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Regulation of Fas Ligand-Induced Apoptosis by TNF Bennett D. Elzey, Thomas S. Griffith, John M. Herndon, Ramon Barreiro, Jurg Tschopp and Thomas A. Ferguson This information is current as of September 29, 2021. J Immunol 2001; 167:3049-3056; ; doi: 10.4049/jimmunol.167.6.3049 http://www.jimmunol.org/content/167/6/3049 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Regulation of Fas Ligand-Induced Apoptosis by TNF1

Bennett D. Elzey,* Thomas S. Grifﬁth,2* John M. Herndon,* Ramon Barreiro,* Jurg Tschopp,‡ and Thomas A. Ferguson3*†

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly .isolated Fas؉ lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2؊/؊ T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1؊/؊ T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1؊/؊ 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2؊/؊ 2,4,6-trinitrophenyl- Downloaded from coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites. The Journal of Immunology, 2001, 167: 3049Ð3056.

mmune privileged sites are areas that maintain a unique re- tion of immune deviation prevent the activation of dangerous cel- http://www.jimmunol.org/ lationship with the immune system. Whereas most organ sys- lular immune reactions that could damage vision or lead to I tems can tolerate inﬂammatory and immune reactions, areas autoimmunity. such as the eye, brain, and reproductive organs tend to prohibit The Fas/FasL system is not only critical for immune privilege (3, 6, immunity to prevent the damaging sequelae associated with im- 7), but also is important in the development of the immune response, mune responses to foreign invaders (1, 2). In the eye, constitutive homeostasis of the immune system, and maintenance of self-tolerance Fas ligand (FasL)4 expression helps maintain immune privilege by (8, 9). Additionally, Fas-mediated apoptosis is involved in T cell cy- ϩ inducing apoptotic cell death of Fas lymphoid cells that enter in totoxicity, tumorigenesis, and liver disease (10Ð12). Fas is a type I

response to infection (3). Studies from our laboratory have also membrane protein belonging to the TNFR superfamily, which in- by guest on September 29, 2021 linked this Fas-mediated apoptosis to the induction of immune cludes the p55 (TNFR1) and p75 (TNFR2) TNFRs, CD40, 4-1BB, deviation (i.e., a preferential induction of a Th2-like response over and the family of TNF-related apoptosis-inducing ligand receptors, what would normally be Th1 dominated) (4). In this setting, apo- among others (13, 14). Cross-linking of Fas by FasL or specific Ab ptosis directly leads to the activation of a systemic Th2-type re- initiates the cell death cascade, with the binding of Fas-associated sponse through the interaction between APCs and dead cells, an death domain protein (FADD) to the cytoplasmic domain of Fas being effect mediated by the production of the anti-inflammatory cyto- the first molecular event (15, 16). This facilitates the binding and kine IL-10 in the apoptotic cells before death (5). Thus, the elim- activation of caspase-8 (FADD-like IL-1␤-converting enzyme), fol- ination of potentially damaging inflammatory cells and the induc- lowed by the activation of other downstream caspases, degradation of DNA, and ultimately cell death (17, 18). This process is tightly regulated and depends, in the case of T cells, on prior cellular activation. Interestingly, elements of the immune response (such as IL-2; Ref. Departments of *Ophthalmology and Visual Sciences and †Pathology, Washington University School of Medicine, St. Louis, MO 63110; and ‡Institute of Biochemistry, 19) potentiate Fas-mediated death through the modulation of the ap- University of Lausanne, Epalinges, Switzerland optosis regulatory protein FADD-like IL-1␤-converting enzyme in- Received for publication April 20, 2001. Accepted for publication July 10, 2001. hibitory protein (FLIP) (20). The costs of publication of this article were defrayed in part by the payment of page FLIP was originally described as a viral product (vFLIP) (21, charges. This article must therefore be hereby marked advertisement in accordance 22), but has since been shown to exist in mammalian cells (cFLIP). with 18 U.S.C. Section 1734 solely to indicate this fact. cFLIP mRNA exists as multiple splice variants, but protein ex- 1 This work was supported by National Eye Institute Grants EY06765 and EY08972 pression is limited to a long and short form (cFLIP and cFLIP ) and by a Department of Ophthalmology and Visual Sciences grant from Research to L S Prevent Blindness, Inc. (New York, NY). (21). cFLIPS contains two death effector domains (DED) and is 2 Current address: Department of Urology, University of Iowa, Iowa City, IA 52242. structurally related to vFLIP, whereas cFLIPL contains an addi- 3 Address correspondence and reprint requests to Dr. Thomas A. Ferguson, Depart- tional caspase-like domain lacking catalytic activity. The DED of ment of Ophthalmology and Visual Sciences, Washington University School of Med- both FLIPL and FLIPS enable them to bind to the DED of FADD icine, 660 South Euclid, Box 8096, St. Louis, MO 63110. E-mail address: and caspase-8, preventing their recruitment to the death domain of [email protected] Fas. This prevents formation of the death-inducing signaling com- 4 Abbreviations used in this paper: FasL, Fas ligand; FADD, Fas-associated death domain protein; FLIP, FADD-like IL-1␤-converting enzyme inhibitory protein; AC, plex and inhibits apoptosis (23, 24). The resistance of naive T cells anterior chamber; AICD, activation-induced cell death; vFLIP, viral FLIP; cFLIP, to Fas-mediated death has been attributed to high intracellular lev- cellular FLIP; DED, death effector domain; TNCB, 2.4.6-trinitro-1-chlorobenzene; TNBS, 2,4,6-trinitrobenzenesulfonic acid; sFasL, soluble FasL; TNP, 2,4,6-trinitro- els of FLIP, which decrease following cellular activation and IL-2 phenyl; TRAF, TNFR-associated factor. production, thereby making the T cells susceptible to Fas-mediated

death (20, 23). In addition to protecting cells from Fas-induced Tween and the TNF standard or test samples were added (100 ␮l/well). apoptosis, FLIP can protect cells from apoptosis induced by TNF, Plates were incubated overnight at 4¡C, wells were washed four times with ␮ NF receptor-related apoptosis-mediated protein, and TNF-related PBS/Tween, and 100 l/well polyvalent goat anti-recombinant murine TNF (diluted 1:500 in DMEM-10% FCS; provided by Dr. K. Sheehan, apoptosis-inducing ligand. FLIP has also been found in heart, skel- Washington University) was added for1hatroom temperature. Wells etal muscle, and kidney (23) where it is presumed to function as an were washed four times, followed by the addition of 100 ␮l/well peroxi- antiapoptotic factor to help maintain organ homeostasis. Expres- dase-conjugated rabbit anti-goat IgG (diluted 1/2000 in DMEM-10% FCS; sion in tumors promotes tumor growth presumably by blocking the ICN Pharmaceuticals, Costa Mesa, CA) for1hatroom temperature. Wells were washed four times with PBS/Tween and 100 ␮l/well ABTS (0.1 M cell death pathway (25, 26). The importance of FLIP was recently sodium citrate, 1 mM ABTS, 0.016% (v/v) H O ) was added. Upon color demonstrated by the embryonic lethality of targeted deletion of 2 2 development, the plate was analyzed on an ELISA plate reader at A414. this molecule (27). In our studies of FasL-induced death in the eye, we have ob- In situ TUNEL staining served that apoptotic cell death occurs rapidly in both activated and freshly isolated lymphoid cells (3, 4). Since Fas-mediated ap- At various times following AC injection of TNP-coupled cells, eyes were removed, fixed in Formalin, and processed for paraffin sectioning. Ten-mi- optosis is tightly regulated, we suspected that other factors might crometer sections were mounted onto microscope slides and incubated over- contribute to this process. In the present study, we examined the night at 55¡C. Sections were then deparaffinized by washing for 5 min in role of the proinflammatory cytokine TNF in the regulation of xylene twice, 5 min in absolute ethanol twice, 3 min in 95% ethanol, 3 min in Fas-mediated apoptosis in the eye. Our results show that TNF, via 70% ethanol, and 5 min in PBS. Protein present in the sections was digested ␮ binding to TNFR2, increases the sensitivity of T cells to Fas-me- with 20 g/ml proteinase K for 20 min at room temperature. Following four washes in distilled water, endogenous peroxidase was quenched with 2.0% diated death by modulating the expression of a molecule that reg- Downloaded from H2O2 for 5 min at room temperature and sections were washed twice in PBS. ulates apoptosis. Labeling of 3Ј-OH fragmented DNA ends was performed with an in situ apoptosis detection kit (ApopTag; Oncor, Gaithersburg, MD) following package Materials and Methods instructions. Detection of labeled ends was done with the kit-supplied anti- digoxigenin-peroxidase Ab and development of diaminobenzidine substrate Mice (Vector Laboratories, Burlingame, CA). BALB/c and C57BL/6 were purchased from the National Cancer Institute. Ϫ Ϫ Ϫ Ϫ / / http://www.jimmunol.org/ TNFR1 , TNFR2 , and normal littermate mice were purchased from RT-PCR The Jackson Laboratory (Bar Harbor, ME). In all in vivo experiments, groups consisted of five or more animals and were used at 6Ð10 wk of age. Total RNA was isolated with TRIzol reagent (Life Technologies, Gaith- Experiments were repeated at least three times with similar results. ersburg, MD) as per the manufacturer’s instructions. RNA samples (1 ␮g each) were tested for DNA contamination by 30 cycles of PCR with mouse Reagents ␤-actin primers. After it was shown there was no DNA contamination, 2,4,6-Trinitro-1-chlorobenzene (TNCB) was purchased from Eastern cDNA synthesis was performed using an RNA PCR kit (PerkinElmer, Nor- Chemical (Smithtown, NY). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) walk, CT) with the supplied oligo(dT)16 primer. Reverse transcription was was purchased from Sigma (St. Louis, MO). Hamster Ab to murine TNF performed using a thermal program of 25¡C for 10 min, 42¡C for 30 min, (TN3.19.12) was provided by Dr. R. Schreiber (Washington University, St. and 95¡C for 5 min. PCR were performed using the following primers: ␤ Ј Ј Louis, MO). Recombinant murine TNF was purchased from Genzyme -actin (forward, 5 -TGGAATCCTGTGGCATCCATGAAAC-3 ; re- by guest on September 29, 2021 Ј Ј (Cambridge, MA). Jo2 (anti-Fas/CD95), anti-CD3-FITC, and streptavi- verse, 5 -TAAAACGCAGCTCAGTAACAGTCCG-3 ); and FLIP (for- Ј Ј Ј din-PE were obtained from BD PharMingen (San Diego, CA). Goat anti- ward, 5 -CAAGATAGCCAAGGACAAGAG-3 ; reverse, 5 -GATGGAT Ј Ј TNFR Abs (TNFR1 and TNFR2) and TUNEL stain kit for flow cytometry GTCTTCACCAGTG-3 ); Bax (forward, 5 -TTTATGGAGGGGTCCG Ј Ј Ј (catalogue no. TA5354) were purchased from R&D Systems (Minneapolis, GGGA-3 ; reverse, 5 -TGTCCAGCCCATGATGGTTCT-3 ) giving ␤ MN). Anti-goat conjugated with FITC or PE was purchased from Jackson products of 349, 307, and 420 bp, respectively. -Actin PCR cycle con- ImmunoResearch (Broomall, PA). The anti-FLAG M2 mAb for aggregat- ditions were 95¡C for 1 min, 55¡C for 1.5 min, and 72¡C for 2 min for 22 ing FLAG-tagged soluble FasL (sFasL) (28) was purchased from Eastman cycles. FLIP PCR cycle conditions were 95¡C for 1 min, 61¡C for 1 min, Kodak (Rochester, NY). and 72¡C for 1.5 min for 35 cycles. Bax PCR cycle conditions were 94¡C for 2 min, 65¡C for 1.5 min, and 72¡C for 1.5 min for 35 cycles. Samples 2,4,6-Trinitrophenyl (TNP) coupling of T cells were resolved on a 2% agarose gel and visualized with ethidium bromide. T cells were isolated from spleen cell suspensions and coupled with TNP as previously described (29, 30). TNP-T cells were used for all anterior Western blot for FLIP chamber (AC) injections, while TNP-spleen cells were used in the RT-PCR Cellular proteins from 4 ϫ 105 cells were separated by SDS-PAGE on a analysis for FLIP. 10% polyacrylamide gel under reducing conditions and then transferred to AC inoculations nitrocellulose membranes by semidry electroblotting. Ponceau S (Sigma) staining of the nitrocellulose confirmed the equal loading of proteins. Mice were lightly anesthetized with Metofane (methoxyflurane) and injec- Membranes were blocked overnight with 5% nonfat dry milk in PBS and tions were done under a dissecting microscope. Each intracameral injection incubated with anti-FLIP (AL 109) (20, 23) at a dilution of 1/400 for 1 h. contained 5 ϫ 105 TNP-T cells or 2.5 ϫ 104 PFU HSV-1 (KOS) in a Membranes were washed three times and then incubated with peroxidase- 0.005-ml volume using a 0.25-ml Hamilton Microliter syringe (Hamilton, conjugated goat anti-rat IgG (Amersham, Arlington Heights, IL) for 1 h. Reno, NV) fitted with a 33-gauge needle. The membranes were washed thoroughly with PBS/0.05% Tween 20 and developed by chemiluminescence according to the manufacturer’s protocol Tissue extraction and TNF ELISA (ECL; Amersham Pharmacia Biotech, Piscataway, NJ). At various times following AC injection of TNP-coupled spleen cells, eyes were removed and added to a 1.5-ml microcentrifuge tube. To each sample, Induction of immune deviation 0.5 ml of 0.1% Tween 20 in PBS (PBS/Tween) was added and the samples were ground using a plastic pellet pestle. Samples were quick frozen in Immune deviation was induced as described elsewhere (29). Briefly, TNP-

liquid N2, thawed in a 37¡C water bath, and ground again. Samples were coupled spleen cells were injected into the AC, and then the mice were then centrifuged for 5 min at 13,000 ϫ g. Supernatants were removed for immunized 48 h after AC injection with 0.050 ml of 1% TNCB in acetone/ TNF determination. olive oil (3:1) applied to shaved abdominal skin. Five days later, mice were TNF protein levels were quantitated using a sandwich ELISA developed challenged with 0.033 ml of 10 mM TNBS in PBS in the right footpad and with two anti-TNF mAb (31). Ninety-six-well ELISA plates (Immulon II; 0.033 ml of PBS in the left footpad. Values are expressed in micrometers Dynatech Laboratories, Chantilly, VA) were coated with 3.5 ␮g/ml puri- (ϮSE) and represent the difference between the right footpad (Ag chal-

ﬁed TN3 19.12 mAb (30) diluted in coating buffer (0.1 M NaHCO3/0.1 M lenge) and the left footpad (PBS challenge). Background values represent Na2CO3)for2hat37¡C. Wells were blocked with DMEM-10% FCS for the difference between the challenged and unchallenged foot in 30 min at room temperature. Plates were washed three times with PBS/ unimmunized mice. The Journal of Immunology 3051

Isolation of ocular inflammatory cells immune deviation to the Ags encountered in the AC (4, 5). The Twenty-four or 48 h following AC injection of HSV-1, mice were enucle- production of TNF is also an important event in this phenomenon ated and eyes were minced between the ends of two frosted glass slides in since TNF mRNA levels peak shortly after AC injection and the HBSS without Ca2ϩ/Mg2ϩ containing 10 mM EDTA. Large pieces were coinjection of a neutralizing mAb to TNF with the Ag blocked removed by filtration through 70-␮m cell strainers and mononuclear cells immune deviation (32). Since members of the TNF family coop- were washed three times with HBSS. Following wash, cells were counted erate in the induction of apoptosis (33, 34), we tested for a link and yields were typically 1 ϫ 106/injected eye. Cells were then analyzed by flow cytometry. between TNF production and FasL-induced death. Following AC injection of TNP-T cells, TNF protein levels in- Flow cytometry (FACS) creased rapidly over the first 2Ð3 h before falling to near normal FACS staining was performed by standard protocols. Briefly, for surface levels by 9 h (Fig. 1). In situ TUNEL staining 24 h following AC labeling, 1 ϫ 105 spleen cells/ml were incubated with 20 ␮lof10␮g/ml injection showed numerous apoptotic cells (Fig. 2A), confirming goat anti-TNFR1 or TNFR2 for 30 min at 4¡C. Cells were washed twice in previous observations (4). However, when a neutralizing anti-TNF staining buffer (2% FCS in PBS plus 0.01% NaN3) and resuspended in 20 ␮l of donkey anti-goat FITC (15 ␮g/ml) for 30 min at 4¡C. Cells were then mAb was coinjected with the TNP-T cells, apoptosis was pre- washed twice with staining buffer, resuspended in 400 ␮l of 2% formal- vented (Fig. 2B). The role of TNF in cell death was then examined dehyde, and collected on a BD Biosciences FACSCalibur (Mountain View, using TNP-T cells from TNFR1Ϫ/Ϫ or TNFR2Ϫ/Ϫ mice. Although CA). In some cases, cells were also stained with anti-CD3-PE and analyzed TNFR1Ϫ/Ϫ TNP-T cells underwent apoptosis in the AC much like for CD3 and TNFR expression. To measure TUNEL-positive lymphocytes the normal TNP-T cells, TNFR2Ϫ/Ϫ TNP-T cells did not (Fig. 2, in the eye, cells were isolated and surface stained as described above. Cells were then fixed, permeabilized, incubated with TdT and biotinylated dNTP C and D, respectively). per kit instructions (R&D Systems) and stained with streptavidin-PE. All Immune deviation following lymphoid cell injection into the eye Downloaded from events were analyzed using CellQuest software. requires both cell death (4) and TNF (32). Since TNFR2Ϫ/Ϫ Cell death assay (DO11.10 T cell hybridoma) TNP-T cells do not undergo apoptosis in the AC of the eye (Fig. 2D), a prediction is that immune deviation cannot be induced with DO11.10 cells (4 ϫ 105/ml) were labeled for2hat37¡C with 5 ␮Ci/ml 3 ϫ 4 these cells. To test this, we compared the ability of normal, [ H]thymidine. Following three washes in culture medium, cells (2 10 / Ϫ/Ϫ Ϫ/Ϫ well of a 96-well plate) were incubated with medium alone, murine TNF, TNFR1 and TNFR2 TNP-T cells to induce immune devi- Jo2, or TNF plus Jo2 for 16 h at 37¡C. Unfragmented DNA was collected ation in normal B6 mice. Upon AC injection, we found that TNP-T http://www.jimmunol.org/ by filtration through glass fiber filters (Packard Instrument, Meriden, CT) cells from the normal and TNFR1Ϫ/Ϫ mice induced immune de- using a Filtermate 96 cell harvester (Packard Instrument) and counted on a viation, whereas those from TNFR2Ϫ/Ϫ or TNFR1/2Ϫ/Ϫ mice did Microplate scintillation counter (Packard Instrument). Percent DNA frag- not (Fig. 3). mentation was determined by the formula (cpm without RPE cells Ϫ cpm with RPE cells) divided by the cpm without RPE cells (ϫ100). Treatments Previous studies showed that activated T cells do not express were done in triplicate. detectable TNFR1 (35). Thus, one possible explanation for our results is that only TNFR2 is expressed on the cells we inject, thus Cell death assays (spleen cells) the dominance of TNFR2. When we examined freshly isolated Spleen cells (1 ϫ 106/ml) were cultured with TNF or goat anti-mouse spleen cells for expression of TNFRs, only low levels of TNFR2 TNFR (R1 or R2) for6hat37¡C in 1 ml of RPMI 1640 with 0.5% normal were detected (Fig. 4A). Therefore, the apparent preference for by guest on September 29, 2021 mouse serum. Cells were collected, concentrated by centrifugation, and added to 96-well plates (Immunlon II; Dynatech Laboratories) containing TNFR2 may be because TNFR1 is simply absent from the lym- immobilized Jo2. Cells were incubated for an additional 16 h and cell death phoid cell population. was determined by trypan blue exclusion. Immobilized Jo2 was prepared To determine whether TNFR2 signaling is crucial for inducing by first coating plates with 5 ␮g/ml rabbit anti-goat Ab for2hat37¡C. apoptosis in cells infiltrating the eye, we needed to examine a Following three washes with PBS, 5 ␮g/ml Jo2 was added for2hat37¡C. Cells were added following a three times wash with PBS. Death was as- situation where both receptors are expressed on the inflammatory sessed by trypan blue exclusion. infiltrate. Following ocular injection of the HSV-1, we have shown For T cell killing by sFasL and TNF, CD4ϩ T cells were obtained from CD8Ϫ/Ϫ spleen cells by purification over antimouse Ig columns. Typical yields were 85% CD4ϩ T cells (data not shown). Cells were placed in 96-well flat-bottom culture plates in triplicate wells (2 ϫ 105/well) in 100 ␮l of complete RPMI 1640 (10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, 10 mM HEPES, 1 mM sodium pyruvate, and antibiotics) and incubated overnight with 100 ng/ml murine TNF in a 5% CO2 humidified 37¡C incubator. Wells were then given FLAG-tagged sFasL (100 ng/ml) for 1⁄2 h before adding aggregating anti-FLAG Ab (M2, 2 ␮g/ml) and incubating for24hat37¡C. To measure apoptotic cell death, subdiploid DNA analysis was performed. Cells were collected and washed in buffer (2% FCS in PBS plus 0.01% NaN3) and fixed at room temperature for 30 min in EtOH. Cells were then resuspended in 500 ␮l of buffer plus 50 ␮g/ml propidium iodide. Apoptosis was determined by gating on the population containing subdiploid DNA using CellQuest software after data acquisition on a BD Bio- sciences FACSCalibur. Statistical analysis Significant differences between groups were evaluated using a two-tailed Student’s t test ( p Ͻ 0.01). Results TNF and lymphocyte apoptosis within the eye FIGURE 1. TNF production following AC inoculation. Mice were in- Studies from our laboratory have shown activated and freshly iso- jected AC with 5 ϫ 105 TNP-T cells and at various times the eyes were lated lymphoid cells entering the AC of the eye rapidly undergo removed and soluble extracts made with PBS/Tween 20. TNF levels were apoptotic cell death (3, 4). This death is induced by the FasL ex- determined in an ELISA by comparison to a known standard. Each point pression on ocular tissues and is a prerequisite for the induction of represents the mean of four eyes Ϯ SE. 3052 FasL-INDUCED APOPTOSIS AND TNF Downloaded from http://www.jimmunol.org/

FIGURE 2. Apoptosis following AC inoculation of TNP-T cells. Normal B6 mice were injected AC with 5 ϫ 105 TNP-T cells and 24 h later the eyes were removed and processed for in situ TUNEL stains as described previously (3Ð4). A, B6 TNP-T cells; B, TNP-T cells plus 0.5 ␮g of anti-TNF; C, TNP-T cells from TNFR1Ϫ/Ϫ mice; and D, TNP-T cells from TNFR2Ϫ/Ϫ mice. IR, iris; C, cornea.

that infiltrating cells are apoptotic within 48 h (3) and this apo- facilitated only in the presence of an active TNFR2. Collectively, by guest on September 29, 2021 ptosis is required for the induction of immune deviation to viral results in Figs. 1Ð5 suggest that TNF signaling through TNFR2 is Ags (4). Data in Fig. 4, B and C, show that wild-type cells entering an integral part of FasL-induced apoptosis of cells in the eye. the eye in response to HSV-1 express both TNFR1 and TNFR2 at When cells lack TNFR2, they do not undergo apoptosis and they significant levels. We then examined apoptosis in TNFR1Ϫ/Ϫ and do not induce immune deviation because they fail to receive a TNFR2Ϫ/Ϫ mice and found that only cells invading the TNFR1Ϫ/Ϫ “sensitizing” signal from TNF. eye were TUNELϩ, whereas there was little or no apoptosis in the Ϫ/Ϫ eyes of TNFR2 mice (Fig. 5). Thus, FasL-induced cell death is TNF and Fas cooperate to induce cell death To further explore the role of TNF in regulating Fas-mediated apoptosis, we tested whether TNF could alter the sensitivity of cells in vitro to Fas-mediated death. When T cell hybridoma DO11.10 cells (high expressers of both TNFRs by FACS analysis; data not shown) were cultured with TNF, minimal apoptosis was observed (Fig. 6A). Similarly, culturing the cells with anti-Fas Ab (Jo2) resulted in a modest level of killing only at the highest concentration tested. However, when noncytotoxic concentrations of TNF and anti-Fas were used together, a substantial increase in cell death was observed. This suggests that TNF and Fas can cooperate to induce cell death in this hybridoma. Results in Fig. 7 extend these observations to splenic cells using TNF or agonistic Abs to the TNFRs. In Fig. 7A, only the combination of TNF and anti-Fas resulted in measurable cell death, while data in Fig. 7B show that inclusion of anti-TNFR2 (but not anti-TNFR1) at Ϫ/Ϫ FIGURE 3. Immune deviation induced by TNP-T cells. B6, TNFR1 , 0.1 and 1.0 ␮g/ml sensitized cells for death on anti-Fas Ab. Although Ϫ/Ϫ Ϫ/Ϫ TNFR2 , or TNFR1/2 TNP-T cells were AC injected into B6 mice 1.0 ␮g/ml anti-TNFR1 with anti-Fas appears to protect cells from 48 h before immunization with 1% TNCB on shaved abdominal skin. Five death, the data did not reach statistical significance. Data in Table I days later, mice were challenged with 0.033 ml of 10 mM TNBS in PBS extend these observations to the natural ligand for Fas and show that in the right footpad and 0.033 ml of PBS in the left footpad. Measurements ϩ (micrometers Ϯ SE) were taken 24 later and represent the difference be- only in the presence of TNF and sFasL do CD4 T cells undergo tween the right footpad (Ag challenge) and left footpad (PBS challenge). significant apoptosis as measured by subdiploid DNA analysis. We Background (Bkg) values represent the difference between challenged and conclude from these data that FasL-induced apoptosis of lymphoid unchallenged sites in naive B6 mice. cells in the eye is assisted by TNF via the TNFR2 receptor. The Journal of Immunology 3053 Downloaded from http://www.jimmunol.org/

FIGURE 5. Apoptosis following AC inoculation of HSV-1. TNFR1Ϫ/Ϫ or TNFR2Ϫ/Ϫ mice were injected in the AC with 2.5 ϫ 104 PFU HSV-1 (KOS). Twenty-four hours later, the eyes were removed and processed to measure TUNELϩ cells vs TNFR expression as described in Materials and Methods. Numbers in quadrant corners indicate the percentage of cells by guest on September 29, 2021 present in the area.

apoptosis (37, 38). Another potent regulator of cell death is FLIP, which is a crucial repressor of apoptosis (23). Fig. 6B shows that incubation of DO11.10 hybridoma T cells with TNF significantly decreased the levels of FLIP. In addition, mRNA levels for the pro-apoptotic molecule Bax were elevated following treatment of FIGURE 4. Expression of TNFR1 and TNFR2. A, Spleen cells were cells with TNF (Fig. 6C) (mRNA levels for Bcl-2 and Bcl-xL were obtained from normal B6 mice and stained for TNFR1 and TNFR2. unchanged; data not shown). TNFR1Ϫ/Ϫ (B) or TNFR2Ϫ/Ϫ (C) mice were injected in the AC with 2.5 ϫ Treatment of normal spleen cells with TNF or agonistic anti-TNFR 104 HSV-1. Eyes were removed 48 h later, infiltrating mononuclear cells were isolated, and expression of TNFR1 and TNFR2 was determined by Abs also revealed modulation of the expression of Bax and FLIP. flow cytometry. Treatment of B6 spleen cells with anti-TNFR2 increased the level of Bax in these cells (Fig. 8A). Whereas FLIP transcripts were detectable in untreated B6, TNFR1Ϫ/Ϫ, and TNFR2Ϫ/Ϫ spleen cells, incubation with TNF resulted in the disappearance of FLIP mRNA only in B6 or Effect of TNF on cell death molecules TNFR1Ϫ/Ϫ spleen cells (Fig. 8B). FLIP mRNA was still detectable in Ϫ Ϫ Next, we explored the potential mechanisms by which TNF sig- the TNFR2 / spleen cells after incubation with TNF, further sup- naling through TNFR2 could facilitate apoptosis in lymphoid cells porting the hypothesis that signaling through TNFR2 is necessary for via Fas. Possible mechanisms for this effect could be increased Fas sensitization to Fas-mediated apoptosis. (Note: levels of Bcl-2 or expression, increased expression of pro-apoptotic molecules, Bcl-xL mRNA remained unaltered after incubation with agonistic and/or down regulation of antiapoptotic molecules. When we ex- TNF or anti-TNFR Abs (data not shown).) amined Fas expression, we observed that TNF did not significantly increase the cell surface expression of Fas measured by flow cy- Discussion tometry (data not shown). Recently, we demonstrated that Fas-FasL interactions were critical We then considered molecules that regulate apoptosis. Bcl-2 for maintaining immune privilege in the eye. The FasL expressed family members are known to regulate cell death by repressing throughout the eye kills infiltrating inflammatory cells before they

(i.e., Bcl-2, Bcl-xL, Mcl-1, and A1) or promoting (i.e., Bax, Bcl- can damage the eye, thereby helping to preserve vision (3). More- xS, Bad, and Bak) the apoptotic process (36). These molecules over, cell death induced by FasL is important for the induction of form homodimers and heterodimers that are key to the induction of immune deviation following Ag encountered in the eye (4, 5). 3054 FasL-INDUCED APOPTOSIS AND TNF

FIGURE 6. Effect of TNF on DO11.10 cells. A, DO11.10 cells (1 ϫ 106 cells/ml) were cultured with 0, 6, or 25 ng/ml TNF for 6 h. Cells were Downloaded from washed and added to wells coated with indicated concentrations of Jo2. Percent DNA fragmentation was determined 16 h later. B, DO11.10 cells (1 ϫ 106 cells/ml) were cultured for 6 or 16 h with 25 ng/ml TNF and cellular proteins from 4 ϫ 105 cells were subjected to Western blot for

FLIP. A 55-kDa band representing FLIPL was noted (mFLIP, mouse FLIP). Band intensity was determined by densitometry. Lane 1, Untreated; http://www.jimmunol.org/ lane 2,TNF6h;andlane 3, TNF 16 h. C, DO11.10 cells (1 ϫ 106 cells/ml) were cultured for 6 or 16 h with 25 ng/ml TNF and RT-PCR was performed for Bax. Lane 1, Untreated; lane 2,TNF6h;andlane 3, TNF 16 h.

Since the cell death in the eye is rapid and takes place in activated, as well as unactivated, cells we asked whether other molecules FIGURE 7. Cell death induced by TNF and anti-Fas. Spleen cells from produced in the ocular microenvironment might affect sensitivity B6 mice were treated with indicated concentration of TNF (A) or goat anti-mouse TNFR1 or TNFR2 (B) for 6 h. Cells were collected, concen- to apoptosis. The studies presented here focused on the effect of

trated by centrifugation, and added to 96-well plates (Immulon II; Dyna- by guest on September 29, 2021 TNF and TNFR signaling. We demonstrate that TNF, acting tech Laboratories) containing immobilized Jo2. The amount of cell death through the TNFR2, increases the susceptibility of T cells to FasL- was determined 16 h later by trypan blue exclusion. induced death. These results further demonstrate that noncytotoxic levels of TNF may regulate Fas-mediated apoptosis through the modulation of two important intracellular regulators of apoptosis. In both splenic T cells and inflammatory cells responding to The sensitizing signal that TNF transmits to the cell down-regu- HSV-1, the activity of TNF observed was mediated through the lates an antiapoptotic protein (FLIP), while up-regulating a pro- p75 (or TNFR2) receptor. Although the exclusive expression of apoptotic one (Bax), enabling a signal through Fas to activate the TNFR2 may explain the results using splenic T cells, this expla- cellular apoptotic pathway. nation is not sufficient to account for apoptosis of inflammatory Previous studies from our laboratory have revealed that TNF cells responding to HSV-1. These cells express both receptors but mRNA levels rapidly increase over the first 2Ð3 h after AC injection are killed by FasL following the interaction of TNF with TNFR2. of TNP-spleen (32). In the present studies, TNF protein levels were The dominance of TNFR2 may reflect the higher affinity of found to follow similar kinetics after AC injection, with intraocular TNFR2 for the ligand (40). This may have relevance to immune levels peaking 2 h after injection before returning to background lev- privilege by allowing lower levels of TNF to sensitize lymphoid els by 8Ð9 h. These kinetics were similar to those found in the skin cells for death in the eye. An effect mediated through a high-af- during the initiation of a contact sensitivity reaction, where TNF can finity receptor would also provide a way to sensitize cells that are be detected within 0.5Ð1 h following Ag application (39). It is inter- early in activation with the relatively low levels of TNF available esting that TNF protein levels increase almost immediately after cell injection into the AC. We have previously observed that TNF mRNA production was the result of the interaction of T cells with ocular Table I. Effect of TNF on CD4ϩ T cellsa tissue (32). Thus, a possible scenario might be that ocular cells pro- duce TNF in response to lymphoid cell invasion leading to increased TNF sensitivity of the lymphocytes to killing by constitutive FasL. Another No Treatment (100 ng/ml) sFasL Both possibility is that the lymphoid cells make TNF in response to other % subdiploid 33.2 Ϯ 5.8 39.5 Ϯ 8.8 48.7 Ϯ 4.5 70.1 Ϯ 6.2 elements in the ocular milieu, leading to their destruction by FasL. A DNA third situation would be relevant to inflammatory reactions in the eye a ϩ Ϫ/Ϫ where lymphoid cells entering the eye in response to infection pro- CD4 T cells were obtained from CD8 spleen cells by purification over antimouse Ig columns. Cells were placed in 96-well flat-bottom culture plates in duce TNF. Under normal circumstances, this cytokine would promote triplicate wells (2 ϫ 105/well) in 100 ␮l of complete RPMI 1640 with 50 mM 2-ME, the immune reaction (39); however, in the immune privileged site and appropriate wells were incubated overnight with 100 ng/ml murine TNF in a 5% CO2 humidified 37¡C incubator. sFasL (100 ng/ml) was then added to appropriate constitutive FasL rapidly kills TNF-sensitized lymphocytes before wells and incubated for 24 h at 37¡C. Subdiploid DNA content was determined by damage can be done. propidium iodide staining of cells fixed in EtOH. The Journal of Immunology 3055

action of Fas and TNFR2 (present studies, data not shown). To our knowledge, this is the first demonstration that cooperation between signals delivered to the cell through TNFR2 and Fas has relevance in vivo, i.e., to the maintenance of immune privilege and the induction of immune tolerance in the eye. Our data show that cells infiltrating the AC at 24 or 48 h are susceptible to TNFR2/Fas-mediated death; however, their exact activation status is unknown. This may be an important consider- ation because it has been reported that T cells rapidly and tran- siently shed TNFRs during the first stage of Ag responsiveness (52). Our data do not address this; however, cells entering the eye in our model seem to be universally susceptible to death via Fas if they are expressing TNFR2. Our results show that TNF affects several components of the cell death machinery. TNF-TNFR2 interaction alters the expression of the intracellular regulators of apoptosis, Bax and FLIP. Bax is a pro-apoptotic member of the Bcl-2 family of proteins that regu- lates cell death through the formation of homodimers and heterodimers with Bcl-2. It has been suggested that the ratio of Bcl- Downloaded from 2:Bax determines cell survival or death following an apoptotic stimulus (36Ð38, 53). Following incubation with TNF, Bax mRNA levels rapidly increase, suggesting that the amount of Bcl-2 within the cell could no longer prevent Bax from forming homodimers and promoting cell death. We have previously demon-

strated that Bcl-xL overexpression in T cells inhibits apoptosis of http://www.jimmunol.org/ these cells in the eye (4). In this instance, excess Bcl-xL probably inhibited Bax homodimer formation and blocked cell death. FIGURE 8. Effect of TNF on spleen cells. A, Spleen cells (1 ϫ 106/ml) We also observed that TNF, acting through the TNFR2, caused from B6 mice were cultured for 6 h with 1 ␮g/ml agonistic Ab to TNFR1 a decrease in the intracellular levels of FLIP mRNA and protein. or TNFR2. RT-PCR was then performed for bax. B, Spleen cells (1 ϫ The expression of FLIP in T cells depends on the degree of cellular 106/ml) from B6, TNFR1Ϫ/Ϫ, or TNFR2Ϫ/Ϫ mice were cultured for 6 h activation, with high levels present in naive T cells as well as T with 25 ng/ml TNF and RT-PCR was performed for FLIP. spl, spleen; KO, cells early in the activation process (23). FLIP levels decline with knockout. prolonged activation, in direct correlation with the T cell’s increased sensitivity to Fas-mediated apoptosis. FLIP has also been by guest on September 29, 2021 found to play a role in AICD of lymphocytes (20). AICD is an in the eye (Fig. 1; Ref. 32). Thus, TNFR2 may have a specific role important mechanism of self-tolerance mediated by Fas-FasL in- in pro-apoptotic and anti-inflammatory responses leading to the teractions and enhanced by IL-2 (19). Interestingly, analysis of the protection of immune privileged sites. biochemical mechanisms of IL-2-enhanced Fas-mediated T cell The two TNF receptors are coexpressed in many tissues, includ- apoptosis revealed that IL-2 induces expression of FasL and si- ing hemopoietic cells, although membrane expression is indepen- multaneously suppresses the transcription and expression of FLIP dently regulated and may differ depending on the cell type (41Ð (20). Our observation that FLIP transcription decreased with TNF 43). It is now known that these receptors activate distinct signal signaling through TNFR2 is similar to these observations with pathways and can function independently (44Ð46). However, IL-2. Thus, these cytokines may play parallel roles in terminating studies have shown that they can cooperate to induce cell death an immune response. In the periphery, the growth-promoting cy- (34, 47), but the mechanism is unclear. It has been suggested that tokine IL-2 sensitizes T cells for death through suicide and fratri- TNFRs cooperate at the level of receptor-ligand interactions (40) cide. This permits an immune response but prevents excessive im- (e.g., the ligand-passing model); however, this does not account mune reactions that could lead to autoimmunity. In immune for the critical role of intracytoplasmic signaling in the function of privileged sites, the proinflammatory cytokine TNF sensitizes cells both receptors. Recent studies have suggested that the TNFR-as- entering the eye for death by constitutive FasL expression. This sociated factor (TRAF) 1/TRAF-2 binding domain in TNFR2 co- prevents the evolution of the immune response to protect the func- operates with TNFR1 to induce apoptosis of PC60 and HeLa cells tion of the visual axis. (34, 47). Although it is possible that TRAF2 could recruit inhib- When considering the mechanisms of immune privilege, it is itory molecules such as FLIP away from Fas, our studies show that important to realize that multiple factors are responsible for this FLIP synthesis is inhibited by the TNF-TNFR2 interaction. biological phenomenon, including immunosuppressive cytokines TNFR2 has been shown to induce the proliferation of submaxi- (54, 55), neuropeptides (56), proinflammatory cytokines (32), and mally activated naive cells, but apoptosis late in the activation FasL (3). We initially examined a number of these factors (includ- process (48, 49). A recent report showed that CD8ϩ T cells are ing TGF-␤ and vasoactive intestinal peptide) to determine their resistant to activation induced cell death (AICD) if they do not complicity with FasL for the induction of cell death in the eye. express TNFR2 (50). Although it has been reported that activated These mediators had no effect on the induction of Fas-mediated CD8ϩ T cells are most sensitive to apoptosis via the TNFRs (35, death in our system (data not shown). The antiproliferative prop- 50, 51), we have observed that nearly all mononuclear cells en- erties of these molecules (particularly TGF-␤) are likely the un- tering the eye, whether they are CD4ϩ, CD8ϩ, or non-T cells, are derlying reason for their contribution to immune privilege (54). killed via the Fas/TNFR2 pathway. Similarly, freshly isolated The only factor we have found thus far that promotes FasL-in- splenic CD4ϩ and CD8ϩ T cells are killed in vitro by the inter- duced death in the eye is the proinflammatory cytokine TNF, 3056 FasL-INDUCED APOPTOSIS AND TNF where it functions with FasL to protect the eye from the damaging uble form is associated with downregulation of its proapoptotic activity and loss effects of immune and inflammatory reactions. of liver toxicity. J. Exp. Med. 187:1205. 29. Ferguson, T. A., J. C. Waldrep, and H. J. Kaplan. 1987. The immune response and the eye. II. The nature of T suppressor cell induction in anterior chamber Acknowledgments associated immune deviation. J. Immunol. 139:352. 30. Ferguson, T. A., J. Hayashi, and H. J. Kaplan. 1989. The immune response and We thank Veronica Franklin for assistance with the tissue sectioning. the eye. III. Anterior chamber associated immune deviation can be adoptively transferred by serum. J. Immunol. 143:821. References 31. 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