Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha

Bernhard Saile, Nina Matthes, Katrin Neubauer, Christoph Eisenbach, Hammoudeh El-Armouche, Joszef Dudas, and Giuliano Ramadori

Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, 37075 Göttingen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 ± 2% of the activated HSC (day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor-beta downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-alpha (TNF-alpha ) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-alpha stimulation reveal that activated HSC and rMF belong to different cell populations.

CD95 receptor; CD95 ligand; transforming growth factor-beta


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF HEPATIC STELLATE cells [HSC; so called since the consensus conference of 1996 (1)], also known as Ito cells, lipocytes, vitamin A-storing cells, perisinusoidal cells, and liver pericytes, is supposed to be one of the most important steps in liver tissue repair and in development of liver fibrosis. Activation describes the morphologic transformation of a vitamin A-storing quiescent cell through an intermediate state (transitional HSC) to a myofibroblast-like activated HSC. Activated HSC are thought to represent the major matrix-producing cell during repair after acute damage and liver fibrogenesis in case of chronic damage. However, activated HSC show characteristics commonly known for smooth muscle cells and myofibroblasts. In vitro and in vivo they have been shown to produce the intermediate filaments vimentin, desmin, and the myofilament smooth muscle alpha -actin (SMA) (10, 13, 33, 38, 39, 41, 46, 52).

Conversely, other cell types of the fibroblast lineage (ie., interstitial fibroblasts, vascular myofibroblasts, and bile duct epithelial cells) have also been shown to be of particular fibrogenic importance, especially in early stages of cholestatic and serum-induced hepatic fibrosis models (4, 12, 14, 18, 47-49, 53). Although a method for isolation of rat liver myofibroblasts (rMF) has already been described in 1985 by Leo et al. (30) and a few other in vitro studies (3, 17, 40) exist demonstrating that activated HSC and rMF may be regarded as cell populations of different origin, both having fibrogenic potential, the possible involvement of the rMF in the fibrosis process has often been disregarded, mainly because rMF has been thought to be the result of a transdifferentiation of the HSC. This may be due to the lack of specific markers to differentiate activated HSC from other fibroblastic cells in vivo. Because activated HSC express SMA (39), SMA-positive cells detected in damaged rat and human livers are thought to be activated HSC. Furthermore, SMA gene expression is considered an identification marker for HSC in vitro. Knittel et al. (25) recently strongly suggested that rat HSC cultures may contain few fibulin-2-/SMA-positive cells whose increase in number during time leads to the overgrowth of nonproliferating HSC. Because this could be the explanation for the supposed transdifferentiation, establishment and characterization of rMF primary culture were necessary.

HSC are known to undergo CD95-mediated spontaneous apoptosis when they are activated (42). To study the mechanisms underlying the survival advantage of myofibroblasts in vitro, we analyzed primary cultured HSC as well as primary and long-term cultured rMF with respect to apoptosis behavior. Our data show that, whereas rMF can be passaged several times, HSC developed spontaneous apoptosis within the activation period. This may be due to the phenomenon that rMF possess CD95R but lack CD95L, which, in contrast, is expressed by activated HSC. Further results also show different apoptosis behavior of rMF and activated HSC after treatment with the CD95 agonistic antibody and tumor necrosis factor (TNF-alpha ) stimulation. The higher CD95R expression and the lack of CD95L expression of the rMF cultures and their behavior on cytokine stimulation may be crucial for the survival of these cells in vivo during chronic liver damage. Further studies will allow understanding of the interaction between apoptotic mechanisms and cell cycle control points. It will also be interesting to study the TNF-alpha pathway of apoptosis induction in myofibroblasts compared with its antiapoptotic effect in activated HSC.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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HSC and rMF Isolation: Characterization, Plating, and Culture Conditions

Wistar rats were provided by Charles River (Sulzfeld, Germany) and maintained under 12:12-h light/dark cycles with food and water ad libitum. During research described in this report, all animals received humane care in accordance with institutional and National Institutes of Health guidelines.

HSC were isolated by sequential in situ perfusion with collagenase and pronase, as previously described (19-23, 26, 27). HSC (40 × 106) were obtained as mean per rat.

Cells were plated onto 24-well Falcon plates (Becton Dickinson, Heidelberg, Germany), 35-mm petri dishes (Greiner, Krefeld, Germany), 96-well Falcon plates (Becton Dickinson) or Lab Tek tissue culture slides (Nunc, Naperville, IL) with a density of 30,000 cells/cm2. Cells were cultured in DMEM supplemented with 15% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% L-glutamine. Culture medium was replaced 2 days after plating and then every other day. Cells were kept in culture at 37°C in 5% CO2 atmosphere and 100% humidity.

To evaluate purity of cultures, HSC were tested by immunofluorescence at day 0, day 2 (quiescent HSC/early activated HSC), and day 7 (activated HSC) after plating as described previously (19-23, 26, 27). Contamination with Kupffer cells (ED1 positive) was <2%, and neither endothelial cells nor hepatocytes were detected. With the use of SMA immunoreactivity as an activation parameter, HSC were fully activated after 7 days of primary culture (100% SMA positive). Fibulin-2 positive cells were always <1%. Some of the cultures were kept up to 2 or 3 wk and were either fixed and stored at -20°C until staining was performed or trypsinized and passaged several times.

For isolation of rMF, the liver was enzymatically digested as described above. The nonparenchymal liver cell population was separated by a Nycodenz density gradient, and the fraction consisting of Kupffer cells and sinusoidal endothelial cells was further purified by centrifugal elutriation according to Knook et al. (28) and De Leeuw et al. (8, 9). With the use of a JE-6B elutriation rotor in a J2-21 centrifuge (Beckman Instruments, Palo Altro, CA) at 2,500 rpm, a fraction enriched with rMF was collected at a flow rate of 23 ml/min. rMF (60 × 106) were obtained as mean per rat. HSC containing the typical fat droplets could not be detected by light microscopy. The presence of so-called empty HSC not containing typical vacuoles cannot totally be excluded, however, because empty HSC at least should be positive for one of the marker proteins like desmin, glial fibrillary acidic protein (GFAP), or V-CAM-1 (35), which were undetectable (GFAP) or present in <1.5% (desmin) or 0.5% (V-CAM-1) of the isolated cells. A considerable contamination of the 23-ml/min fraction with empty HSC is unlikely.

Cells of the 23-ml/min fraction were plated onto 24-well Falcon plates (Becton Dickinson) at a density of 30,000 cells/cm2. Cells were cultured in DMEM supplemented with 15% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% L-glutamine. At confluency, usually reached within 7-10 days, cells were released from the culture plates by trypsination and were replated at a 1:4 split ratio. rMF were passaged again at confluency using the same experimental condition. Subcultivation was performed several times.

Flow Cytometric and Fluorescence Microscopic Quantification of Living, Apoptotic and Necrotic HSC or rMF

For quantification of apoptotic cells, we used flow cytometry after trypsination of HSC and rMF (Epics ML; Coulter, Kerfeld, Germany). To detect apoptotic changes, staining with annexin V-FITC/propidiumiodide and Tdt-mediated X-dUTP nick-end labeling (TUNEL) were used (Boehringer Mannheim, Indianapolis, IN). Data obtained by TUNEL labeling were identical to those obtained with the annexin V-FITC/propidium iodide binding.

Western Blot Analysis of SMA, Fibulin-2, CD95, CD95L, and TNF Receptors 1 and 2

Cells at different times after plating were lysed in hot Laemmli buffer (95°C) and processed by SDS-PAGE under reducing conditions according to Laemmli (29). Protein content of cellular lysates was calculated by the Coomassie protein assay (Pierce, Rockford, IL). Proteins were transferred onto Hybond-enhanced chemiluminescense nitrocellulose hybridization transfer membranes according to Towbin et al. (51). Immunodetection was performed according to the enhanced chemiluminescense Western blotting protocol. Antibodies against SMA (Sigma, Munich, Germany), fibulin-2 (Dr. R. Timpl, MPI for Biochemistry, Martinsried, Germany), CD95 (APO-1/Fas), CD95L, TNF receptor (TNFR)1, and TNFR2 (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 2.5 µg/ml solutions, and peroxidase-labeled anti-mouse and anti-rabbit immunoglobulins (DAKO, Copenhagen, Denmark) were each used at a 1:1,000 dilution. Densitometric evaluation of the blots was performed using the program Scion Image version beta 2 (National Institutes of Health).

Northern Blot Analysis of SMA and Fibulin-2

Briefly, 5 µg of total RNA was resolved by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with specific 32P-labeled cDNA probes. Hybridization was performed for 2 h at 68°C using the QuickHyb Kit (Stratagene, La Jolla, CA). Posthybridization washes were performed two times for 15 min each at 60°C in 2× standard saline citrate solution containing 0.1% SDS. Nylon filters were washed, dried, and exposed to X-ray films at -80°C.

Immunocytochemical Detection of SMA, Fibulin-2, CD95R, and CD95L

First transmission pictures of HSC (days 2 and 7) and rMF (passage 4) were taken after marking the regions of interest. After cell cultures were fixed in methanol/acetone (5 min/10 s at -20°C), slides were incubated with specific primary antibodies against SMA (Sigma), fibulin-2 (Dr. R. Timpl, MPI for Biochemistry, Martinsried, Germany), or CD95 and CD95L (Santa Cruz Biotechnology) for 1 h (37°C). For double immunofluorescence staining, slides were subsequently covered with FITC-conjugated antibodies against mouse IgG and tetrarhodamine isothiocyanate-conjugated antibodies against rabbit IgG (Sigma). Nonspecific staining was evaluated by incubation with mouse or rabbit IgG (Sigma) rather than the primary antibody, and maximum fluorescence intensity was subsequently digitally subtracted using a confocal laser scan microscope (Zeiss, Oberhofen, Germany).

Investigation of Soluble CD95 and CD95L.

To investigate whether rMF releases soluble CD95R and CD95L, we performed Western blot analysis. For this purpose, we used concentrated supernatants (30-min vacuum centrifugation) of rMFs that were handled as described above. To functionally prove the possible presence of CD95L in the supernatant, we cultivated HSC in the quiescent phase (day 2) in the presence of supernatants of rMF. Occurrence of apoptosis was then measured by flow cytometry. As a second method to detect soluble CD95L, we performed a sCD95L ELISA (Boehringer Mannheim) according to the manufacturer's protocol using supernatants from rMF and HSC cultures.

Occurrence of Spontaneous and Induced Apoptosis in HSC and rMF

To investigate induction of apoptosis and apoptosis occurring spontaneously in activated HSC (day 7) and rMF, we performed a test using confocal laser scan microscopy (Zeiss) in the time-scan mode. To detect early apoptotic changes, staining with annexin V-FITC was used. To distinguish apoptosis and necrosis, annexin V-FITC and trypan blue, a common dye exclusion test, were employed in parallel for showing membrane integrity after annexin V-FITC binding to cells. In all investigated cases, we could not notice loss of membrane integrity within 30 min after annexin V-FITC binding was detected; but 12 h after initiation of the test, considerable amounts of cells showed membrane leakage indicating secondary necrosis.

Activated HSC and rMF were treated with 3 and 1 ng/ml of CD95 agonistic antibodies, respectively (Bender Med Systems, Ingelheim, Germany). Preliminary dose-response studies performed using different concentrations of CD95 agonistic antibodies (0.1, 0.5, 1, and 3 ng/ml) showed that triggering into apoptosis was obtained in activated HSC by 3 ng/ml and in rMF by 1 ng/ml. As negative controls, HSC and rMF were cultured with and without mouse IgG (Sigma). Apoptosis rates did not differ in the two controls.

Transfection Assay

rMF (primary culture, passages 2, 4, and 6) and activated HSC (day 7) were transfected by modification of the liposome transfection protocol. Briefly, rMF and HSC (day 7) were transfected with FuGENE (Boehringer-Mannheim) according to the manufacturer's protocol with minor modifications by using 10 µg CD95L plasmid DNA (PH Krammer, DKFZ, Heidelberg, Germany) per 2 × 106 cells. Null-plasmid controls were performed to evaluate a possible nonspecific effect of transfection assays on apoptosis of rMF or HSC. All experiments were repeated at least three times, and consistent results were obtained in all cases.

Culture Conditions for Stimulation with Transforming Growth Factor-beta or TNF-alpha

HSC at day 7 of primary culture and rMF at passages 2, 4, and 6 were washed three times with Gey's balanced salt solution and incubated for 20 h in serum-reduced (0.3% FCS) culture medium alone or in the presence of transforming growth factor-beta (TGF-beta ) (1 ng/ml) or TNF-alpha (100 U/ml; Sigma). Optimal concentrations and incubation time of the cytokines used have previously been tested and shown to have no cytotoxic effects (5, 23, 44).

Statistical Analysis

Results are expressed as means ± SD, and the significance of the difference between the means was assessed by the Mann-Whitney U-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of rMF and HSC Isolations

Morphologically freshly isolated rMF do not show lipid droplets around the nuclei present in cytospins from freshly isolated HSC. Whereas freshly isolated rMF were >95% positive for SMA and fibulin-2, in HSC isolations, only a few SMA- and fibulin-2-positive cells could be detected (Fig. 1A). After activation (day 7), HSC express SMA but remain fibulin-2 negative (Fig. 1B). In HSC cultures derived from isolations with low yield of cells (5-7 Mio cells per rat liver marked with an asterisk; Fig. 1C), a contamination with fibulin-2-positive cells at day 2 (also SMA positive) and day 7 of culture could be detected (Fig. 1C). However, even in very pure HSC cultures (average yield of cells: 40-60 Mio cells per rat liver), single fibroblasts could be observed (Fig. 1D).


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Fig. 1.   A: characterization of freshly isolated rat liver myofibroblasts (rMF) and hepatic stellate cells (HSC) by hematoxilin and peroxidase staining. Please note that freshly isolated rMF are positive for fibulin-2 and smooth muscle alpha -actin (SMA). Only a few SMA- and fibulin-2-positive cells can be detected in freshly isolated HSC (arrows). On the other hand, freshly isolated HSC show lipid droplets in the cytoplasm not detectable in rMF. B: the same results could be shown by Western blot analysis. Although activated HSC (day 7) express SMA, they remain fibulin-2 negative. Blots presented are 1 of 3 Western blot analyses of 3 different isolations. C: Northern blot analysis of different HSC and rMF cultures. In HSC cultures of isolations with low yield of cells per rat liver (marked with an asterisk), SMA and fibulin-2 transcripts could be detected revealing contamination with rMF. D: in very pure HSC cultures, single fibroblasts can be detected (SMA and fibulin-2 positive). Magnification: ×200; d, day; P, passage; TRIC, tetrarhodamine isothiocyanate.

Apoptosis of Activated HSC and rMF. Influence of the CD95 System

Different expression of CD95 and CD95L of activated HSC and rMF. Initial experiments showed that rMF could be subcultivated without any problems, whereas fully activated HSC could only be cultured until passage 2. Regarding apoptosis 18 ± 2% of activated HSC (day 7) could be shown to undergo spontaneous apoptosis. In subcultivated HSC cultures 73.4% (passage 1) and 94.3% (passage 2) of evaluable cells showed signs of apoptosis. In contrast to this, rMF cultures showed a maximum of 1.3% apoptotic cells in all investigated cultures (primary culture day 7; passages 2, 4, and 6). Under the influence of 1 ng/ml CD95 agonistic antibodies rMF cultures showed 100% evaluable cells undergoing apoptosis, whereas in HSC cultures (day 7), 34.7% showed apoptosis signs. A concentration of 3 ng/ml of CD95 agonistic antibodies was needed to completely trigger HSC cultures (day 7) into apoptosis (Fig. 2).


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Fig. 2.   Flow cytometric quantification of apoptosis of activated HSC and rMF under serum-reduced conditions (0.3% FCS; 12 h), rMF cultures incubated with 1 ng/ml (6 h) CD95 agonistic antibodies, and activated HSC cultures incubated with 3 ng/ml (10 h) CD95 agonistic antibodies. Data show the percent portion of apoptotic cells per total HSC or rMF population. Values presented are means ± SD of 9 different isolations. Level of significance: *P < 0.05; **P < 0.01.

To further analyze the reason why rMF do not undergo spontaneous apoptosis, CD95R and CD95L expression were studied. The CD95 system has previously been shown to be crucial for spontaneous apoptosis of HSC (42). Therefore, this system could be of importance to differentiate between activated HSC and rMF.

CD95 was detectable in 70% higher amounts in rMF compared with activated HSC. CD95L, however, was not detectable in rMF cultures but is present in activated HSC (day 7) (Fig. 3, A and B).


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Fig. 3.   A: Western blot analysis of CD95 and CD95L. SDS-PAGE (9% polyacrylamide) of HSC and rMF cell lysates (30 µg protein/lane). Blots presented are 1 of 3 Western blot analyses of 3 different isolations. B: densitometric evaluation. Values for the activated HSC [day 2 (CD95R; A) or day 7 (CD95L; B)] were set at 100%. Densitometric values for HSC cultures at days 2 and 7 and the values of the rMF cultures relate to the respective value of the activated HSC. Values presented are means ± SD of 3 Western blot analyses of 3 different isolations. Level of significance: *P < 0.05, **P < 0.01.

CD95 and CD95L gene expression of these cultures was evaluated on the single cell level by immunocytochemistry and compared with HSC. Evaluation of 700 cells of activated HSC cultures (7 different isolations) showed a CD95L positivity of 79 ± 5% after subtraction of maximum fluorescence intensity of the negative controls (Fig. 4). However, taking mean + 2× SD of the fluorescence activity of the negative controls as reference, 100% of the activated HSC could be shown to be CD95L positive, whereas primary rMF (day 7) and HSC (day 2) cultures were CD95L negative.


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Fig. 4.   Expression of CD95 and CD95L in HSC (days 2 and 7) and in rMF (passage 4). Top: transmission images of quiescent HSC, activated HSC, and rMF. Middle and bottom: expression of CD95 and CD95L. Immunofluorescence activities after subtraction of maximum immunofluorescence of the respective negative controls are shown. Magnification: ×400.

Because it is well known that soluble CD95 and CD95L can be released by cells, we investigated this possibility using Western blot and ELISA after protein concentration enrichment of supernatants. Neither CD95 nor CD95L could be detected by Western blot and ELISA. As a third approach, we therefore incubated HSC cultures with supernatants of the rMF cultures and vice versa. However, compared with the control media, the apoptosis rate did not alter in the cultures using the annexin V/propidium iodide and the TUNEL method.

Transfection of rMF (passage 4) with CD95L. To further investigate the role of CD95L in the apoptotic process of rMF, cultures were transfected with a CD95L expressing vector. Whereas control cultures and cultures transfected with the control vector did not show a considerable induction of apoptosis, cultures transfected with the CD95L-cDNA underwent apoptosis (95-100%) within 2 h. This apoptosis could be avoided by treating the cells with CD95-blocking antibodies. To further prove that apoptosis was due to the synthesized CD95L, incubation of rMF with the supernatants of the CD95L-transfected rMF cultures was performed. The supernatant also led to complete apoptosis of untransfected rMF cultures. Simultaneous administration of CD95-blocking antibodies completely prevented these effects (Fig. 5). Supernatants from CD95L-transfected rMF also induced apoptosis in cultures of activated HSC (data not shown). Also, in this case, apoptosis was prevented by the addition of CD95 antagonistic antibodies. Furthermore, the presence of soluble CD95L in supernatants from CD95L-transfected rMF was measurable by ELISA and Western blot. This series of experiments was performed to show that transfection of rMF with the CD95L plasmid truly induced synthesis of soluble and biologically active CD95L.


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Fig. 5.   Transfection of rMF (primary culture and passage 4) with CD95L-cDNA. Tdt-mediated X-dUTP nick-end labeling (TUNEL) staining of control cultures and cultures after transfection with CD95L and cultures under the influence of supernatants of CD95L-transfected cultures are shown. Apoptosis-inducing effect of CD95L transfection can be completely annulled by simultaneous administration of CD95 antagonistic antibodies.

Time dependency of apoptosis induction due to CD95 agonistic antibodies. To investigate the susceptibility of activated HSC and rMF to CD95-mediated apoptosis induction, we used confocal laser scan microscopy in the time-scan mode. By these means, it is possible to detect signs of early apoptosis over a 12-h period by taking one picture every quarter of an hour of the identical field. With the use of CD95-agonistic antibodies (1 ng/ml), rMF cultures [primary culture (day 7), passages 2, 4, and 6] could completely be triggered into apoptosis within 2 h, whereas activated HSC-cultures were more resistant. Although, as shown above, 3 ng/ml of the CD95 agonist is necessary to induce apoptosis of the total culture. The period of time until activated HSC underwent the apoptosis process also was considerably longer (5.5 h; Fig. 6).


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Fig. 6.   Confocal microscopy monitoring of annexin V-FITC binding of activated HSC incubated with 3 ng/ml CD95 agonistic antibodies; rMF P0, rMF P2, rMF P4, and rMF P6 were each incubated with 1 ng/ml CD95 agonistic antibodies. Values presented are means ± SD of 500 cells from 4 different isolations.

Effects of TNF-alpha or TGF-beta on Apoptosis of Activated HSC and rMF. Both TNF-alpha and TGF-beta are fibrogenic mediators that have been shown to exert the function of surviving factors in isolated HSC. In this paper we confirmed previous results obtained on HSC (2, 23, 27, 37). With regard to apoptosis, TNF-alpha exerted effects on rMF that differ from those observed on activated HSC. Whereas both cytokines effected apoptosis inhibition on the latter cell type (44), rMF showed a different but continuous behavior in all investigated cultures. TNF-alpha administration led to a 10- to 20-fold increase in apoptosis in rMF. On the other hand, TGF-beta treatment caused an apoptosis inhibition in all cases. After induction of CD95-mediated apoptosis by 1 ng/ml CD95 (rMF) or 3 ng/ml (HSC) agonistic antibodies (leading to 100% apoptosis of all rMF or HSC cultures), approx 15% of total rMF cultures could be rescued from apoptosis by TGF-beta (Fig. 7).


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Fig. 7.   Flow cytometric quantification of apoptosis of activated HSC and rMF under serum-reduced conditions (0.3% FCS; 20 h) and cultures incubated with transforming growth factor-beta (TGF-beta ; 1 ng/ml) or tumor necrosis factor-alpha (TNF-alpha ; 100 U/ml) alone or in combination with 1 ng/ml (rMF), 3 ng/ml [HSC (20 h)] CD95 agonistic antibodies. Data show the percent portion of apoptotic cells per total HSC or rMF population. Values presented are means ± SD of 3 different isolations. Level of significance: *P < 0.05; **P < 0.01.

TNF-receptor status of activated HSC and rMF. The pleiotropic biological properties of TNF-alpha are signaled through two distinct cell surface receptors [TNF receptor 1 (TNFR1) and TNFR2]. To investigate whether a different TNFR status of rMF and HSC is responsible for the different effect of TNF-alpha on apoptosis, we performed Western blot analysis. It could be shown that the status of TNFR1 and TNFR2 was similar in activated HSC and rMF (primary culture, passages 2, 4, and 6). Moreover, it could be shown that the amount of both receptors is not changed after TNF-alpha stimulation (Fig. 8).


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Fig. 8.   Western blot analysis of TNFR1 and TNFR2. SDS-PAGE (9% polyacrylamide) of activated HSC (day 7) and rMF cell lysates (30 µg protein/lane) with and without TNF-alpha stimulation. One of 3 Western blot analyses of 3 different isolations are presented.

Effects of TGF-beta or TNF-alpha on CD95/CD95L expression of rMF. To evaluate whether changes of the CD95/CD95L system are involved in the effects of TGF-beta or TNF-alpha , we performed Western blot analysis of total proteins. Neither TGF-beta nor TNF-alpha changed expression of CD95 or CD95L (Fig. 9), suggesting that TGF-beta and TNF-alpha regulate apoptosis pathways in rMF downstream of the CD95R and probably independently of the CD95 pathway (caspase 3; data not shown).


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Fig. 9.   Western blot analysis of CD95 and CD95L. SDS-PAGE (9% polyacrylamide) of activated HSC (day 7) and rMF cell lysates (30 µg protein/lane). One of 3 Western blot analyses of 3 different isolations are presented.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Until now, most studies in the field of liver repair and fibrogenesis concentrated on activated HSC, which was regarded as the major matrix synthesizing cell type. Only a few studies (3, 4, 7, 11, 12, 18, 24, 47, 49, 53) demonstrate that other myofibroblastic cells also have fibrogenic potential. So far, differentiation of activated HSC and other liver myofibroblasts hardly has been possible because of morphologic similarities and lack of specific marker proteins. Knittel et al. (25) recently demonstrated differences with respect to expression of cytoskeletal proteins, adhesion molecules, cytokine synthesis, and matrix proteins between HSC and rMF isolated from normal rat liver. The basis of this study was the different growth behaviors of activated HSC and rMF. Although prolonged cultivation of rMF was possible, activated HSC could be maintained in culture only until passage 2, when the first passage was performed at the time of full activation (day 7). In contrast to rMF cultures, a significant proliferation measured as increase of cell number could also not be detected in HSC cultures. Whether the HSC of the hepatic sinusoid increase in number is controversially discussed (15, 31, 32). However, mitosis of myofibroblasts of the periportal and perivenous tracta could be shown (31, 32, 53). We also reported that HSC undergo apoptosis mediated by the CD95/CD95L system when they are activated and demonstrated that data show both TGF-beta or TNF-alpha inhibit apoptosis and DNA synthesis of activated HSC (42, 44). Because survival of myofibroblasts and myofibroblast-like cells seems to be crucial for development of liver fibrosis in chronically damaged liver, it was of great interest to study apoptosis pathways and the effect of two major fibrogenic cytokines on apoptosis in rMF. For this purpose, we established a method to isolate myofibroblasts from rat livers.

Although maximum 2% apoptosis was detectable in the rMF cultures (primary culture day 7, passages 2, 4, and 6), primary cultured HSC showed 18 ± 2% apoptosis at day 7. In subcultivated HSC cultures, an apoptosis frequency of >80% could be detected. Because the CD95/CD95L system is shown to be the major pathway for spontaneous apoptosis of HSC (42), this system was also investigated for rMF. Studies (6, 36, 45, 50) on fibroblast cultures of different organs have shown CD95 expression but conflicting results with regard to CD95L expression and functionality of the CD95/CD95L system. Lack of spontaneous apoptosis in rMF also is not due to the lack of CD95R. In fact, compared with activated HSC (day 7), surprisingly <= 70% higher expression of CD95 could be observed in the rMF cultures. However, whereas CD95L is expressed in activated HSC, it is not detectable in rMF. Probably because of the higher expression of CD95R, triggering of the CD95/CD95L system using agonistic antibodies at the low amount of 1 ng/ml led to apoptosis of >95% of cells of the different rMF cultures within 2 h. A dependency of apoptosis susceptibility and amount of CD95-expression could also be shown for normal skin fibroblasts and keloid fibroblasts (34). Maybe because of the lower amount of CD95R activated, HSC seem to be more resistant to triggered apoptosis. In the presence of 1 ng/ml CD95-agonistic antibodies, the apoptosis rate of activated HSC rose from 18 to 37% but was far below that seen in rMF. A concentration of 3 ng/ml was needed to lead all activated HSC cultures into apoptosis. A higher susceptibility of rMF to CD95-mediated apoptosis could also be deduced from the transfection assays, because supernatants of the CD95L-transfected rMF cultures led to complete apoptosis of rMF cultures but only to a significant increase of apoptosis rate in activated HSC. A greater resistance of activated HSC to CD95-mediated apoptosis could also be shown when regarding time dependency of apoptosis occurrence. Although the rMF cultures could be triggered into apoptosis within 2 h, a period of 5-6 h is needed in case of activated HSC. Both dose- and time-dependent differences in responsiveness to CD95/CD95L-mediated apoptosis might be the result of differences in intracellular apoptotic signaling pathways triggered by CD95 in these two cell populations. This may also be due to differences in antiapoptotic pathways activated in parallel, thus making activated HSC, in part, refractory to external CD95L. In their paper, Gong et al. (16) demonstrated an increment of the susceptibility to apoptosis during the transformation of HSC to myofibroblasts, which contrasts our results. Although the authors described myofibroblasts as activated HSC, the behavior on apoptosis induction due to the CD95 receptor and the lack of CD95 ligand expression more closely resembles our data of rMF than that of activated HSC raising questions about the origin of their "myofibroblasts."

TGF-beta and TNF-alpha are two key factors involved in many processes of tissue repair and in the development of fibrogenic disorders. Both TGF-beta and TNF-alpha inhibit apoptosis of activated HSC (43, 44). Although TGF-beta also effected an apoptosis inhibition in rMF, TNF-alpha stimulated apoptosis. Because the receptor status of HSC and rMF is similar for TNFR1 and TNFR2, it might be speculated that activated HSC and rMF also differ in recruitment and reciprocal influences of apoptosis-regulating pathways. These investigations seem to be of special importance because they could open valuable insights into the mechanisms of fibrogenesis and also offer possibilities for therapeutic approaches. The fact that TNF-alpha is upregulated in early phases of models of acute liver damage and that TGF-beta is predominant in late phases of acute liver damage and in chronic liver damages suggests (together with our in vitro data) that activated HSC may be of special importance in tissue repair after acute damage, whereas cytokine situation in chronic liver damage leads to an environment allowing rMF to grow out and to take over special functions in tissue repair.

Data of the present report strongly suggest that rMF have to be considered as an additional population of nonparenchymal cells. rMF and activated HSC differ in occurrence of spontaneous apoptosis due to different CD95L expression in their susceptibility to CD95-mediated apoptosis and their reaction on TNF-alpha stimulation. Because rMF and activated HSC could not be delimited by conventional markers until now, and both cell types of the fibroblastic lineage share great morphologic similarities, in vitro data obtained from prolonged cultured HSC should be examined critically since the cultures used may consist of rMF grown out from contaminating cells in nonparenchymal cell cultures (compare Fig. 1, C and D). This possibility may also be the reason why many published data attributed to activated HSC demonstrate conflicting results. Furthermore, the results presented in this paper could possibly be translated to other fields where transdifferentiation of epithelial to mesenchymal cells has been supposed (54).


    ACKNOWLEDGEMENTS

The authors are indebted to A. Herbst and N. Nolte for excellent technical assistance.


    FOOTNOTES

This study was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 402 Molekulare und Zelluläre Hepatogastroenterologie, project C6.

Address for reprint requests and other correspondence: G. Ramadori, Dept. of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg August University Göttingen, Robert Koch Straße 40, 37075 Göttingen, Germany (E-mail: gramado{at}med.uni-goettingen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

December 5, 2001;10.1152/ajpgi.00441.2001

Received 15 October 2001; accepted in final form 26 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 283(2):G435-G444
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