Cell Biology International 2001, Vol. 25, No. 5, 485–488doi:10.1006/cbir.2000.0621, available online at http://www.idealibrary.com on

SHORT COMMUNICATION

POSSIBLE ROLE OF FAS ANTIGEN (CD 95) IN HUMAN AMNIOTIC EPITHELIALCELL DEATH: AN IN VITRO STUDY

SANKAR SURENDRAN

National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan

Received 17 November 1999; accepted 12 July 2000

Treatment of human amniotic epithelial (HAE) cells with anti-Fas monoclonal antibody (CH11) at 100 ng/ml or 1 �g/ml for 12 or 24 h increased necrotic cell death. Apoptotic cell deathinduced by this antibody was significantly increased, although far fewer cells underwentapoptosis, as determined by the TUNEL method. This study suggests that Fas antigen is animportant mediator in HAE cell death. � 2001 Academic Press

K: Fas; (CD95); apoptosis; HAE cells; necrosis.

INTRODUCTION

HAE cells have been used for transplantation inpatients with various diseases, including lysosomalstorage (Yeager et al., 1985), because they possesslarge quantities of lysosomal enzymes. However,their in vivo life span after grafting is as short(Sakuragawa et al., 1992) as it is in vitro. Therefore,we have studied the factors which induce HAE celldeath in vitro.

We recently reported that Fas antigen is ex-pressed in cultured HAE cells (Surendran et al.,1999). Fas antigen/CD95/APO1 is believed to in-duce cell death in a variety of cells (Nagata, 1997).Fas is a cell surface receptor belonging to theprotein superfamily nerve growth factor/humancell antigen CD40/TNF receptor (Shima et al.,1995). The Fas/CD95/Apo-1 ligand is a type IItransmembrane protein which binds with its recep-tor to induce apoptosis (Nagata, 1997). Apoptosisdiffers from other types of cell death, such asautophagic degeneration, by being a non-lysosomalmechanism, leading to the formation of apoptoticbodies and cleavage of genomic DNA intohigh molecular weight fragments. These are

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subsequently cleaned into smaller nucleosomalfragments. In contrast, necrosis involves dilation ofendoplasmic reticulum, swelling of mitochondria,loss of plasma membrane integrity and releaseof free intracellular components resulting in aninflammatory reaction (Spreafico et al., 1994). TheFas ligand exists in both membrane bound andsoluble forms. Of the two membrane-bound Fasligand appears to be a more potent inducer ofapoptosis (Tanaka et al., 1998). The Fas antigenhas also been reported to regulate growth in vari-ous cell types by inducing apoptosis (Aggarwalet al., 1995).

The recent in vivo study of Runic et al. (1998)suggests that cells from human amniotic fluidundergo Fas-induced apoptosis. The amnion con-tains not only epithelial cells, but also fibroblasts(Economopoulous et al., 1996), and fibroblasts arereported to undergo apoptosis (Jelaska and Korn,1998). Therefore an in vitro study was requiredto elucidate the role of Fas on human amnioticepithelial cells.

To whom correspondence should be addressed: Dr S. Surendran,Department of Pediatrics cytogenetics, Children’s Hospital, Universityof Texas Medical Branch at Galveston, Texas 77555–0359, U.S.A.E-mail: ssurendr@utmb.edu

MATERIALS AND METHODS

HAE cells were prepared as described previously(Yeager et al., 1985). Briefly, placentae wereobtained from Caesarean sections. The amniotic

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membrane was separated from the placenta,washed with phosphate buffered saline (PBS) andcut into pieces, which were then repeatedlytrypsinized. The epithelial cells were grown inRPMI-1640 medium supplemented with 10% fetalbovine serum at 37�C in 5% CO2 in air at 100%relative humidity. After 24 h the medium waschanged to remove unattached cells, and adheringHAE cells free from fibroblast contamination wereused for the following experiments after a furtherpassage.

HAE cells were seeded at 1�105/dish inmedium, and incubated in the presence or absenceof 100 ng/ml or 1 �g/ml anti-Fas monoclonal anti-body (CH 11; MBL, Nagoya, Japan) as describedin Shima et al. (1995). After 12–24 h treatment,control and treated cells were removed with a cellscraper and tested for necrosis using trypan bluestaining. Apoptotic cell death was determinedusing Terminal deoxynucleotidyl transferase (TdT)mediated dUTP-biotin nick end labeling (TUNEL)and a Mebstain apoptosis kit (MBL, NagoyaJapan). Cells were washed in PBS and fixed in 4%paraformaldehyde at 4�C for 15 min, followed bywashing in PBS containing 0.2% BSA. Cells weresubsequently treated with 0.5% Tween 20 solution,washed in PBS solution and incubated in TdTsolution for 1 h at 37�C. After washing with PBS,cells were incubated in blocking solution for10 min at room temperature. Following Avidin–fluorescein isothiocyanate (FITC) staining, nucleiwere counterstained with propidium iodide, andthe cells visualized by fluorescence microscopyusing filter No. 487909 (Mori et al., 1994). TUNELpositive cells were counted.

In addition to the TUNEL method, DNA frag-mentation in HAE cells was identified by a Hoechststaining method. The Fas antibody-treated anduntreated HAE cells were washed in PBS andfixed in 4% paraformaldehyde at 4�C for 15 min.Subsequently these cells were washed in PBS,and incubated in Hoechst 33258 (Sigma, Tokyo,Japan) at room temperature for 5 min. The stainedcells were washed in PBS, mounted and visualizedby fluorescence microscopy and the number ofapoptotic cells were counted.

Statistical analysis

The percentage of apoptotic or necrotic cell deathwas calculated as being equal to the total numberof apoptotic or necrotic cells�100/total cellnumber (viable+dead cells). Data was analyzed byANOVA and P<0.05 was considered significant.

RESULTS

Treatment of HAE cells for 12 or 24 h with thisantibody induced more cell death via necrosis thanthe control (P<0.001 (Fig. 1A) The increase wasfour-fold at 100 ng and five-fold at 1 �g, withapprox. 60% cells dead. To investigate the possi-bility of apoptotic cell death being caused byanti-Fas antibody, we stained the antibody-treatedand untreated HAE cells using both TUNEL andHoechst staining methods.

The degree of HAE apoptosis induced by alow dose (100 ng/ml) of the monoclonal antibodyapplied for 24 h was significant. A high dose (1 �g/ml) of the antibody also showed significant celldeath via apoptosis (P<0.05) (Fig. 1B). Althoughthis increase was at most 30–70% at 12 h, theapprox. 4.5-fold increase over 24 h was significant.This was on a background of just 1–2% apoptoticcells (making the contribution not much more than4–6% of the total dead cells). However, there wasmore cell death induced by the anti-Fas mono-clonal antibody via necrosis than via apoptosis.

DISCUSSION

Expression of Fas/Apo 1 protein has been demon-strated in different types of cells (Yonehara et al.,1989). Expression of Fas by different epithelial cellshas been observed more recently by immunohisto-chemical staining of human tissue (Leithauseret al., 1993) and by immunofluorescence of cul-tured human amniotic epithelial cells (Surendranet al., 1999). To determine the function of Fasantigen in HAE cells, we incubated these cells withanti-Fas monoclonal antibody (CH-11), whichworks as an agonist to induce cell death in thosecells expressing Fas in vivo and in vitro (Jayanthiet al., 1999). Treatment of HAE cells with anti-Fasantibody induced more cell death via necrosis.Only a few cells underwent apoptosis, although theincrease was significant. The low level of apoptosiscould not be detected by DNA ladder pattern(Wong et al., 1994). The induction of both necroticcell death and apoptosis by anti-Fas antibodysuggests that Fas plays a dual role in humanamniotic epithelial cell death. The induction ofFas-mediated apoptosis is possibly via activationof the caspase pathway. Meanwhile Fas inductionof necrosis is possibly caused by the stimulationof mitochondrial oxygen radical production(Vercammen et al., 1998; Jayanthi et al., 1999). Thein vivo study of Runic et al. (1998) with human fetalmembranes, further supports our finding that Fas

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induces apoptosis in HAE cells. In conclusion, wereport that the occurrence of HAE cell death is atleast partly ascribable to signaling by the Fasantigen, resulting in both necrosis and apoptosis.

Although Fas induction of HAE cell death hasbeen confirmed in vitro, it will be important tostudy the role of Fas antigen in vivo after grafting,because Fas induction of cell death appears to becorrelated to graft rejection (Kato et al., 1997).

ACKNOWLEDGEMENTS

I thank the CBI Manuscript Presentation Servicefor presubmission preparation. I am very grateful

to Dr Norio Sakuragawa for his advice. Thisstudy was supported by National Institute ofNeuroscience, NCNP, Tokyo, Japan.

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Fig. 1. A: Induction of human amniotic epithelial cell death by necrosis was anti-Fas antibody. Human amniotic epithelial cellswere treated with anti-Fas antibody (CH 11) at 100 ng/ml or 1 �g/ml and stained using trypan blue. Necrotic cell death inducedby this antibody was significant. Bar�S.E. (n=9) (***P<0.001). B: Induction of human amniotic epithelial cell death byapoptosis using anti-Fas antibody. Treatment of human amniotic epithelial cells with anti-Fas antibody at 100 ng/ml or 1 �g/mlfor 24 h induced significant apoptotic cell death. Bar�S.E. (n=9) (*P<0.05). Control, �; 100 ng/ml anti-Fas antibody, �;1 �g/ml anti-Fas antibody, .

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