American Joumnal of Pathology, Vol. 151, No. 5, November 1997 Copyright ©) American Society for Investigative Pathology
CD95/CD95L-Mediated Apoptosis of the Hepatic Stellate Cell A Mechanism Terminating Uncontrolled Hepatic Stellate Cell Proliferation during Hepatic Tissue Repair
Bernhard Saile, Thomas Knittel, Nina Matthes, Peter Schott, and Giuliano Ramadori From the University of Gottingen, Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Gdttingen, Germany
During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 80/0 (+ 5%) of "transitional" cells (day 4) and in 180/% (± 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data
demonstrate that apoptosis becomes detectable in parallel with HSC activation, which suggests that apoptosis might represent an important mechanism terminating proliferation of activated HSC. (Am J Pathol 1997, 151:1265-1272)
Both proliferation and apoptosis are the main pillars for cell cultures and homeostasis or development of tissues.1'2 For example, apoptosis is found in the development of the nervous and immune systems.3'4 Certain viral infections lead to initiation of an intrinsic death program, which spares other cells of the same organism from subsequent infection.5 In addition, cytotoxic T lymphocytes and natural killer cells eliminate their targets by apoptosis.6'7 FasIAPO-1/CD95 is known as an important mediator of apoptosis8 9 and acts as an inducer of apoptosis in CD95-expressing cells in response to ligand binding (CD95L).10 CD95 has been shown to be expressed in a wide range of tissues and in liver.11 Upregulation of CD95 could be demonstrated in liver in course of acute or chronic liver damage due to viral infections, alcohol abuse, or xenobiotics. Recently, a down-regulation of CD95 and up-regulation of CD95L in liver tumor cells were described. It was hypothesized that the ligand produced by the tumor cells could induce apoptosis in cytotoxic T lymphocytes by the CD95 system and this mechanism was responsible for the immune escape. 12
The induction of apoptosis underlies various activating and inhibiting processes. The two known major systems are the Bcl-2 and the p53 systems. The major function of Bcl-2 appears to be inhibition of apoptosis acting upstream of interleukin-1,B-converting enzyme protease activation. In addition, Bcl-2 functions as a pro-oxidant that
Supported by the Deutsche Forschungsgemeinschaft SFB 402 (Sonderforschungsbereich 402 Molekulare und Zellulare Hepatogastroenterologie), project C6. Accepted for publication July 31, 1997. Address reprint requests to Dr. G. Ramadori, Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University G6ttingen, Robert-Koch-Stra,Be 40, 37075 G6ttingen, Germany. E-mail:
[email protected]
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regulates levels of reactive oxygen intermediates and controls early entry into apoptosis.1I-15 The ratio of death antagonists (Bcl-2, Bcl-XL, Bcl-w, mcl-1, Al) to agonists (bax, bak, Bcl-Xs, bad, bid) of the Bcl-2-related proteins determines whether a cell will respond to an apoptotic signal. This is, at least in part, mediated by competitive dimerization between selective pairs of antagonists and agonists.16'17 p53 exerts a significant, dose-dependent effect in the initiation of apoptosis but only when induced by agents that cause DNA-strand breakage. The p53dependent induction of apoptosis appears to be composed of two components. The first is the recruitment for p53 transactivation of target genes through sequencespecific DNA binding, whereas the second is an apparent transactivation-independent pathway that bypasses p53 target genes.18'19 The growth-suppressive role of p53 occurs by activating the expression of WAF1 gene (p2lCipl), which is a potent inhibitor of G1 and G2 phase cyclin-dependent kinases, which then leads to failure to phosphorylate pRb (retinoblastoma protein) and subsequent to cell cycle arrest.20'21 These findings are in agreement with the current model of p53 function in which p53 somehow senses DNA damage and arrests cells in either G1 or G2 phase to allow DNA repair to take place. If repair is not successful, p53 may promote cell death by apoptosis. p53 has also been shown to have a direct effect on apoptosis by down-regulating Bcl-2 expression and up-regulating bax expression. However, up-regulation of Bcl-2 expression can block p53-triggered apoptosis but not growth arrest.22'23 Tissue repair in the liver after acute liver damage involves activation of hepatic stellate cells (HSC) to myofibroblastic cells. Their role consists mainly in synthesis of matrix proteins. The fate of activated cells after the completed repair process is unclear. During primary HSC culture, three morphological and functional phases can be distinguished, the resting cell (day 2), the transitional cell (day 4), and the activated cell (day 7). The aim of this study was to investigate the fate of activated HSC in primary culture and in the in vivo model of the acute CCI4-induced liver damage.
Materials and Methods HSC-lsolation, Characterization, and Plating Wistar rats were provided by Charles River (Sulzfeld, Germany) and received care in compliance with the institution's guidelines and the National Institutes of Health Guidelines. HSC were isolated according to the method of De Leeuw et al.24 Purity of HSC preparations was assessed by intrinsic vitamin A autofluorescence, immunocytochemistry using antibodies against desmin and glial fibrillary acidic protein (HSC), the macrophage antigen ED2 (Kupffer cells), and diacetyl-low-density lipoprotein incorporation (sinusoidal endothelial cells).25-30 Relative purities of freshly isolated cells were 85%. Major contaminants were endothelial cells and Kupffer cells.
Cells were plated onto 24-well Falcon plates (Becton Dickinson, Heidelberg, Germany), 35-mm Petri dishes (Greiner, Krefeld, Germany), and Lab Tek tissue culture slides (Nunc, Naperville, IL) commonly with a density of 30,000 cells/cm2. To investigate the influence of cell density on apoptosis induction, we also used cell densities between 7500/cm2 and 60,000/cm2 on 96-well Falcon plates (Becton Dickinson). To evaluate purity of cultures, HSC were tested by immunofluorescence at days 2, 4, and 7 after plating, as described above. In a less than 2% contamination with Kupffer cells (ED2 positive), neither endothelial cells nor hepatocytes were detected. Using a-smooth muscle actin immunoreactivity as an activation parameter, HSC were fully activated after 7 days of primary culture (100% a-smooth muscle actin positive).31
Western Blot Analysis of CD95, CD95L, p53,
BcI-2, and bax Cells at different days after plating were lysed in hot Laemmli buffer (950C) and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions according to Laemmli.32 The protein content of cellular lysates was calculated by Coomassie protein assay (Pierce, Rockford, IL). Proteins were transferred onto Hybond ECL nitrocellulose hybridization transfer membranes according to Towbin et al.33 Immunodetection was performed according to the ECL Western blotting protocol. Monoclonal antibodies against CD95 (APO-1/Fas), CD9SL, p53, Bcl-2, and bax were used at 2.5 ,ug/ml solutions. Peroxidase-labeled antimouse and anti-rabbit immunoglobulins were used at 1:1000 dilutions each.
Investigation of Soluble CD95 and Soluble CD95L To investigate whether HSC release soluble CD95 or soluble CD95L we performed Western blot analysis. For this purpose, we used concentrated supernatants (30minute vacuum centrifugation) of HSC that were handled as described above. To further support our Western blot data, we cultivated HSC in the resting and transitional phase in presence of supernatants of activated cultures. The occurrence of apoptosis was then measured by flow cytometry.
Apoptosis Detection and Time Dependency of Occurrence of Apoptotic Signs To investigate induction of apoptosis, blocking apoptosis, and apoptosis occurring spontaneously at day 2, 4, and 7, we performed a test using confocal laser scan microscopy (Zeiss, Oberkochen, Germany) in the timescan mode. To detect early apoptotic changes, staining with annexin V-fluorescein isothiocyanate (FITC) was used because of its high affinity to phosphatidylserine.34 Phosphatidylserine is normally situated on the inner leaf-
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let of the plasma membrane. In course of cell death, phosphatidylserine is translocated to the outer layer of the membrane, ie, the external surface of the cell.35 This occurs in early phases of apoptosis while the cell membrane itself remains intact. In contrast to apoptosis, necrosis is accompanied by loss of cell membrane integrity and leakage of cellular constituents into the environment. Therefore, to distinguish apoptosis and necrosis, trypan blue, a common dye exclusion test, and annexin V-FITC were used in parallel to show membrane integrity after annexin V-FITC binding to cells. In all investigated cases, the loss of membrane integrity was not noticed within 30 minutes after annexin V-FITC binding was detected. But 12 hours after initiation of the test, considerable amounts of cells showed membrane leakage, which indicates secondary necrosis. The biochemical hallmark of apoptosis is the degradation of genomic DNA caused by endonucleases. To further support our observations we used the TUNEL method (TdT-mediated X-dUTP nick end labeling), considered to be a sensitive assay, to visualize DNA damage on a single-cell basis as a second method to detect apoptotic cells.36 Data obtained by TUNEL labeling were identical to those received from the annexin V-FITC binding.
Flow-Cytometric Quantification of Apoptotic, Living, and Necrotic HSC For quantification of apoptotic cells, we used flow cytometry after trypsination of HSC (Epics ML, Coulter, Kerfeld, Germany). Measurement of annexin V binding was performed simultaneously with a dye-exclusion test using propidium iodide to discriminate between apoptosis and
necrosis.37
BrdU-ELISA for Cell Proliferation To measure cell proliferation we used a BrdU labeling and detection ELISA-kit (Boehringer Mannheim, Germany), quantitating BrdU incorporated into newly synthesized DNA of replicating cells. Cultures were incubated with BrdU for 12 hours, 370C, in presence of agonistic and antagonistic CD95 antibodies. Two different control groups were used. The first group was treated without antibodies, but to the second group, an antibody was added that recognizes CD95 but does not stimulate nor block CD95. The results of both control groups did not differ. BrdU uptake was measured according to the standard ELISA protocol. The data presented are normalized on the total cell number of the cultures (alive + apoptotic cells) measured by flow cytometry.
Induction of Acute Liver Damage Rats were administrated carbon tetrachloride/corn oil solution (50%, volume to volume) orally according to Yokoi
I
P0
2
20
3
3*
Figure 1. Western blot analysis. Sodium dodecyl sulfate-polyacrylanmide gel electrophoresis (90/o polyacrylamide) of HSC cell lysates (30 g of protein! lane) day 2 (lanes 1 and 1 *), day 4 (lanes 2 and 2*), and day 7 (lanes 3 and 3*) after plating. Controls (lanes 1, 2, 3) and cultures incubated with CD95 agonistic antibody (lanes I 2*, 3*~) This is one of 5 Western blot analysis _
*
of 5 different cell isolations.
et al.38 Acute liver damage was induced by administration of a single dose of 150 gl 00141100 g of body weight. Control animals were given corn oil only. At 3, 6, 9,12, 24, 48, 72, and 96 hours after treatment, respectively, two animals were killed, liver was perfused with saline solution (0.9%), removed, and snap-frozen in liquid nitrogen for immunohiStoChemistry.
Results Induction of CD95/CD95L Expression during HSC Activation and Lack of Evidence for Soluble CD95 and Soluble CD95L Western analysis showed increasing amounts of CD95 during cultivation. However, CD95L was detectable in increasing amounts at days 4 and 7 of culture but not at day 2. In addition, CD95 and CD95L protein concentrations were not modulated by CD95 agonistic antibodies (Figure 1). Neither soluble CD95 nor soluble CD95L could be detected by Western blot analysis. Incubating cultures in the resting and transitional phases with supernatants of activated cultures did not change the apoptosis rate of the cultures, as well.
Spontaneous Apoptosis of Transitional and Activated HSC Time Dependent Induction of Apoptosis due to CD95 Agonistic Antibodies and Inhibition of Apoptosis through Antagonistic Antibodies To investigate the mechanism of apoptosis occurring spontaneously and in response to agonistic and antagonistic CD95 antibodies, we performed a study using confocal laser scan microscopy and flow cytometry. In contrast to spontaneous apoptosis being detectable in cultures at days 4 (8 ± 5%) and 7 (18 ± 8%) after plating, no spontaneous apoptosis was evident day 2 (Figure 2 and 3a; Table 1). The administration of antagonistic CD95 antibodies led to a complete inhibition of spontaneous apoptosis at days 4 and 7 during the investigated period (12 hours) (Table 1). Moreover, even cells showing a weak enhancement of fluorescence intensity due to an-
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Figure 2. Occurrence of spontaneous apoptosis in course of activation of primary cultured HSC. In the upper line, the transmission images of HSC in the different activation phases are presented. In the lower line, apoptotic cells are identified by TUNEL. Magnification, X400.
nexin V at the beginning of the experiment, which indicates a very early state of apoptosis, were fully restored (Figure 3B). This finding indicates that apoptosis in the early state is a process that is reversible when the activating pathway (CD95) is blocked. Using CD95 agonistic antibodies at each point of time, more than 95% of cells could be carried into apoptosis (Table 1). At days 2 and 4, apoptosis is initiated within 2 to 4 hours, whereas apoptosis at day 7 is not initiated until 6 to 8 hours (Figure 3C). The discrepancy between higher amounts of CD95 receptors and delayed apoptotic response prompted us to investigate the two apoptosis regulating systems, namely the Bcl-2 and the p53 systems.
Expression of Bcl-2, bax, and p53 in Course of Activation of HSC and Modulation through CD95 Agonistic Antibodies Using Western blot analysis, Bcl-2 could not be detected in HSC cultures at day 2 but was found in small amounts after administration of CD95 agonistic antibodies. In parallel to activation, increasing amounts of Bcl-2 were shown at days 4 and 7 of cultivation. A weak induction of Bcl-2 was found at day 7 in response to CD95 agonistic antibodies. The apoptosis-agonist bax could be detected at days 2 and 4 in similar amounts and was considerably up-regulated at day 7. At no time could any regulation due to agonistic CD95 antibodies be observed. Like
Bcl-2, p53 could not be detected in control cultures at day 2 but was found after induction of apoptosis by CD95 activating antibodies. At day 4, a considerable up-regulation of p53 was shown due to CD95 agonistic antibodies. Spontaneous occurrence of p53 at day 7 was higher than at day 4 but was not modulated by CD95 agonistic antibodies (Figure 4).
Effect of CeOl Density and Modulation of the CD95/CD95L-System on Proliferation of HSC Although in initial experiments, apoptosis was uniformly present in areas with high and low cell densities, nevertheless the effect of different cell densities was analyzed in more detail in subsequent studies. BrdU-incorporation was analyzed by plating the cells at different cell densities (7500 cells/cm2 to 60,000 cells/cm2). The overall proliferation profile showed similar kinetics during primary culture both at low and high cell densities. A massive increment of BrdU-uptake was observed at day 4 and significantly (P < 0.01) decreased at day 7, which demonstrates declining proliferation potency (Figure 5). By administration of agonistic CD95 antibodies, a nearly complete inhibition of BrdU-uptake at each time point was observed. However, blocking CD95 led to increased BrdU-uptake at day 4(4 to 9%, P < 0.05) and day 7(7 to 17%, P < 0.05) (Figure 6). Even here, no dependency on cell density was evident. In summary, decreasing prolif-
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Detection ofApoptotic HSC after Acute Liver Damage in Vivo To investigate whether or to which extent our in vitro findings are of direct relevance for tissue repair in vivo we used the model of acute liver damage. HSC were detected by staining with antibodies against desmin or glial fibrillary acidic protein. HSC, identified by glial fibrillary acidic protein or desmin positivity, were present in the necrotic tissue in increasing numbers. At 24 hours after intoxication, the total number of HSC reached maximal levels due to HSC proliferation. At later stages (96 hours), HSC (desmin positive cells) decreased in number and showed signs of apoptosis (TUNEL positive), which was absent in HSC at 24 hours (Figure 7). Specific immunofluorescence signals of CD95 and CD95L could also first be detected at this point of time, and apoptotic HSC are situated in the same regions with high CD95 and CD95L fluorescence signals. These observations match well with our in vitro data that apoptosis of HSC was prominent in the transitional stage of the cultures and even stronger in the fully activated stage.
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Our data demonstrate that HSC in primary culture undergo spontaneous apoptosis. The process is mediated by the APO-1/Fas/CD95 system. In fact, the occurrence of spontaneous apoptosis of HSC in course of activation is accompanied by an increased expression of CD95L by the HSC themselves. This is demonstrated by the complete inhibition of apoptosis by a CD95 blocking antibody. The observations that HSC do not produce soluble CD95
or
soluble CD95L and that
even
single-situated
HSC show apoptotic changes suggest that HSC could self-activate their own death. Although activated HSC possess more CD95 and CD95L, compared with HSC in the resting and transitional phase, they show a higher resistance to CD95-mediated apoptosis induction. To explain this finding, we studied the apoptosis regulating systems, the Bcl-2 and the p53 systems. Both Bcl-2 and p53 could be found in increasing amounts in parallel to activation but were not detectable in the
tim Qh)
Figure 3. Annexin V-FITC binding of primary rat H SC at day 2o(), day 4c(e), and day 7 (A) after plating. Error bars demonstra eight different HSC isolations. A: Examples for sporntaneous apoptosis of cells at day 4 and day 7 of culture in comparison to ce lls at day 2. B: Cultures at day 4 and day 7 incubated 12 hours with CD95 anttagonistic antibodies. Only cells showing no or weak annexin V-FITC bindir igat the beginning of the experiment were evaluated. (*) demonstrates an example for possible regeneration of cells. C: Administration of CD9)5 agonistic antibodies to cultures at days 2, 4, and 7. Only cells showing na o annexin V-FITC binding at the beginning and apoptotic signs at the endI of the experiment were evaluated.
erative potency after day 4, up-regulI ation of proliferation after blocking CD95, and independenice from cell density support the hypothesis that HSC thennselves initiate their own suicide program. Cell-cell conta .cts do not seem to be necessarily required.
resting
cells.
Whereas only a weak induction of Bcl-2 occurred in response to CD95 agonistic antibodies at day 7, no change of
Bcl-2
amount could be
observed at day 4. The
Bcl-2-
related protein bax was detected in similar amounts at days 2 and 7 and was up-regulated in activated HSC (day 7). The bax expression
not
modulated through
incubation of CD95 agonistic antibodies. Although p53 was
considerably up-regulated by
CD95 agonists at day
4, no modulation was found at day 7. Bcl-2 inhibits apo-
ptosis by a still not well explained mechanism. One possibility is an inhibition of apoptosis induction by the inhibition of cytochrome c release out of mitochondria that is necessary for initiation of the apoptosis program.39 However, the formation of the heterodimer Bcl-2/bax is necessary for this effect, and the production of bax is dependent on the presence of p53. Additionally, p53 has been shown to down-regulate Bcl-2-production. By these
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Flow Cytometric Quantification of Apoptosis of Controls and Cultures Incubated with CD95 Agonistic Antibodies or CD95 Antagonistic Antibodies
Table 1.
Day 2 Induction (%)
Blockage (%)
Controls
Cells
Controls (%)
Alive Apoptotic Necrotic
97.6 1.2 1.2
4 95.7 0.3
98.9 0.3 0.8
Blockage (%)
Controls (%)
Day 7 Induction (%)
Blockage
(%)
Day 4 Induction (%)
90.2 8.3 1.5
1.0 98.9 0.1
95.7 3.3 1.0
80.4 18.1 1.5
1.5 97.7 0.8
83.7 14.4 1.9
(%)
The data show the percental portion of alive, apoptotic, and necrotic cells per total HSC population in the different phases of cultivation (resting (day 2); transitional (day 4); activated (day 7)). The data of control cultures and cultures under influence of CD95 agonistic and CD95 antagonistic antibodies for 12 hours are presented for each phase of cultivation.
apoptosis-inducing and -inhibiting activities of p53 could be shown.40 However, in our system the bax expression does not seem to be coupled with p53. In fact, up-regulated p53 at day 4 due to stimulation of the CD95 receptor did not result in up-regulated bax. Nevertheless, our data fit well with the observation that upregulated Bcl-2 blocks p53-induced apoptosis.22 However, p53 seems to have no effect on Bcl-2 production, as means,
Tl
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2
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Figure 4. Western blot analysis. Sodium dodecyl sulfate-polyacrylaimiide gel electrophoresis (11% polyacrylamide) of HSC cell lysates (30 ,ug of protein/ lane) day 2 (lanes 1 and 1*:), day 4 (lanes 2 and 2*), and day 7 (lanes 3 and 3*) after- plating. Controls (lanes 1, 2, 3) and cultures incubated with CD95 agonistic antibody (lanes 1 *, 2*, 3*). This is one of 5 Western blot analyses of 5 different cell isolations.
1000-
_
75OelOe1s/cm
well. First, up-regulation of p53 at day 4 in response to CD95 agonistic antibodies offers an explanation for breakage of the Bcl-2-induced apoptosis inhibition. Next, up-regulated Bcl-2 and bax at day 7 could lead to formation of higher amounts of antiapoptotic Bcl-2/bax heterodimers that possibly overcome the proapoptotic bax/ bax homodimers. Finally, this could lead to the lower resistance to CD95 mediated apoptosis induction at day 4 compared with day 7. In total, the data demonstrate a nonlymphatic cell that physiologically expresses both CD95 and CD95L in increasing amounts during spontaneous activation. In parallel with the occurrence of CD95L, apoptotic HSC could be detected. As spontaneous apoptosis could completely be inhibited by blocking CD95, our study provides evidence for the assumption that this system is the major tool for initiation of apoptosis. In the process of tissue repair, we were able to detect apoptotic HSC 4 days after initiation of acute liver damage. These apoptotic HSC are the same that express high amounts of CD95 and CD95L. This suggests that HSC self-activate cell death when their
M controls M cutures + CD95 antagonistic Ab em cukures + CD95 agonistic Ab
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time of culture (d) Figure 5. Percental changes of BrdU-uptake/10 cells during 12 houLrs at different days of cultures. Absorbance values (A.,,,,,9A92,,,,,)/109 cells were set as 100% for each cell density at day 2 and valLies for CLltuLres at day 4 and dlay 7 relate to the respectise salcie at day 2.
time
of culture (d)
Figure 6. Measurement of BrdU uptake during 12 hours of cultures (30,000 cells/cm2) without (controls) and with simultaneous administration of CD95 antagonistic or CD95 agonistic antibodies. Absorbance values of controls (A , ,A9,,,2,,,,) were set as 100% for each time point, and values for cuLltures tinder influLence of antibodies relate to the respective control culture.
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|(
i
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96h aftei damage
241i after damage 1-RI( - Dcsmiin
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-
(CID95L
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Figure 7. Detection of apoptotic HSC during tissue repair after induction of acute liver damage. Sequential liver sections at different time points after induction of acute liver damage were stained with TRIC-desmin (identification of HSC) and TUNEL (identification of apoptotic cells). Thus, specific immunofluorescence of CD95 and CD95L was not able to be detected until 96 hours after induction of acute liver damage, only sections of this time point are presented. Magnification of the control sections and the sections 24 hours after induction of acute liver damage, X400; magnification of the sections 96 hours after induction of acute liver damage, X800. Examples of apoptotic HSC are marked with arrows.
work is no more needed. In chronic liver diseases, chronic inflammation due to reiterative tissue damage could be responsible for the synthesis of growth factors,41'42 which in turn could overcome apoptotic induction, and so contribute to the maintenance of the fibrosis process. Furthermore, production of CD95L by HSC could be responsible for apoptotic death of cytotoxic T lymphocytes in chronic viral hepatitis, inhibiting viral elimination.
7.
8. 9.
10.
Acknowledgments We thank S. Bierkamp and A. Grundmann for excellent technical assistance.
11. 12.
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19. 20. 21.
22.
23. 24.
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