PURPOSE. TO investigate the mechanism(s) involved in human fetal retinal pigment epithelium .... heat-inactivated fetal calf serum (GIBCO, Life Technologies,.
Human Fetal Retinal Pigment Epithelial Cells Induce Apoptosis in the T-Cell Line Jurkat Lilt Farrokh-Siar,1 Kourous A. Rezai,1 Roshanak Tolouei Semnani,2 Samir C. Patel,1 J. Terry Ernest,1 ErikJ. Peterson,5 Gary A. Koretzky,5 and Gijs A. van Seventer2 investigate the mechanism(s) involved in human fetal retinal pigment epithelium (HFRPE)-mediated T-cell death.
PURPOSE. TO
METHODS. Pure HFRPE cells were isolated and cultured. Normal and interferon (IFN)-y-activated HFRPE from early and late in vitro passages were incubated with cells from the human T-cell leukemia line Jurkat Qkt). Cultures were pulsed with [3H]-thymidine to measure Jkt cell proliferation. Jkt cells were evaluated for apoptosis either by staining with an ethidium bromide/acridine orange mixture (AO/EB) or with Annexin V-phycoerythrin. The role of Fas ligand (FasL) molecule in HFRPE-mediated apoptosis was assessed by using a mutant Jkt cell line (DD3), which is deficient in Fas-mediated signaling. The involvement of the antiapoptotic human gene bcl-x, was determined by using Jkt cells that were stably transfected with bcl-x,. The role of cell-cell contact in the induction of apoptosis was evaluated in a transwell system in the presence or absence of neutralizing antibodies against IFN-y and tumor necrosis factor (TNF)-a.
HFRPE cells inhibited the proliferation of Jkt cells by inducing apoptosis through a FasL-independent pathway. Passaging and IFN-y activation strengthened the inhibitory effect of HFRPE cells on the proliferation of Jkt cells. At lower HFRPE passages (P2), bcl-x,, overexpression rescued the HFRPE cell-mediated apoptosis. The separation of the cells by the transwell system did not affect the HFRPE cell-mediated suppression. This suppressive effect was not mediated by the secretion of IFN-y or TNF-a molecules.
RESULTS.
HFRPE cells suppressed the proliferation of Jkt cells by inducing apoptosis. HFRPE cells induced a stronger inhibitory effect on Jkt cells at higher in vitro passages. The HFRPE-induced apoptosis was not mediated through the FasL/Fas pathway or through the secretion of the apoptosis-inducing cytokines IFN-y and TNF-a. The bcl-x, gene may play a role in preventing HFRPE cell-induced apoptosis in Jkt cells. These combined results suggest that the HFRPE-mediated suppression of primary T cells may also be mediated by the induction of apoptosis. Therefore, the retinal pigment epithelium may play a role in the induction of immune privilege in the subretinal space. (Invest Ophthalmol Vis Set. 1999;40:1503-1511) CONCLUSIONS.
I
mmune privileged sites are defined as anatomic sites in which immunogenic tissues survive for extended periods of time in an immunocompetent host.1 It is well documented that the eye is an immune privileged site.2'3 A significant proportion of cornea! grafts are accepted indefinitely when transplanted in allogenic eyes.4 Recently, it was reported that human fetal retinal pigment epithelium (HFRPE) cells transplanted into the subretinal space of human eyes with intact Bruch's membrane survived for an extended period and experienced a low rejection occurrence.5
From the 'Department of Ophthalmology and Visual Science; the Department of Pathology, and The Committee on Immunology, University of Chicago, Illinois; and the 'Department of Internal Medicine and Physiology, University of Iowa College of Medicine. Supported by Research to Prevent Blindness, New York, New York. Submitted for publication January 14, 1999; accepted February 12, 1999. Proprietary interest category: N. Reprint requests: Lili Farrokh-Siar, Department of Ophthalmology and Visual Science, University of Chicago, 939 East 57th Street, Chicago, FL 60637. 2
investigative Ophthalmology & Visual Science, June 1999, Vol. 40, No. 7 Copyright © Association for Research in Vision and Ophthalmology
We and others recently showed that HFRPE cells can induce apoptosis in T cells, contributing to the immune privilege of the eye.6'7 Apoptosis can be defined as a complex regulatory process that controls cell death by intrinsic preprogrammed mechanisms.8 It is an evolutionary conserved process used to eliminate cells that are not needed or that are potentially dangerous to the organism. Apoptosis is an important pathway for the maintenance of cellular homeostasis, especially in the immune system.9 The mechanism of HFRPE-mediated T-cell apoptosis is not well defined. Jorgensen et al.7 have recently suggested that it was mediated through the FasL/Fas pathway and that it is cell- cell contact dependent. However, the recent observation by Smith et al.10 casts doubt on the validity of the anti-FasL reagent used in these studies. In light of these contrasting results, we performed a more detailed evaluation of the mechanism(s) involved in the HFRPE-mediated T-cell apoptosis by using human T-cell lines. Furthermore, we investigated one of the intracellular signaling pathways involved in apoptosis. One of the important apoptosis-regulating genes is the bcl-x gene, which belongs to the bcl-2 gene family.'' Transcripts of the bcl-x gene are pro1503
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vided in two forms: bcl-xr, an anti-apoptotic gene; and bcl-xs, a proapoptotic gene. It has been shown that under certain conditions the stable transfection of bcl-x, prevents the apoptotic cell death in T cells. 1213 In this study, we examine in detail the mechanism(s) that may be involved in the induction of T-cell death. The human T-cell leukemia line Jurkat (Jkt) was used as a model for activated human T cells. Jurkat cells have been widely studied for signaling pathways and receptors representing activated human T cells.14 l5 The role of passaging, HFRPE-Jkt ratio, cellcell contact, and the soluble factors interferon (IFN)-y and tumor necrosis factor (TNF)-a on the suppressive effect of HFRPE cells was examined. The role of the FasL/Fas pathway was evaluated by using the mutant Jkt cell line DD3, which is deficient in Fas-mediated signaling.16 Last, the role of bcl-xL overexpression in rescuing the HFRPE-mediated apoptosis was evaluated using stably transfected bcl-x, Jkt cells. MATERIALS AND METHODS
Isolation of HFRPE Cells HFRPE cells were obtained from human fetal eyes at 18 to 22 weeks of gestational age (Anatomic Gift Foundation, Woodbine, GA). Microdissection was performed under sterile conditions using a dissecting microscope. The eyes were opened by a circumferential incision just above the ora serrata near the limbus, and the anterior segment and lens were separated. The posterior segment of the eye was cut into four quadrants and placed in a petri dish containing Dulbecco's minimal essential medium (DMEM; Sigma, St. Louis, MO).17 The neural retina and any remaining vitreous were removed. Sheets of RPE cells were separated from the choroid using fine forceps and immediately placed into a petri dish containing phosphate-buffered saline (PBS) without Ca2+/Mg2+. After the separation of all four quadrants, the sheets were trypsinized (0.25% trypsin; Sigma) for 15 minutes. Growth medium consisting of DMEM (Sigma), 15% fetal bovine serum (Sigma), and a 1% solution of antibiotics and L-glutamine (Sigma) was added, and the content was centrifuged at 2000 rpm. The supernatant was discarded, and the cells isolated from each eye were resuspended with growth medium into one well of a 24-well plate (Becton Dickinson, Lincoln Park, NJ) and incubated for 1 week in 95% air/5% CO2 at 37°C. The cells were trypsinized and resuspended into a larger culture flask. The cultures were examined on a daily basis, and the growth medium was changed twice a week. At confluence the cells were subcultured by trypsinization. The purity of the cultures was also evaluated with anti-pan cytokeratin antibody (Sigma).
Preparation of HFRPE Cells HFRPE cells at second, third, fifth, and sixth passages were used for the experiment. All the assays were performed on two populations of HFRPE cells, activated and nonactivated. Activated HFRPE cells were obtained by incubation with 1000 U/ml of IFN-y (Pharmingen, San Diego, CA) for 72 hours before each experiment. Before each assay, the cultured cells were washed with PBS without Ca2+/Mg2+ and were incubated with versene/EDTA (GIBCO, Life Technologies, Grand Island, NY) for 30 minutes. Enzymatic dissociation was avoided for possible modulation of the cell surface molecules. The dissociated cells were centrifuged at 2000 rpm for 5 minutes and
IOVS, June 1999, Vol. 40, No. 7 counted after resuspension with a hemocytometer (Fisher Scientific, Pittsburgh, PA). For the assays, the cells were irradiated with 2500 Gy QL Shepherd and Associates, Glendale, CA).
Preparation of Jkt Cells Four different Jkt cell lines were used throughout this study. ThefirstJurkat cell line Qkt) was generated in our laboratory as a subclone of the Jurkat cell line, which is available from the parental cell line of American Tissue Culture Cell Line (ATCC).15 The second Jkt cell line was generated by their stable transfection with the neomycin-resistant gene and the bcl-xL antiapoptotic human gene (bcl-xL/]kt). The third Jkt cell line, which served as the control, was only transfected with the neomycin-resistant gene (Neo/Jkt). Both bcl-xL/]kt and Neo/Jkt cells were generously provided by C. G. Thompson and M. Vander Heiden (Department of Medicine, University of Chicago, Chicago, IL). The fourth Jkt cell line was generated by Peterson et al.16 through a chemical mutagenesis and subsequent selection with the anti-Fas mAb CH11 (DD3/Jkt). This DD3/Jkt cell line was deficient in Fas-mediated signaling secondary to a mutational loss in the cytoplasmic death domain of Fas and, consequently, were resistant to Fas-mediated apoptosis. All the Jkt cells were cultured in standard growth medium RPMI 1640 (Biowhittaker, Walkersville, MD) supplemented with 20 mM glutamine (Biowhittaker, Walkersville, MD), 10% heat-inactivated fetal calf serum (GIBCO, Life Technologies, Grand Island, NY), 100 IU/ml penicillin, 100 /xg/ml streptomycin (Biowhittaker) in 95% air/5% CO2 at 37°C. The culture media of Neo/Jkt and bcl-x,/Jkt cells also contained 0.5 mg/ml G418 (Sigma) for selection purposes. To maximize the number of viable cells, Jkt cells were subcultured 72 hours before each assay and were always kept at densities below 1 X 106 cells/ml.
Jkt Cell Proliferation Assays Assay I: Standard Cultures. In this assay the suppressive effect of HFRPE cells was examined on Jkt cells at different concentrations and passages. Normal or IFN-y-activated HFRPE cells at passages P2, P3, P5, or P6 and at concentrations of 25,000, 12,500, or 6,250 were incubated with 50,000 Neo/ Jkt cells. The cells were cultured in 96-well-plate tissue culture clusters with flat-bottomed wells (Costar, Cambridge, MA) in growth medium consisting of RPMI 1640 (Biowhittaker) supplemented with 20 mM glutamine (Biowhittaker), 10% heatinactivated fetal calf serum (GIBCO), 100 IU/ml penicillin, and 100 /Ltg/ml streptomycin (Biowhittaker) in 95% air/5% CO2 at 37°C. After an incubation period of 48 hours, the cultures were pulsed with a [3H]-thymidine solution (5 mCi/ml, 2 mCi/mmol specific activity; New England Nuclear, Boston MA) for 24 hours before harvesting on glass fiber filters. Incorporation of the radioactive label was measured by liquid scintillation counting and was expressed as the arithmetic mean counts per minute (cpm) of triplicate cultures. In each figure, 100% indicates the proliferation of Jkt cells alone (served as a control), which was generally 350,000 ± 30,000 cpm. Assay II: Transwell System. In this assay the HFRPE cells were separated from the Jkt cells by an anopore membrane. Fifty thousand Jkt cells were placed in the wells of a 96-well-plate tissue culture cluster (Costar). The plate was centrifuged at 2000 rpm for 3 minutes. Tissue culture inserts with 0.2 jam anopore membranes (Nalgene Nunc International, Naperville, IL) were inserted into the wells of the 96-well-
JOVS, June 1999, Vol. 40, No. 7 plate, and 12,500 normal or IFN-7-activated HFRPE cells were added into the top chamber of the transwells. In each figure, 100% indicates the proliferation of Jkt cells alone (served as a control), which was generally 350,000 ± 30,000 cpm. Assay m: Neo/Jkt Cells and bcl-x, /Jkt Cells. The rescue effect of bcl-x, overexpression on HFRPE cell-mediated apoptosis in Jkt cells was examined in this assay. Third (P3) or sixth (P6) passage normal or IFN-7-activated HFRPE cells were incubated with bcl-xL/)kt cells. Neo/Jkt cells served as control. The cells were incubated in a HFRPE-to-&c/-Jc//Jkt ratio of 1 to 4. The cultures were incubated and pulsed similar that in assay I. Incorporation of the radioactive label was measured by liquid scintillation counting and was expressed as the arithmetic mean cpm of triplicate cultures. In each Figure 100% indicates the proliferation of Jkt cells alone (served as a control), which was generally 350,000 ± 30,000 cpm. Assay IV: Fas-Signaling Deficient Jurkat Cells (DD3/ Jkt). The role of FasL in the HFRPE-mediated T-cell apoptosis was evaluated by incubation of normal or IFN-7-activated HFRPE cells with DD3/Jkt cells at a ratio of 1 to 4. Jkt cells served as control. The resistance of DD3/Jkt cells to FasL-mediated apoptosis was examined by human anti-Fas mAb (clone CH-11). Both DD3/Jkt cells and the control Jkt cells were cultured in the presence of 0.1 ju-g/ml human anti-Fas mAb (clone CH-11; Kamiya Biomedicals, Seattle, WA). After 24 hours of incubation the apoptosis rate was evaluated with flow cytometry (FACScan) using Annexin V-PE (R & D, Minneapolis, MN) staining, which binds to phosphatidyl serine of the cell membrane. Staining procedures were performed according to the manufacturer's instructions (R & D). In each figure, 100% indicates the proliferation of Jkt cells alone (served as a control), which was generally 350,000 ± 30,000 cpm.
Ethidium Bromide and Acridine Orange Staining Five hundred thousand or 125,000 IFN-7-activated adherent HFRPE cells (P3) were cocultured with 500,000 Neo/Jkt or bcl-xf/Jkt cells in 24-well plates for 72 hours. The supernatant (containing the nonadherent Jkt cells) was carefully removed and centrifuged at 1500 rpm for 10 minutes. The cell pellet was resuspended in 400 (xl culture media. Twenty-five microliters of the cell suspension was mixed with 2 JLL1 of ethidium bromide (Sigma) and acridine orange (Sigma) mixture in a 5-ml tube (Falcon, Franklin Lakes, NJ). A glass slide was covered with 15 /xl of the stained cells. The cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) with a fluorescein isothiocyanate filter set. A total of 200 cells were counted per sample. The percentage of apoptotic cells was calculated by adding the total number of apoptotic living (AL) and apoptotic dead (AD) cells, divided by the total of 200 cell count. Photographs were taken with a Kodak Film 320 Tungsten, EPJ 135-36 (Rochester, NY).
Blocking Conditions The role of the two major soluble factors IFN-7 and TNF-a in HFRPE-mediated T-cell suppression was evaluated with neutralizing anti-human IFN-7 and anti-human TNF-a antibodies. HFRPE cells were cultured with Jkt cells in a transwell system as described above (Assay II). Neutralizing anti-human IFN-7 and anti-human TNF-a antibodies (R & D) were added at a concentration of 10 /xg/ml. The proliferation of the Jkt cells was assessed as explained above.
HFRPE Cells Induce Apoptosis in Jkt Cells
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FIGURE 1. Phase-contrast photomicrograph of cultured human fetal retinal pigment epithelium (magnification, X5). The cells formed an intact monolayer with cuboidul-hexagonal morphology.
The paired f-test was used for the statistical analysis. P ^ 0.05 was accepted as significant.
RESULTS HFRPE Cell Cultures Pure sheets of HFRPE cells were isolated from fetal eyes. The cells expressed a cuboidal-hexagonal shape and were pigmented. Cultured cells attached to the culture plate and proliferated to form a tight monolayer (Fig. 1). Although the pigmentation decreased with passaging, the cultured HFRPE cells maintained their homogenous epithelial morphology throughout all passages. The anti-pan cytokeratin staining showed that all cultured cells expressed cytokeratin, indicating their epithelial origin (data not shown). Jkt Cell Proliferation Assays Assay I: HFRPE Cells Suppressed the Proliferation of Jkt Cells. In this assay, different passages (P2-P6) of normal or IFN-7-activated HFRPE cells were incubated with Jkt cells. At second passage (P2), normal HFRPE cells induced an average of 20%, and IFN-7-activated HFRPE cells induced an average of 40% inhibition of Jkt cell proliferation (Fig. 2, condition P2). The inhibitory effect of TFN-7-activated HFRPE cells was significantly increased with passaging (conditions P3-P6). At the third passage a total suppression of Jkt proliferation was achieved (condition P3). In contrast, the inhibitor)' effect of nonactivated HFRPE cells was only slightly increased with passaging. Statistical analysis with the paired /-test indicated that only IFN-7-activated HFRPE cells from passages 3 and later were significantly inhibiting Jkt proliferation (P
