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(The FASEB Journal. 1999;13:1950-1960.)
© 1999 FASEB

Fas (CD95, APO-1) antigen expression and function in murine liver endothelial cells: implications for the regulation of apoptosis in liver endothelial cells

JOSÉ E. CARDIER*1, TARA SCHULTE{dagger}, HEIDE KAMMER{dagger}, JENNY KWAK{dagger} and MARISABEL CARDIER{ddagger}

* Laboratorio de Fisiopatologia, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas 1020-A, Venezuela;
{dagger} Hipple Cancer Research Center, Dayton, Ohio 45439-2092, USA; and
{ddagger} Hospital Risquez, Caracas, Venezuela

1Correspondence: IVIC-Medicina Experimental, Apartado Postal 21827, Caracas 1020A, Venezuela. E-mail: jcardier{at}medicina.ivic.ve


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Fas up-regulates the expression...
 DISCUSSION
 REFERENCES
 
The Fas (CD95, APO-1) receptor is a membrane-associated polypeptide that can mediate apoptosis in various cell types. Although Fas receptor is expressed in endothelial cells (EC), little is known about its function in these cells. The expression of Fas by liver endothelial cells (LEC) suggests that upon stimulation, apoptosis may occur in these cells. We show that Fas is highly and constitutively expressed in cloned murine liver endothelial cells (LEC-1). In contrast, FasL expression was not detected at the protein and mRNA level in these cells. Our results show that Fas ligation in LEC-1 induces apoptotic cell death, indicating that Fas receptor is functional in these cells. The doses of Fas agonist required to induce LEC-1 apoptosis were higher than those used previously in other cells, including hepatocytes, suggesting that LEC-1 are highly resistant to the Fas apoptotic pathway. TNF treatment of LEC-1 induced up-regulation of Fas receptor on these cells. In contrast, TNF did not induce the expression of FasL on LEC-1. An increased susceptibility to Fas-mediated apoptosis was observed in TNF-treated LEC-1. Enhanced susceptibility to Fas-mediated apoptosis was also observed in LEC-1 pretreated with actinomycin D, suggesting that transcription of message coding for protective proteins is necessary to protect these cells against Fas-mediated apoptosis. Up-regulation of VCAM-1 and ICAM-1 was observed in LEC-1 treated with a dose of Fas agonist that does not induce apoptosis. To our knowledge, this is the first report that Fas mediates apoptosis in LEC, suggesting that apoptosis of these cells may participate in the liver damage observed in animals after receiving anti-Fas mAb or soluble FasL. Our findings also suggest that the Fas/FasL system may transduce activating signals independently of cell death in LEC-1.—Cardier, J. E., Schulte, T., Kammer, H., Kwak, J., Cardier, M. Fas (CD95, APO-1) antigen expression and function in murine liver endothelial cells: implications for the regulation of apoptosis in liver endothelial cells.


Key Words: Fas-L • LEC-1 • liver • HUVEC


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 Fas up-regulates the expression...
 DISCUSSION
 REFERENCES
 
THE FAS (CD95, APO-1)/FAS LIGAND (FasL) system constitutes an important cellular pathway regulating the induction of apoptosis in a wide variety of tissues (1) . Fas is a 45 kDa cell surface protein belonging to the tumor necrosis factor (TNF) receptor family (2 , 3) . FasL is a 40 kDa type II transmembrane protein belonging to the TNF family (4) . Fas is expressed on a wide variety of cell types such as T and B cells, monocytes, neutrophils, eosinophils, macrophages, keratinocytes, and endothelial cells (EC) (2 , 3 , 5 , 6) . In contrast, the surface expression of FasL appears to be more limited and often requires cellular activation (7) . The Fas cytoplasmic domain contains a segment known as the `death domain' homologous to that present within the cytoplasmic domain of the TNF receptor p55 (TNFRp55) (8) . Despite the similarity observed between both Fas and TNFRp55, current evidence indicates that induction of apoptosis by these two receptors proceeds along different pathways (9 , 10) .

The Fas/FasL system has been shown to play a critical role in the regulation of the immune system by controlling processes such as clonal deletion of autoreactive T cells (11) and elimination of target cells by cytotoxic T and natural killer (NK) cells (12) . Physiologically, FasL-bearing inflammatory cells (i.e., activated T cells, neutrophils, and monocytes) may induce apoptosis upon interaction with Fas-bearing target cells (13 , 14) . A biologically active soluble form of FasL may also interact with Fas-bearing cells, contributing to systemic tissue injury during inflammation (15) .

The EC are involved in multiple processes such as development, inflammation, and wound healing. During these processes, proliferation, followed by EC death, is observed (16) . Signals promoting apoptosis of EC have not been well characterized. In contrast to the role of the Fas/FasL system in lymphocytes, much less is known about its expression and function in EC. Data obtained in in vitro models showed that activation of Fas did not trigger apoptosis in human umbilical vein EC (HUVEC) (6) . However, using EC from other sources, it was shown that Fas may mediate apoptosis (17 , 18) . Taken together, these results suggest there is a differential sensitivity to Fas-mediated apoptosis between EC from different sources. On the other hand, in vivo experiments in a murine model showed the development of inflammatory angiogenesis induced by local stimulation of Fas (19) . All of these results suggest that Fas may have a biological function in some types of EC.

Activation of Fas with agonist anti-Fas monoclonal antibodies (mAb) or by its natural ligand, FasL, leads to apoptosis in susceptible tissues (1 , 20) . The liver is highly sensitive to induction of Fas-mediated apoptosis, which may be related to the fact that Fas is constitutively expressed in liver cells (3) . Thus, mice injected with anti-Fas mAb or soluble FasL die after 6 h due to liver damage (20) . The main mechanism postulated is hepatocyte apoptosis, since these cells express Fas and because in vitro experiments have shown that FasL induces apoptosis in hepatocytes (20 , 21) . However, because liver endothelial cells (LEC) also express Fas (21) , we hypothesize that the Fas/FasL system might also be involved in the regulation of apoptosis in these cells. In this study, we tested this hypothesis by using cloned murine LEC (LEC-1) and examining their susceptibility to Fas-mediated apoptosis. We also examined other LEC-1 responses to Fas ligation involving regulation of cellular adhesion molecule (CAM) expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 Fas up-regulates the expression...
 DISCUSSION
 REFERENCES
 
Reagents
Purified (endotoxin and azide-free) hamster anti-mouse Fas mAb (clone Jo2), FITC-labeled hamster anti-mouse Fas mAb (clone Jo2), purified FITC-labeled or unlabeled hamster immunoglobulin G (IgG) isotype, biotinylated hamster anti-mouse FasL mAb, streptavidin-phycoerythrin (Sav-PE), and FITC or PE-conjugated rat anti-mouse intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM) mAb were from PharMingen (San Diego, Calif.). Actinomycin D (ActD) was purchased from ICN (Aurora, Ohio). Recombinant annexin V-FITC was from PharMingen. Recombinant murine tumor necrosis factor {alpha} (rmTNF-{alpha}) was purchased from R&D Systems (Minneapolis, Minn.). TNF-{alpha} was resuspended in 10% human serum albumin (Sigma, St. Louis, Mo.), stored at -20°C, and used at an optimal concentration of 25 ng/ml.

LEC-1 isolation and cloning
The cloning and characterization of LEC-1 have been reported (22 23 24) . LEC-1 express a full range of cell lineage-specific markers: von Willebrand Factor (VIII/vWF), CD34, fms-like tyrosine kinase (flt), fetal liver kinase-1 (flk-1), platelet endothelial cell adhesion molecule (PECAM), ICAM-1, and VCAM-1 (22 , 24) . Two clones of LEC-1 were selected because they maintained stable functional and phenotypical characteristics after 10 passages. LEC-1 clones were maintained in Iscove's modified Dulbecco's medium (IMDM) (BioWhittaker, Walkersville, Md.) -10% fetal calf serum (BioWhittaker) and used for experiments after the cells reached 90% confluence.

Flow cytometric analysis of Fas and FasL expression on LEC-1 clones
LEC-1 were grown to 90% confluence, the supernatant was removed, and the cells were harvested and incubated with FITC-anti-Fas mAb or biotinylated mAb anti-murine FasL, followed by Sav-PE. Simultaneous negative control staining reactions were performed by incubating the cells with the FITC-labeled hamster IgG isotype or with biotinylated hamster IgG isotype, followed by Sav-PE. After two washes with phosphate-buffered saline (PBS), the cells were fixed with 1% paraformaldehyde in PBS. To examine the effects of TNF on Fas and FasL expression, LEC-1 were incubated with TNF at 25 ng/ml for 18 h in serum-free medium (X-vivo; BioWhittaker). LEC-1 were then harvested, washed, and analyzed for Fas and FasL expression as described above. Data collection and analysis of the fluorescent intensities were carried out using a FACSort (Becton Dickinson, San Jose, Calif.). Ten thousand events were acquired and analyzed using the Lysis II software program.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis of FasL expression in LEC-1
Total RNA was isolated from monolayers of LEC-1 clones using TRI Reagent (Molecular Research Center, Cincinnati, Ohio). One microgram of the total RNA was reverse-transcribed in a total volume of 20 µl in buffer containing 50 mmol/L TRIS-HCL (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgCl2, 10 mmol/l dithiothreitol, 10 mmol/l deoxynucleotide mixture, 100 pmol/l random hexamer oligonucleotides, and 200 U Moloney's murine leukemia virus reverse transcriptase (Perkin Elmer, Branchburg, N.J.). PCR amplification of the cDNA was then performed using specific oligonucleotides for the detection of FasL transcripts. The primers for murine FasL were (5__-CACTCAAGGTCCATCCCTCTG-3__ (sense) and 5__-TAGCTGACCTGTTGGACCTTGC-3__ (antisense). Primers for ß-actin were 5__-CATGATCTGGGTCATCTTTTCACGGTTTGGC-3__ (sense) and 5__-ATGGATGATGATATCGCTGCGCTGGTCGTC -3__ (antisense). PCR conditions were 1 min of denaturation at 95°C, annealing at 55°C for 1 min, and extension at 72°C for 1 min for 35 cycles. Analysis of the PCR products was performed by comparing them with the predicted PCR fragment size after ethidium bromide staining of the PCR products separated by electrophoresis in a 1% agarose gel.

Fas-mediated cell death
We studied the role of Fas/FasL pathway on LEC-1 viability by using anti-Fas mAb, which induces apoptosis in susceptible cells (1 , 25 , 26) . For this purpose, LEC-1 were plated in 96-well plates at 1 x 103 cells per well in X-vivo. The cells were allowed to attach to the bottom of the wells and purified (endotoxin and azide-free) anti-Fas mAb at 1 to 50 µg/ml or isotype-matched IgG was added to each well. The cells were further incubated at 37°C and cell viability was measured by a colorimetric assay using MTS (Promega, Madison, Wis.). For MTS assay, 20 µl of a stock solution of MTS was added to each well. After 3 h of incubation, the bioreduction of MTS by viable cells into a soluble formazan, reflecting the number of viable cells per well, was measured at 490 nm. Cell viability at each anti-Fas mAb concentration was indicated as a percentage of the control. The possible synergistic effect of anti-Fas mAb with TNF on LEC-1 viability was also investigated by culturing these cells with TNF at 25 ng/ml and anti-Fas mAb. Likewise, we examined the effect of anti-Fas mAb on LEC-1 viability in the presence of ActD. LEC-1 apoptosis was confirmed by morphology. Morphological changes of apoptosis in LEC-1 were defined as diminution in cell volume and cell nucleus and as chromatin condensation (27) .

Delayed addition assays
We investigated the effect of delayed addition of anti-Fas mAb to TNF-treated LEC-1. LEC-1 were preincubated with TNF at 25 ng/ml for 12 h at 37°C and 5% CO2. Each well was washed to remove TNF; anti-Fas mAb or isotype control was added and the cells were incubated for 24 h. Cell viability was measured by a colorimetric assay as described above.

Flow cytometric analysis of LEC-1 apoptosis by annexin staining
Fas-mediated LEC-1 apoptosis was confirmed by flow cytometry using annexin staining. This technique detects changes in the surface of the cell plasma membrane occurring earlier than the nuclear changes associated with apoptosis (DNA fragmentation) (28) . Briefly, LEC-1 were untreated or treated for 12 h with either anti-Fas mAb alone or in combination with TNF. After incubation, specific binding of annexin V-FITC was performed by incubation of LEC-1 (5 (105 cells) in 50 µl of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) containing 5 µl of annexin V-FITC for 30 min at room temperature. Nonspecific binding was determined by incubating LEC-1 in calcium-free binding buffer (10 mM HEPES, 140 mM NaCl, 10 mM EDTA, pH 7.4) containing 5 µl of annexin V-FITC. After incubation the cells were analyzed by flow cytometry. Ten thousand events were acquired (excluding cell debris) and analyzed using the Lysis II software program.

CAM expression on Fas-treated LEC-1
The effect of low doses of anti-Fas mAb (at which no apoptosis is observed on LEC-1) on the expression of CAM was examined. LEC-1 were grown to 90% confluence, the supernatant removed, and the cells cultured in X-vivo alone or containing anti-Fas mAb at 1 µg/ml for 24 h at 37°C in a 5% CO2 atmosphere. After incubation, cells were harvested and stained for analysis of VCAM-1 and ICAM-1 expression by flow cytometry.

Statistical analysis
Results are reported as mean ± standard error (SE) from triplicate wells in all assays. We tested the data from the experiments for statistical significance using the analysis of variance. The level of significance was less than 0.05. All experiments were repeated at least three times and have documented reproducibility.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
Fas up-regulates the expression...
 DISCUSSION
 REFERENCES
 
Expression of Fas and FasL on LEC-1 clones
We examined the level of expression of Fas on the surface of LEC-1 clones (A12 and C7) by flow cytometry. A12 and C7 clones both constitutively express Fas, as shown by the high expression of Fas detected on the surface of these two LEC-1 clones (Fig. 1 ). Similar results were obtained with freshly isolated LEC-1 (not shown). Expression of FasL was not detected by either flow cytometry (Fig. 2 A) or RT-PCR (Fig. 2B ).



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  		Figure 1. Fas expression on murine LEC-1. Flow cytometric analysis was performed to detect surface expression of Fas. A12 and C7 LEC-1 clones were incubated with an FITC-labeled anti-Fas mAb (thin line) or an FITC-labeled control IgG isotype mAb (black profile). Results are representative of at least three independent experiments, all with similar results.



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  		Figure 2. LEC-1 cells do not express FasL either at protein or mRNA level. A) Flow cytometric analysis of FasL expression on LEC-1 clones. A12 and C7 LEC-1 clones were incubated with biotinylated anti-FasL mAb (thin line) or biotinylated IgG isotype, followed by Sav-PE (black profile). B) RT-PCR analysis of FasL expression on LEC-1 clones. RT-PCR was performed on 1 µg total RNA obtained from the A12 clone and C7 clone. Reverse-transcription and amplification were performed as described in Materials and Methods with the primers specific for FasL. MW = molecular markers; - CTRL = negative control, PCR reaction without RNA template; ß-actin, internal control; CTTL = RNA from murine cytotoxic T cell line (CTTL), positive control; A12 and C7 = LEC-1 clones.

Fas and FasL expression on TNF-treated LEC-1
Since LEC-1 clones constitutively express Fas, we investigated whether TNF, which induces LEC-1 to express different molecules (unpublished data), could up-regulate the expression of Fas on the LEC-1 surface. A significant increase in Fas expression was observed when these cells were treated with TNF for 18 h (Fig. 3 ), indicating that TNF up-regulates the expression of Fas on LEC-1. In contrast, TNF did not induce the expression of either membrane (Fig. 3) or cytoplasmic (not shown) FasL in LEC-1, as assessed by flow cytometry.




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  		Figure 3. Effect of TNF on LEC-1 Fas and FasL expression. LEC-1 cells (A12 clone) were cultured for 18 h with TNF (25 ng/ml) or medium alone (CTRL) and Fas expression was analyzed by flow cytometry. A12 LEC-1 clones were stained with an FITC-labeled anti-Fas mAb or an FITC-labeled isotype control IgG mAb (black profile). For FasL expression, A12 clone was incubated with biotinylated anti-FasL mAb (thin line) or with biotinylated IgG isotype, followed by Sav-PE (black profile). Data are representative of at least three independent experiments, all with similar results. Similar results were observed with the C7 clone.

Effect of anti-Fas mAb on LEC-1 viability
We examined whether Fas might transduce apoptotic signals in LEC-1. We cultured LEC-1 clones in the presence of three different concentrations of anti-Fas mAb (1, 10, and 50 µg/ml). After 24 h of culture, anti-Fas mAb induced a significant reduction in LEC-1 viability in a dose-dependent manner under serum-free conditions (Fig. 4 ). Morphological changes associated with apoptosis were observed in LEC-1 treated with anti-Fas mAb (Fig. 5 C). No other significant changes were observed in viability of LEC-1 cultured for 48 h (data not shown).



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  		Figure 4. Effect of anti-Fas mAb on LEC-1 cell viability. LEC-1 (A12 clone) were seeded in 96 wells at 1000 cells/well under serum-free conditions. Purified anti-Fas mAb at 1, 10, and 50 µg/ml were added to the cultures. After 24 h, the viability of LEC-1 was assayed using a colorimetric method (see Materials and Methods). The absorbance of the reaction was read at 490 nm. The results are expressed as the means ± SE. Results are representative of three separate experiments. The decrease in viability was statistically significant for anti-Fas mAb at 10 and 50 µg/ml with respect to the control (*P<=0.05). Similar results were observed with the C7 clone.



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  		Figure 5. Morphological changes in LEC-1 treated with anti-Fas mAb. LEC-1 were cultured for 24 h under serum-free conditions with anti-Fas mAb alone or in combination with TNF at 25 ng/ml. A) LEC-1 culture at 0 h. B) Isotype mAb-treated LEC-1 x 24 h. C) LEC-1 undergoing apoptosis (indicated by arrows) after 24 h of treatment with anti-Fas mAb (10 µg/ml). D) LEC-1 after 24 h of treatment with TNF (25 ng/ml). Greater confluence of LEC-1 monolayers can be seen after TNF treatment. E) A higher proportion of LEC-1 undergo apoptosis compared with panel C after 24 h of treatment with anti-Fas mAb (10 µg/ml) and TNF (25 ng/ml). Original magnification x20. Ap = apoptosis.

Effect of TNF on Fas-mediated LEC-1 apoptosis
Since TNF and Fas belong to the death factor family, we investigated whether TNF could increase the Fas-induced apoptosis of LEC-1. LEC-1 were incubated under serum-free conditions in the presence of TNF at 25 ng/ml and anti-Fas mAb at 10 µg/ml for 48 h. After 48 h of culture and under serum-free conditions, untreated-LEC-1 viability was always greater than 90% as determined by trypan blue exclusion dye (not shown). Greater than 50% reduction of LEC-1 viability was induced by TNF and anti-Fas mAb compared with the effect exerted by each alone and with respect to the untreated LEC-1 (Fig. 6 , Fig. 5E ). These results suggest that signals transduced from Fas and TNF receptor may synergize to induce apoptosis on LEC-1. LEC-1 apoptosis was confirmed by flow cytometry analysis using annexin-V staining. This technique detects apoptotic changes in the cell membrane surface that occur earlier than DNA fragmentation. Annexin-positive cells were detected at 13%, 10%, and 11% in LEC-1 cultures maintained under serum-free conditions or treated with TNF or anti-Fas mAb for 12 h, respectively (Fig. 7 ). Treatment with anti-Fas mAb and TNF for 12 h induced more than a twofold increase in the percentage of annexin-positive cells compared with untreated, TNF-, or anti-Fas mAb-treated LEC-1 (Fig. 7) . Less than 2% of annexin-positive cells were detected in recently harvested LEC-1 (data not shown).



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  		Figure 6. Effect of TNF on anti-Fas mAb-mediated LEC-1 death. LEC-1 (A12 clone) were seeded in 96 wells at 1000 cells/well under serum-free conditions in the presence of TNF (25 ng/ml), anti-Fas mAb (10 µg/ml), or both. After 48 h, the viability of LEC-1 was assayed using a colorimetric method (see Materials and Methods). The absorbance of the reaction was read at 490 nm. The results are expressed as the means ± SE. Results are representative of three separate experiments. The decrease in viability was statistically significant for anti-Fas mAb with respect to the untreated LEC-1 (*P<=0.05) and anti-Fas mAb plus TNF with respect to each alone and the untreated LEC-1 (**P<=0.001). Similar results were observed with the C7 clone.



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  		Figure 7. Flow cytometric analysis of LEC-1 apoptosis. LEC-1 cells were untreated or treated for 12 h with either anti-Fas mAb (10 µg/ml) or TNF (25 ng/ml) alone or in combination. After incubation, the cells were harvested and incubated with annexin V-FITC and analyzed by flow cytometry. Ten thousand events were acquired and analyzed using the Lysis II software program. Anti-Fas mAb plus TNF treatment increased the percentage of LEC-1 undergoing apoptosis. The M1 and M2 gates demarcate annexin V-FITC negative (nonapoptotic cells) and positive (apoptotic cells) populations, respectively. Results are representative of at least three independent experiments, all with similar results.

To examine whether pretreatment with TNF could increase the susceptibility of LEC-1 to Fas-induced apoptosis, experiments of delayed addition of these factors were performed (Fig. 8 ). Serum-free cultures were either untreated or treated with TNF or anti-Fas mAb for 12 h. After incubation, culture medium was removed and each well was washed and then supplemented with medium, anti-Fas mAb or TNF. A further and significant reduction in the viability (more than 50%), was observed after delayed addition of anti-Fas mAb (Fig. 8) to LEC-1 pretreated with TNF, compared with LEC-1 treated with anti-Fas mAb alone. In contrast, the delayed addition of TNF to LEC-1 cultures pretreated with anti-Fas mAb did not induce significant changes in viability compared with those cultures treated with anti-Fas mAb alone (Fig. 8) . These results indicate that TNF increases the susceptibility of LEC-1 to Fas-mediated apoptosis.



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  		Figure 8. Effect of TNF pretreatment on Fas-mediated apoptosis on LEC-1. LEC-1 were cultured without (control) or with TNF at 25 ng/ml or with anti-Fas mAb at 10 µg/ml for 12 h. After incubation, each well was washed; medium (control, CTRL), anti-Fas mAb, or TNF-{alpha} was added and the cells were incubated for 24 h. The viability of LEC-1 cells was evaluated by the MTS assay (see Materials and Methods). The results are expressed as the means ± SE. Results are representative of three separate experiments. The decrease in viability was statistically significant in the groups of delayed addition of anti-Fas mAb on untreated LEC-1 compared with control (P<=0.05) and in the groups of delayed addition of anti-Fas mAb on TNF-pretreated LEC-1 compared with the groups of delayed addition of anti-Fas mAb on untreated LEC-1 and control (P<=0.05 and P<=0.001, respectively). Similar results were observed with the C7 clone.


   Fas up-regulates the expression of VCAM-1 and ICAM-1 on LEC-1
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Fas up-regulates the expression...
DISCUSSION
 REFERENCES
 
Since Fas is expressed at high levels on the LEC-1 surface, we examined whether anti-Fas mAb (at doses that do not alter their viability) could modulate some biological functions of LEC-1. The expression of VCAM-1 and ICAM-1 was investigated on LEC-1 treated with anti-Fas mAb at 1 µg/ml for 24 h (Fig. 9 ). Upon treatment with anti-Fas mAb, there was a marked increase in the expression of both VCAM and ICAM-1 on LEC-1, indicating that signals transduced through Fas may up-regulate the expression of adhesion molecules on these cells.



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  		Figure 9. Effect of anti-Fas mAb on the expression of adhesion molecules on LEC-1. Cells were cultured for 18 h with 1 µg/ml of anti-Fas mAb (anti-Fas) or with medium alone (black profile) and then stained with PE- or FITC-conjugated rat mAb against mouse ICAM-1, VCAM-1 or with an isotype control IgG mAb (thin line). Ten thousand events were acquired and analyzed using the Lysis II software program. Results are representative of at least three independent experiments, all with similar results.

Effect of anti-Fas mAb on LEC-1 viability in the presence of ActD
Since inhibition of RNA synthesis by ActD has been shown to enhance anti-Fas mediated cell death in various cell types (29 30 31) , we investigated the effect of anti-Fas mAb on LEC-1 viability in the presence of this drug. ActD at 10 ng/ml caused a small but significant reduction of LEC-1 viability. However, anti-Fas mAb in the presence of ActD caused a significant reduction of LEC-1 viability compared with either control, ActD, or anti-Fas mAb alone (Fig. 10 ). Thus, ActD enhances the susceptibility of LEC-1 to anti-Fas treatment.



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  		Figure 10. Effect of anti-Fas mAb on LEC-1 viability in the presence or absence of ActD. LEC-1 were seeded in 96 wells at 1000 cells/well under serum-free conditions in the presence of ActD (10 ng/ml) and anti-Fas mAb (10 µg/ml). The viability of LEC-1 was measured by MTS assay (see Materials and Methods) after 24 h of culture and compared with incubation with isotype control. The results are expressed as the mean ± SE. Results are representative of three separate experiments. The decrease in viability was statistically significant for anti-Fas mAb and ActD with respect to the control (*, P<=0.05 and P<=0.01, respectively) and anti-Fas mAb plus ActD with respect to each alone and with respect to the isotype control (*P<=0.01). Similar results were observed with the C7 clone.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Fas up-regulates the expression...
 DISCUSSION
REFERENCES
 
The binding of FasL to Fas stimulates an intracellular cascade that leads to the induction of apoptosis in a wide variety of target cells (1) . Fas-mediated apoptosis is one of the primary mechanisms used in the elimination of activated lymphocytes and the maintenance of homeostasis (8) . It has also been implicated in T and NK cell-mediated cytotoxicity of tumor cells (9) . Although Fas is expressed in EC (6) , little is known about the role of the Fas/FasL system on these cells. Previous in vitro reports suggest that Fas ligation does not lead to induction of EC apoptosis (6) ; however, there is some in vivo evidence suggesting that the Fas/FasL system may be involved in inducing the death of EC in different organs (17 , 18 , 32) . There are reports that animals receiving anti-Fas mAb, which acts as an agonist of Fas, or soluble FasL die 6 h after injection due to liver necrosis (20) . It has been proposed that the main mechanism involved in this effect is the death of hepatocytes because these cells express Fas and in vitro experiments have shown that FasL induces apoptosis in hepatocytes (20 , 21) . However, apoptosis of hepatic sinusoid EC (LEC) may also contribute to the liver damage observed in animals injected with Fas agonists. The fact that LEC express Fas makes it possible that activation of this receptor on these cells leads to the induction of apoptosis. To investigate this hypothesis, cloned LEC (LEC-1) were analyzed for Fas and FasL expression and for their relative sensitivity to Fas-induced apoptosis. Here we show for the first time that in vitro activation of Fas in LEC-1 induces apoptotic signals in these cells.

Using two clones of LEC-1, our study shows that these cells constitutively express high levels of Fas. These results are in agreement with previous reports that show that hepatic sinusoidal EC are Fas positive (21) . However, we show here that murine LEC-1 express higher levels of Fas on their surface than those reported previously (21) . The level of Fas expression on LEC-1 is also higher than that reported in other cells (5) . This may be explained by the fact that we measured Fas expression in a homogeneous clone of cells. The fact that Fas expression is increased in LEC-1 treated with TNF indicates that Fas expression may be up-regulated in these cells by proinflammatory cytokines. Similar results have been reported using other cells (1 , 3 , 6) . These results may have clinical implications for some liver inflammatory diseases since up-regulation of Fas by proinflammatory cytokines such as TNF could increase the susceptibility of LEC to Fas-mediated apoptosis (see below).

In contrast to the high expression of Fas by LEC-1, FasL is not constitutively expressed in these cells either at the mRNA or the protein level, as assessed by PCR and flow cytometry analysis. These results agree with previous reports showing that FasL expression is relatively limited to several cell types (5 , 7) . Although earlier reports have shown the possibility that FasL expression may be up-regulated in LEC (21) , our results show that the expression of FasL is not up-regulated in LEC-1 by the proinflammatory cytokine TNF, as occurs with Fas.

The high level of Fas expression on the LEC-1 surface suggests that it may play some biological function in these cells. We show here that anti-Fas mAb induces a significant decrease in the viability of LEC-1 in a dose-dependent manner. The observed magnitude of LEC-1 apoptosis differs from that reported for hepatocytes and other cells, such as neutrophils and mast cells (5 , 33) , in that LEC-1 are more resistant to the Fas-induced apoptotic pathway. High resistance of EC to Fas-mediated apoptosis has also been previously reported (6) . The high resistance of LEC-1 to Fas-mediated apoptosis may constitute a protective effect to prevent cell death and subsequent vascular pathology due to Fas-induced death signals. However, our results showing that in vitro activation of Fas in LEC-1 induces apoptosis of these cells demonstrate that Fas is functional in these cells and that it may play a role in hepatic sinusoidal endothelial cell apoptosis. Fas-mediated LEC apoptosis may contribute to the liver tissue injury observed after soluble FasL or anti-Fas mAb injection (20) . In vivo, a role for Fas in liver endothelial cell injury has also been suggested from Fas ligand-defective (gld) mice and Fas-deficient (lpr) mice (34) .

Our in vitro studies provide evidence that TNF increases the susceptibility of LEC-1 to Fas-mediated apoptosis. Although in our system TNF does not induce LEC-1 apoptosis, our results show that simultaneous activation of the TNF receptors and Fas on LEC-1 induces a significant and synergistic decrease in the viability of these cells compared with the effect exerted by anti-Fas mAb alone. An increase in the sensitivity to Fas-mediated apoptosis has also been reported in B cells stimulated with CD40L, another member of the TNF family (35) . Since the 55 kDa TNF receptor and Fas, which share structural homology and a cell death inducing domain (9 , 10) , both mediate apoptosis analogously but by an independent mechanisms (9 , 10) , we postulate that the two receptors may synergistically increase intracellular death signals in LEC-1. Therefore, it will be interesting to determine the intracellular changes in LEC-1 associated with the sensitizing effect of TNF to Fas-mediated killing.

Another possible mechanism by which TNF may increase the susceptibility of LEC-1 to Fas-mediated apoptosis may be due to TNF up-regulation of cell surface Fas on these cells. The observation that prestimulation of LEC-1 with TNF results in an enhanced susceptibility of these cells to Fas cytotoxicity supports this hypothesis. A similar finding has recently been reported in cultured glioma cells in which an increase in Fas expression resulted in an increased susceptibility to anti-Fas treatment (36) . However, there are also reports showing that up-regulation of Fas on other EC (i.e., HUVEC) induced by pretreatment of these cells with proinflammatory cytokines did not result in apoptosis (6) . Taken together, these results suggest a differential function of Fas among EC.

An important question for both the Fas/FasL system and endothelial cell research suggested by our findings is whether the in vitro apoptotic effect induced by TNF and Fas ligation on LEC-1 also occurs in vivo. It is well known that in vivo, EC activation in response to TNF plays a key role in numerous inflammatory conditions (37) . There is evidence showing that early induction of TNF during certain viral infections plays a key role in promoting hepatic inflammatory responses and mediating liver damage (38) . Likewise, there are reports showing Fas expression in hepatic tissues during certain liver inflammatory diseases (39 40 41 42 43 44) . Moreover, hepatic failure has been observed in patients with high levels of soluble FasL (15) . We propose that in those clinical hepatic conditions associated with high levels of TNF, LEC apoptosis mediated by Fas may contribute to liver damage. Under these conditions, Fas-FasL interaction could occur between LEC expressing high levels of Fas and FasL-bearing inflammatory cells (i.e., activated T cells, neutrophils, and monocytes) and/or biologically active soluble FasL (1 , 5 , 14 , 15 , 19 , 45 , 46) . In fact, it has been proposed that both FasL-bearing inflammatory cells and biologically active soluble FasL may represent a pathological pathway to promote injury in Fas-susceptible tissues (1 , 19) .

FasL and Fas belong to the TNF and TNF receptor families, respectively (2 3 4) . We have demonstrated that TNF is a potent activator of LEC-1. It enhances adhesion molecule expression and cytokine production by LEC-1 (data not shown). We show here that activation of Fas with a dose of anti-Fas mAb that does not induce LEC-1 apoptosis results in transduction of some activating signals in these cells. Our studies show that ligation of Fas with a low dose of anti-Fas mAb (1 µg/ml) up-regulates the expression of VCAM-1 and ICAM-1 on LEC-1. These results suggest that Fas may transduce cell activation signals in LEC-1 independently or as an alternative to cell death, and may be explained by activation through Fas of transcription factors unrelated to apoptosis. Activation signals mediated by Fas have been reported in T cells (47 , 48) . Other members of the TNF-receptor family, i.e., TNFRp55, TNFRp75, and CD40, also have biological functions other than cytotoxic signals (47 48 49 50) . Regarding the up-regulation of VCAM and ICAM-1 by Fas activation, it may be speculated that the Fas/FasL system may be involved in recruiting inflammatory cells by increasing the adhesion of these cells, in those cases associated with inflammatory liver disease. Other in vivo studies support the hypothesis of the possible role of Fas in promoting inflammatory cell recruitment (19) . Additional LEC-1 functions such as production of nitric oxide were not affected by anti-Fas mAb (data not shown).

Intracellular signals triggered by Fas/FasL pathways have been reported (51 52 53) . Recent studies have shown that activation of caspases (ICE family of cysteine proteases) seems to be a common intracellular pathway in most apoptotic process (51) . It has been shown that caspases plays an essential role in Fas-mediated apoptosis (52 , 53) . Previous reports using ActD in Fas-sensitive cells also suggest that there are intracellular molecules associated with the protection of cells from Fas-mediated apoptosis (29 30 31) . We show in this work that ActD-treated LEC-1 have an increased susceptibility to Fas-mediated apoptosis compared with the effect exerted by ActD alone. A similar mechanism for eliciting apoptosis has been postulated in hepatocytes (9 , 54)

In conclusion, we have shown that LEC-1 constitutively express high levels of Fas. Ligation of Fas induces LEC-1 apoptosis, indicating that Fas is functional on these cells. These findings suggest an important role for the Fas/FasL system in the regulation of apoptosis of hepatic sinusoidal EC that may contribute to liver damage. TNF up-regulates the expression of Fas on LEC-1 and increases the susceptibility of LEC-1 to Fas-mediated apoptosis; thus, this cytokine may regulate different biological functions of the Fas/FasL system on LEC-1. The data presented here not only demonstrate that signals transduced through Fas may be involved in inducing apoptosis in LEC-1, but that they can also be involved in activating LEC-1 biological functions such as up-regulation of CAM expression.


   ACKNOWLEDGMENTS
 
We are extremely grateful for the ideas and suggestions provided by Dr. Emilio Barberá-Guillem. The authors also wish to thank to Dr. Egidio Romano, Dr. Peter Taylor, Dr. Andrés Soyano, and Dr. Connie-Erickson Miller for critical reading and for stimulating discussions. The authors thank Alexandra Hidalgo for her assistance in the preparation of this manuscript. This work was supported by Minster Machine Company Foundation, Mathile Family Foundation, and Sigma Beta Sorority.


   FOOTNOTES
 
Received for publication September 18, 1999. Revised for publication May 26, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 Fas up-regulates the expression...
 DISCUSSION
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