver sections from ob/ob mice exhibited massive liver injury. Activity of caspase 3 was significantly more enhanced in livers of ob/ob mice after Jo2 challenge. The increased sensitivity was confirmed in vitro by using ob/ob-derived primary hepatocytes. CD95 expression was similar in ob/ob and control mice. However, hepatocytes from ob/ob mice revealed a decreased mitochondrial membrane potential, suggesting that mitochondria play a potential role in this increased susceptibility. Juergen Siebler and Markus Schuchmann contributed equally to the work.     

Inhibition of mouse hepatocyte apoptosis via anti-Fas ribozyme

AIM: To investigate the effects of anti-Fas ribozyme on Fas expression and apoptosis in primary cultured mouse hepatocytes. METHODS: Mouse hepatocytes were isolated by using collagenase irrigation. A hammerhead ribozyme targeting the Fas mRNA was constructed, and transfected into mouse hepatocytes via Effectene. Then Fas expression in mouse hepatocytes was detected by RT-PCR and western blotting. After being treated with anti-Fas antibody (JO2), hepatocytes viability was measured with MTT assay. Caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to Annexin V-FITC apoptosis detection kit. RESULTS: Fas expressed in primary mouse hepatocytes. Fas expression in hepatocytes transfected with anti-Fas ribozyme was decreased remarkably and correlated with the resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. CONCLUSION: Anti-Fas ribozyme can remarkably decrease the Fas expression in mouse hepatocytes, thus inhibit Fas-mediated apoptosis in hepatocytes. It is suggested that anti-Fas ribozyme could significantly increase the resistance of transplanted hepatocytes to apoptosis and improve the survival of transplanted hepatocytes.     

The superoxide dismutase mimetic MnTBAP prevents Fas-induced acute liver failure in the mouse.

BACKGROUND & AIMS: Acute liver failure (ALF) of viral origin results from massive hepatocyte apoptosis induced by the interaction between Fas expressed on hepatocytes and Fas ligand on activated T lymphocytes. Because Fas-induced apoptosis of hepatocytes involves mitochondrial damages and potential reactive oxygen species (ROS) overproduction, we investigated whether manganese III tetrakis (5,10,15,20 benzoic acid) (MnTBAP), a nonpeptidyl mimic of superoxide dismutase (SOD), can inhibit Fas-induced ALF. METHODS: An agonist anti-Fas monoclonal antibody was used to induce hepatocyte apoptosis in vitro and ALF in vivo. RESULTS: Preventive and curative treatments by MnTBAP significantly increased survival rates and significantly reduced aspartate aminotransferase levels and parenchymal lesions. ROS generation was suggested by those beneficial effects and significant increases in SOD and Gpx activities after anti-Fas injection. Flow cytometry of isolated hepatocytes incubated with anti-Fas monoclonal antibody showed that ROS production was associated with the collapse of transmembrane potential and loss of cardiolipin content. After injection of anti-Fas monoclonal antibody, mitochondrial Bcl-2 was decreased, cytochrome c released, and caspase-3 activated. Mitochondrial alterations and their consequences were abrogated by MnTBAP. CONCLUSIONS: ROS are key executioners in Fas-induced hepatocyte apoptosis. This finding explains why a nonpeptidyl mimic of SOD can cure ALF in a model of viral hepatitis, pointing out the potential interest of this molecule in humans.     

Role of angiotensin‐1 receptor blockade in cirrhotic liver resection

Background: The regeneration capacity of cirrhotic livers might be affected by angiotensin‐1 (AT1) receptors located on hepatic stellate cells (HSC). The effect of AT1 receptor blockade on microcirculation, fibrosis and liver regeneration was investigated. Materials and methods: In 112 Lewis rats, cirrhosis was induced by repetitive intraperitoneal injections of CCl₄. Six hours, 3, 7 and 14 days after partial hepatectomy or sham operation, rats were sacrificed for analysis. Animals were treated with either vehicle or 5 mg/kg body weight losartan pre‐operatively and once daily after surgery by gavage. Microcirculation and portal vein flow were investigated at 6 h. The degree of cirrhosis was assessed by Azan Heidenhein staining, activation of HSC by desmin staining, apoptosis by ssDNA detection and liver regeneration by Ki‐67 staining. Changes in expression of various genes important for liver regeneration and fibrosis were analysed at 6 h and 3 days. Haemodynamic parameters and liver enzymes were monitored. Results: Losartan treatment increased sinusoidal diameter, sinusoidal blood flow and portal vein flow after partial hepatectomy (P<0.05), but not after sham operation. AT1 receptor blockade resulted in increased apoptosis early after resection. HSC activation was reduced and after 7 days, a significantly lower degree of cirrhosis in resected animals was observed. Losartan increased the proliferation of hepatocytes at late time‐points and of non‐parenchymal cells early after partial hepatectomy (P<0.05). Tumour necrosis factor (TNF)‐α was significantly upregulated at 6 h and stem cell growth factor (SCF) was downregulated at 3 days (P<0.05). Conclusion: Losartan increased hepatic blood flow, reduced HSC activation and liver fibrosis, but interfered with hepatocyte proliferation after partial hepatectomy in cirrhotic livers.     

Liver regeneration in heparin-binding EGF-like growth factor transgenic mice after partial hepatectomy

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized in the form of a membrane-anchored precursor (proHB-EGF), which subsequently is processed proteolytically to mature HB-EGF. This study describes the effects of HB-EGF on liver regeneration after 70% partial hepatectomy in proHB-EGF transgenic mice with liver-specific expression. Methods & Results: No significant differences in liver/body weight ratios and in bromodeoxyuridine (BrdU)-labeling index (the ratios of BrdU-positive hepatocyte nuclei) were found between adult transgenic and wild-type mice. However, in regenerating liver after partial hepatectomy, transgenic mice had higher liver/body weight ratios than wild-type mice and at 120 hours reached a level equal to that before partial hepatectomy. The BrdU-labeling index was about 5 times higher in the livers of transgenic mice compared with the wild type (51.5% vs. 10.2%, respectively; P < 0.01) at 48 hours after partial hepatectomy. Activation of microtubule-associated protein kinase after partial hepatectomy was higher and earlier in the transgenic mice as compared with the wild-type mice. Soluble HB-EGF was increased in the liver (at 8 min) after partial hepatectomy, indicating that the shedding of proHB-EGF occurred after partial hepatectomy. CONCLUSIONS: The transgenic expression of HB-EGF accelerates the proliferation of hepatocytes after partial hepatectomy, suggesting that HB-EGF functions as a hepatotrophic factor in vivo.     

Interleukin 1beta protects mice from Fas-mediated hepatocyte apoptosis and death.

BACKGROUND & AIMS: Fas-mediated apoptosis is one of the major death processes of hepatocytes in liver diseases. The aim of this study was to determine whether interleukin (IL)-1beta regulates the Fas-mediated apoptotic process of differentiated hepatocytes in vivo. METHODS: IL-1beta was injected into Balb/cA mice 5 hours before lethal challenge with agonistic anti-Fas administration. Survival and hepatocyte apoptotic process of these mice were examined. RESULTS: IL-1beta pretreatment prolonged animal survival in a dose-dependent manner, and 500 ng of IL-1beta completely protected mice from lethality. Both serum alanine aminotransferase value and hepatic DNA fragmentation were significantly suppressed by IL-1beta pretreatment. IL-1beta affected neither hepatic distribution of anti-Fas antibody nor Fas expression levels on hepatocytes but significantly suppressed Fas-induced activation of hepatic caspase 3-like protease. Suppression of Fas-induced activation of the caspase by IL-1beta was diminished by coadministration with D-galactosamine and reversed by coinjection with an excess amount of uridine. CONCLUSIONS: These results suggest that IL-1beta suppresses Fas-mediated hepatocyte apoptosis by inducing molecule(s) that suppress the apoptosis control machinery upstream of caspase 3. This observation raises the possibility that IL-1beta acts as a negative regulator of Fas-mediated hepatocyte apoptosis during liver injury.     

Opioid receptor blockade reduces Fas-induced hepatitis in mice

Fas (CD95)-induced hepatocyte apoptosis and cytotoxic activity of neutrophils infiltrating the injured liver are two major events leading to hepatitis. Because it has been reported that opioids, via a direct interaction, sensitize splenocytes to Fas-mediated apoptosis by upregulating Fas messenger RNA (mRNA) and modulated neutrophil activity, we assumed that opioids may participate in the pathophysiology of hepatitis. Using the hepatitis model induced by agonistic anti-Fas antibody in mice, we showed that opioid receptor blockade reduced liver damage and consequently increased the survival rate of animals when the antagonist naltrexone was injected simultaneously or prior to antibody administration. Treatment of mice with morphine enhanced mortality. Naloxone methiodide-a selective peripheral opioid antagonist-had a protective effect, but the absence of opioid receptors in the liver, together with lack of morphine effect in Fas-induced apoptosis of primary cultured hepatocytes, ruled out a direct effect of opioids on hepatocytes. In addition, the neutralization of opioid activity by naltrexone did not modify Fas mRNA expression in the liver as assessed with real-time quantitative polymerase chain reaction. Injured livers were infiltrated by neutrophils, but granulocyte-depleted mice were not protected against the enhancing apoptotic effect of morphine. In conclusion, opioid receptor blockade improves the resistance of mice to Fas-induced hepatitis via a peripheral mechanism that does not involve a down-modulation of Fas mRNA in hepatocytes nor a decrease in proinflammatory activity of neutrophils.     

Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice

Although Fas stimulation has been reported to cause outer mitochondrial membrane rupture in Jurkat cells, the mechanism of this effect is debated, and it is not known if outer membrane rupture also occurs in hepatocyte mitochondria. We studied the in vivo effects of Fas stimulation on ultrastructural lesions and mitochondrial function in mice. Four hours after administration of an agonistic anti-Fas antibody (8 microg/animal), caspase activity increased 5.4-fold. Nuclear DNA showed internucleosomal fragmentation, whereas supercoiled mitochondrial DNA was replaced by circular and linear forms. Mitochondrial cytochrome c was partly released into the cytosol. Ultrastructurally, mitochondrial lesions were observed in both apoptotic hepatocytes (with nuclear chromatin condensation/fragmentation) and nonapoptotic hepatocytes (without nuclear changes). In nonapoptotic cells, outer mitochondrial membrane rupture allowed herniation of the inner membrane and matrix through the outer membrane gap. In apoptotic hepatocytes, the matrix became electron-lucent and no longer protruded through the outer membrane gap. Mitochondria clustered around the nucleus, whereas rough endoplasmic reticulum cisternae became peripheral. In liver mitochondria isolated after Fas stimulation, the membrane potential decreased, whereas basal respiration increased. Pretreatment with either z-VAD-fmk (an inhibitor of caspases) or cyclosporin A (a permeability transition inhibitor) totally or mostly prevented mitochondrial outer membrane rupture, membrane potential decrease, cytochrome c release, and apoptosis. In conclusion, in vivo Fas stimulation causes caspase activation, mitochondrial permeability transition (decreasing the membrane potential and increasing basal respiration), mitochondrial matrix expansion (as shown by matrix herniation), outer mitochondrial membrane rupture, and cytochrome c release.     

Role of Valpha 14 NKT cells in the development of impaired liver regeneration in vivo

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.     

Prolonged, but not acute, glutathione depletion promotes Fas-mediated mitochondrial permeability transition and apoptosis in mice

Glutathione depletion either decreased or increased death-receptor-mediated apoptosis in previous studies. Comparison of the durations of glutathione depletion before death-receptor stimulation in these studies might suggest a different effect of prolonged versus acute thiol depletion. We compared the effects of the prolonged glutathione depletion caused by a sulfur amino acid-deficient (SAA(-)) diet and the acute depletion caused by a single dose of phorone on hepatic apoptosis triggered by the administration of an agonistic anti-Fas antibody. The chronic SAA(-) diet did not affect hepatic Fas or Bcl-XL, but increased p53 and Bax, and exacerbated Fas-mediated mitochondrial membrane depolarization, electron-microscopy-proven outer mitochondrial membrane rupture, cytochrome c translocation to the cytosol, and caspase 3 activation. These effects were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. The SAA(-) diet increased internucleosomal DNA fragmentation, the percentage of apoptotic hepatocytes, serum alanine transaminase (ALT) activity, and mortality after Fas stimulation. Despite a similar decrease in hepatic glutathione, administration of a single dose of phorone 1 hour before the anti-Fas antibody did not change p53 or Bax, and did not enhance Fas-induced mitochondrial permeability transition and toxicity. However, 4 repeated doses of phorone (causing more prolonged glutathione depletion) increased Bax and Fas-mediated toxicity. In conclusion, a chronic SAA(-) diet, but not acute phorone administration, increases p53 and Bax, and enhances Fas-induced mitochondrial permeability transition and apoptosis. Thiol depletion could cause oxidative stress that requires several hours to increase p53; the latter induces Bax, which translocates to mitochondria after Fas stimulation.     

Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or gamma radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or gamma radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.     

In vivo hepatic HB-EGF gene transduction inhibits Fas-induced liver injury and induces liver regeneration in mice: a comparative study to HGF

BACKGROUND/AIMS: It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF. METHODS: Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration. RESULTS: Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice. CONCLUSIONS: HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.     

Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein and are refractory to Fas-mediated apoptosis

OBJECTIVE: The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. METHODS: The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. RESULTS: CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. CONCLUSION: These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.