Apoptosis-Inducing Ligand (TRAIL), and This Information Is Current As TNF-Α During T Cell Activation of September 25, 2021

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Differential Regulation of the Expression of CD95 Ligand, Receptor Activator of Nuclear Factor-κB Ligand (RANKL), TNF-Related Apoptosis-Inducing Ligand (TRAIL), and This information is current as TNF-α During T Cell Activation of September 25, 2021. Ruoxiang Wang, Liying Zhang, Xiaoren Zhang, Jose Moreno, Xunyi Luo, Mehrdad Tondravi and Yufang Shi J Immunol 2001; 166:1983-1990; ; doi: 10.4049/jimmunol.166.3.1983 Downloaded from http://www.jimmunol.org/content/166/3/1983

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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. Differential Regulation of the Expression of CD95 Ligand, Receptor Activator of Nuclear Factor-␬B Ligand (RANKL), TNF-Related Apoptosis-Inducing Ligand (TRAIL), and TNF-␣ During T Cell Activation1,2

Ruoxiang Wang,* Liying Zhang,* Xiaoren Zhang,* Jose Moreno,† Xunyi Luo,* Mehrdad Tondravi,† and Yufang Shi3* Members of TNF superfamily are characterized by their ability to inﬂict apoptosis upon binding to their cognate receptors in a homotrimeric manner. These proteins are expressed on different cell types under various conditions. However, the mechanisms governing the expression of these molecules remain elusive. We have found that the TCR signal can elicit the expression of receptor ␬ ␣

activator of NF- B ligand (RANKL), TNF- , CD95L, and TNF-related apoptosis inducing ligand (TRAIL) in T cell hybridoma Downloaded from A1.1 cells, thus allowing us to examine the expression pattern of these molecules under precisely the same conditions. We have previously reported that CD95L expression requires both protein kinase C (PKC) translocation and Ca2؉ mobilization and is inhibited by cyclosporin A, and dexamethasone. We demonstrate now that activation-induced expression of RANKL is mediated -by Ca2؉ mobilization. PKC activation does not induce RANKL expression nor does it synergize with the Ca2؉ signal. Activation induced RANKL expression is blocked by cyclosporin A, but not by dexamethasone. The expression of TNF, in contrast, is ␣ 2؉ mediated by PKC, but not by Ca . TNF- expression is not inhibited by cyclosporin A, but is sensitive to dexamethasone. A1.1 http://www.jimmunol.org/ cells constitutively express TRAIL at low levels. Stimulation with anti-CD3 leads to an initial reduction and subsequent increase in TRAIL expression. TRAIL induction is not inhibited by cyclosporin A, but highly sensitive to dexamethasone. Therefore, expression of the TNF superfamily genes is regulated by distinct signals. Detailed understanding of the regulatory mechanisms could provide crucial information concerning the role of these molecules in the modulation of the immune system. The Journal of Immunology, 2001, 166: 1983–1990.

he TNFR superfamily, a group of type I transmembrane through their cognate receptors (5). Although the biological role of glycoproteins, represents a growing family of receptors some of these ligand-receptor pairs remains obscure, a number of T fundamental for cell growth, differentiation, and apoptosis studies have revealed insights into the mechanisms by which the ex- by guest on September 25, 2021 during development and pathophysiological processes. Several family pression of the TNF superfamily genes is regulated. TNF-␣ is pro- members have been identified in mammalian cells. Each member of duced by a variety of cell types in response to diverse stimuli. The the TNFR family binds to its distinct cognate ligand(s) (1, 2). The regulation of TNF-␣ is best appreciated in response to signals from identification of this family of proteins is based on the shared homol- the TCR ligation. It has been reported that the production of TNF-␣ ogies in their cysteine-rich extracellular ligand-binding domains and in T cell clones depends on Ca2ϩ mobilization induced by TCR stim- intracellular effector (death) domains (3). These receptors transmit ulation (6). This Ca2ϩ-dependent induction of TNF-␣ is blocked by their signals mainly via the interactions of their intracellular domains cyclosporin A (6). In B cells, activation through Ag receptor or via the with intracellular proteins bearing death domain-containing proteins CD40 pathway also induces NF-AT-dependent expression of TNF-␣ (4). The discovery of these domains has shed light on the overall that is blocked by cyclosporin A/FK506, although the promoter bind- architecture of the pathway leading from receptor ligation to the ac- ing site of NF-AT may be different from that in T cells (7–9). More- tivation of signaling cascade and eventually to biological functions over, transfection of a cDNA encoding a constitutively active form of such as apoptosis, differentiation, proliferation, and cell survival. calcineurin is sufficient to activate the TNF-␣ promoter (10). There- Concomitantly, the ligands for these receptors, now referred to as fore, the induction of TNF-␣ expression by the TCR activation signals the members of the TNF superfamily, have been identified as a series in lymphocytes is dependent on Ca2ϩ mobilization and the activation of type II transmembrane glycoproteins that bind to and signal of NF-AT. On the other hand, it has been shown that the TNF-␣ promoter can be activated by phorbol esters (PMA) or LPS in human mononuclear cells, a process dependent on NF-␬B (11–13). Interest- Departments of *Immunology and †Tissue Biology, Jerome H. Holland Laboratory, American Red Cross, Rockville, MD 20855 ingly, the existence of ␬ elements in the promoter of TNF-␣ gene is ␣ Received for publication March 27, 2000. Accepted for publication October 31, 2000. not responsible for PMA- and LPS-induced TNF- production (14). ␣ The costs of publication of this article were defrayed in part by the payment of page TNF- promoter analysis revealed that the PMA-responsive element charges. This article must therefore be hereby marked advertisement in accordance is activated by AP-1 (15, 16). with 18 U.S.C. Section 1734 solely to indicate this fact. Our previous studies have demonstrated that activation-induced 1 This work was supported by National Institutes of Health Grants AI43384, expression of CD95 ligand (CD95L)4 requires both protein kinase CA76492, and AR44089. 2 This is publication number 107 of the Department of Immunology, Holland Labo- ratory of the American Red Cross. 4 Abbreviations used in this paper: CD95L, CD95 ligand; AICD, activation-induced 3 Address correspondence and reprint requests to Dr. Yufang Shi, Department of cell death; FasL, Fas ligand; OPG, osteoprotegerin; PKC, protein kinase C; RANKL, Immunology, Jerome H. Holland Laboratory of the American Red Cross, 15601 receptor activator of NF-␬B ligand; TMB-8, 8-(diethylamino)-octyl-3,4,5-trimethoxy- Crabbs Branch Way, Rockville, MD 20855. E-mail address: [email protected] benzoate hydrochloride; TRAIL, TNF-related apoptosis-inducing ligand.

2ϩ C (PKC) translocation and Ca mobilization (17). The expression mM L-glutamine, 50 mM 2-ME, 10% heat-inactivated FBS (Sigma, St. of CD95L can be blocked by either PKC inhibitors or blockers of Louis, MO), and 10 mM gentamicin. Cell cultures were incubated at 37°C 2ϩ in humidified atmosphere with 5% CO /95% air. Ca mobilization (17). The same finding has also been reported 2 by others (18). A recent study has demonstrated that the expression Reagents of CD95L is mediated by PKC␪ (19). In addition, it has also been Ab to CD3 was produced by a hamster B cell hybridoma, 145-2C11⑀ (from shown that CD95L expression is sensitive to cyclosporin A, reti- Dr. Jeffery Bluestone, University of Chicago, Chicago, IL). PMA, N-(2- noic acid, and dexamethasone (17, 20–22). Promoter analysis has guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), 1-(5-isoquinoline- revealed that the activation of CD95L transcription requires sulfonyl)-2-methylpiperazine (H7), GO¨ 6976, bisindolylmaleimide I, Cal- NF-␬B (23, 24), NF-AT (25, 26), Nurr-77 (27), ALG-4 (28), Egr-3 phostin C, and ionomycin were purchased from Calbiochem (San Diego, CA). An inhibitor for intracellular Ca2ϩ mobilization, 8-(diethylamino)- (29), and AP-1 (30). Several of these transcription factors have octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), dexamethasone, been shown to directly regulate CD95L transcription in response to and retinoic acid were obtained from Sigma. Cyclosporine was a gift from TCR activation. In addition, we have reported that activation-in- Novartis Pharmaceuticals (East Hanover, NJ). FR901228 was obtained duced CD95L expression requires the participation of the protoon- from Fujisawa Pharmaceutical Company (Osaka, Japan). rDR5 was kindly cogene c-myc (31). provided by Dr. Youhai Chen (University of Pennsylvania, Philadelphia, ␬ PA). Anti-murine CD95L, MFL3, was purchased from PharMingen (La Receptor activator of NF- B ligand (RANKL)/TNF-related ac- Jolla, CA), and rOPG was from R&D Systems (Minneapolis, MN). Other tivation-induced cytokine (TRANCE) has been shown to be ex- chemicals used were the purest grade available from Sigma. pressed in T cells and other cell types (32–34). Its major function TCR activation has been attributed to the regulation of skeletal homeostasis acting in concert with its decoy receptor osteoprotegerin (OPG) (35, 36). A1.1 cells were activated with anti-CD3 coated on tissue culture plastic. Downloaded from Therefore, this molecule has also been referred to as OPG ligand Plastic coating was conducted by overnight incubation with 0.05 M Tris- HCl (pH 9) containing 2.5 ␮g anti-CD3. Unbound Ab was washed away (OPGL)/ODF (osteoclast differentiation factor). Because this mol- with PBS. Alternatively, the cells were also activated with the combination ecule is expressed on activated T cells and dendritic cells (35, 37), of PMA plus ionomycin. Activation was conducted for different periods, its principal function in the immune system has been suggested to and samples were harvested for analysis. be involved in T cell-dendritic cell interaction during an immune TRAIL-mediated apoptosis in breast cancer cells response (32, 38, 39). Interestingly, the expression of RANKL in http://www.jimmunol.org/ B cells is also found to be induced by CD40L (35). Surprisingly, A1.1 cells (2 ϫ 106/ml) were stimulated with plastic-bound anti-CD3 for RANKL is also crucial for early lymphocyte development and 6 h. Supernatant was collected at 6 h postculture. Breast cancer cells (MDA-231, 80% confluence in 24-well plate) were treated with cyclohex- lymph node organogenesis (40). imide (8 ␮g/ml) for 2 h and then incubated with supernatant of activated TNF-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand) A1.1 cells at 1/1 dilution with or without blocking reagents (DR5 at 16 is another member of TNF cytokine family. It shares homology ␮g/ml, or OPG at 500 ng/ml). Cells were collected at 16 h after treatment with CD95L and also activates rapid apoptosis in many types of and assessed for apoptosis by DNA content analysis. cancer cells (41). TRAIL is expressed in a wide variety of tissues. DNA content analysis It can bind to several receptors including those containing a cyto-

Flow cytometric analysis was employed to assay cellular DNA content, in by guest on September 25, 2021 plasmic death domain and those lacking a functional cytoplasmic which apoptotic cells were shown as a hypodiploid peak. After treatments, death domain (decoy receptors) (1, 42). Most tissues are resistant splenocytes were fixed with 70% ethanol for 30 min at 4°C, followed by to TRAIL cytotoxic action due to the presence of decoy receptors two washes with PBS. The fixed splenocytes were then incubated in PBS (1). In T cells, TRAIL expression induced by activation is blocked containing propidium iodide (Sigma) at 50 ␮g/ml and RNase (Boehringer by cyclosporin A, rapamycin, and inhibitors of phosphatidylinosi- Mannheim, Indianapolis, IN) at 0.1 mg/ml at room temperature for 30 min. DNA content was determined by flow cytometry on FACScan (Becton tol-3 kinase, PKC, and protein tyrosine kinases (43). In addition, Dickinson, San Jose, CA). The FL2 intensity was plotted as histograms on activation-induced TRAIL expression is enhanced by type I IFNs a linear scale. (44, 45). Northern blotting Therefore, it is clear that the expression of these genes is dif- ferentially regulated. However, the information regarding the reg- Total RNA was isolated with affinity columns (Qiagen, Chatsworth, CA), ulatory mechanisms of these genes is derived from various cell according to the protocol recommended by the manufacturer. RNA samples were fractionated on 1% agarose/2.2 M formaldehyde denaturing gel, types in response to different stimuli. In the present study, we and transferred onto Nytran membrane (Schleicher & Schuell, Keene, NH). employed monoclonal T cell hybridoma A1.1 cells and examined The DNA probes were labeled with [32P]dCTP by random priming (Boehr- the expression of TNF-␣, TRAIL, RANKL, and CD95L. We have inger Mannheim) according to manufacturer’s instructions. Prehybridiza- found that the expression of these proteins follows different kinet- tion and hybridization were conducted at 42°C in a solution containing 5ϫ ϫ ics in response to TCR stimulation. Interestingly, these genes ex- SSC (10 SSC is 1.5 M NaCl, 0.15 M sodium citrate), 2.5 mM EDTA, 0.1% SDS, 5ϫ Denhardt’s solution, 2 mM sodium pyrophosphate, 50 mM hibited different responses to inhibitors known to modulate TCR sodium phosphate, and 50% formamide. After washing with 0.2ϫ SSC, signals. Taken together, these studies begin to clarify the diverse 0.1% SDS at 56°C for 1 h, hybridization signals were detected by biochemical events modulating the expression of genes of TNF autoradiography. superfamily. More detailed analysis of the expression patterns of these ligands and receptors on lymphocyte subpopulations will be Results necessary to define their different roles in immune activation and Distinct expression kinetics of TNF family genes following TCR suppression. activation The expression of the genes in the TNF family has been studied in Materials and Methods various cellular systems in response to different stimuli. However, Cells due to the inconsistency of the experimental systems, it is difficult to conclude the expression kinetics of these genes. The T cell Murine T cell hybridoma A1.1 cells were originally obtained from Dr. hybridoma system has been employed as a model system to in- Bhagirath Singh (University of Alberta, Edmonton, Canada) (46). They were recloned and selected for the expression of TCR and the responsive- vestigate activation-induced cell death (AICD) in T cells (47–49). ness to activation signals. A1.1 cells were maintained in RPMI 1640 me- It has been shown that AICD in this model system depends on dium (Life Technologies/BRL, Gaithersburg, MD), supplemented with 2 activation-induced expression of CD95 and CD95L (20, 50). In the The Journal of Immunology 1985

other than CD95L also participate in the induction of apoptosis. We employed OPG fusion protein (a decoy receptor of RANKL and TRAIL), DR5 fusion protein (an inhibitor of TRAIL) (52), anti-TNF, CD95 fusion protein, and anti-CD95L. We found that both CD95 fusion protein and anti-CD95L completely blocked activation-induced apoptosis. Blocking of TNF, RANKL, and TRAIL does not have any effect on anti-CD3-induced apoptosis (Fig. 2). The concentrations of OPG, anti-TNF, and DR5 used in our studies all blocked the biological activity of their respective FIGURE 1. The expression kinetics of TNF family members upon anti- ligands (data not shown). Therefore, even though several members CD3 stimulation. T cell hybridoma A1.1 cells were stimulated with anti- of the TNF family proteins are expressed in A1.1 cells upon TCR CD3 for different times. Total RNA was isolated from samples collected at activation, only CD95L participates in the induction of apoptosis. 30-min intervals. The same RNA blot membrane was analyzed by Northern blotting for the expression of CD95L, TRAIL, RANKL, and TNF-␣. Nevertheless, this cellular system provides us with a model to ex- GAPDH and IL-2 were included as controls. amine the regulatory mechanisms controlling TCR signal-mediated expression of TNF family members.

present study, we show that activation through the TCR also in- ϩ ␣ Differential requirement for PKC activation and Ca2

duces the expression of TNF- , RANKL, and TRAIL. Therefore, Downloaded from this T cell hybridoma system provides us with a unique opportu- mobilization in TCR-activated expression of the genes of the nity to investigate the expression kinetics of various TNF family TNF family members after activation through the TCR. We stimulated A1.1 Signals generated by ligating the TCRs initiate a cascade of phos- cells with plastic-bound anti-CD3. Total RNA was isolated from phorylation and enzyme activation, including the activation of these cells at half-hour intervals for a maximum of 9.5 h and trans- multiple tyrosine kinases, phospholipases, PKC, and inﬂux of ferred onto blotting membrane. The same membrane was exam- 2ϩ 2ϩ

Ca (53–55). Ca inﬂux and the activation of PKC have been http://www.jimmunol.org/ ␣ ined for the expression of CD95L, TRAIL, RANKL, TNF- , IL-2, shown to be the key events of activation-induced cytokine produc- and GAPDH by Northern blot analysis (Fig. 1). The induction of tion and proliferation in T cells, because the combination of PMA IL-2 indicates that the plastic-bound anti-CD3 activated this T cell ϩ and Ca2 ionophores could completely mimic the signals from the hybridoma, and GAPDH reveals equal loading of the total RNA ϩ TCR (55, 56). Furthermore, inhibition of the Ca2 -dependent cal- samples. CD95L starts to appear at 3.5–4 h after stimulation. The cineurin phosphatase by cyclosporin A results in immune suppres- expression of TNF-␣ could be observed as early as 1.5 h postac- sion (57), and the inhibition of apoptosis (48, 58). We tested the tivation. In contrast, the expression of RANKL was observed at 2ϩ 2.5 h after activation. Interestingly, A1.1 cells constitutively ex- requirement for PKC translocation and Ca mobilization in the regulation of the expression of the TNF family genes. We activated press TRAIL at low levels. Upon activation, there is an initial by guest on September 25, 2021 reduction and then an increase in the level of TRAIL. Therefore, A1.1 cells with either PMA or ionomycin or their combination for our studies revealed differential expression of various TNF family 4 h and examined the expression of different genes by Northern genes in the same cell in response to the same stimulus, indicating blot analysis. Similar to our previous report (17), the expression of that these genes are regulated by different mechanisms. CD95L is induced only minimally by ionomycin. PMA treatment does not have any effect on CD95 expression. CD95 expression The expression of CD95 and CD95L is solely responsible for was, however, dramatically enhanced when treated with both PMA activation-induced apoptosis and ionomycin (Fig. 3). Interestingly, TRAIL is constitutively ex- Previous studies have demonstrated that activation-induced apo- pressed at low levels. This expression is inhibited by atni-CD3 ptosis in T cell hybridomas is mediated by the interaction between treatment for 4 h. The inhibitory effect by anti-CD3 is most likely CD95 and CD95L, as it could be blocked by soluble rCD95 protein through the activation of PKC, because ionomycin treatment did (51). Because other members of the TNF family are also induced not alter TRAIL expression, while PMA did. With regard to the in A1.1 cells upon activation, we determined whether the proteins expression of RANKL, we found that the RANKL expression is

FIGURE 2. Activation-induced apoptosis in T cell hybridoma A1.1 cells is solely mediated by CD95-CD95L interaction. A1.1 cells were activated on anti-CD3-coated plastic in the presence or absence of anti-FasL (40 ng/ml), Fas-Fc (10 ng/ml), DR5 (10 ␮g/ml), OPG (200 ng/ml), or anti-TNF (50 ␮g/ml). Cells were harvested at 12 h after activation, and cellular DNA content was analyzed by ﬂow cytometry upon propidium iodide staining. 1986 DIFFERENTIAL REGULATION OF GENES IN THE TNF SUPERFAMILY

CD95, expression (17). Similar to our ﬁnding of the role of ionomycin in the regulation of CD95L, we show that TCR activation- induced RANKL expression is also blocked by TMB-8. However, this inhibitor has no effect on TNF-␣ expression (Fig. 3). Thus, the effect of Ca2ϩ mobilization on the induction of RANKL and CD95L is exerted through the cyclosporine-sensitive calcineurin pathway. Interestingly, the constitutive expression of TRAIL is inhibited by TMB-8, but not by cyclosporine, indicating a role of calcineurin-independent Ca2ϩ in maintaining TRAIL expression.

Differential effect of dexamethasone on TCR activation-induced expression of TNF family genes Dexamethasone, a synthetic glucocorticoid hormone, is one of the best-known immunosuppressants. It has been used to control au- toimmunity, inﬂammation, and rejection of transplanted organs and tissues (60). Its immunosuppressive effect has been attributed FIGURE 3. The requirement of PKC activation and Ca2ϩ mobilization to the inhibition of TCR-stimulated activation, proliferation, ex- for the expression of different members of the TNF family. A1.1 cells were activated with anti-CD3, ionomycin (100 nM), PMA (30 nM), or both pression of cell surface molecules, and the production of soluble PMA and ionomycin for 4 h. Total RNA was isolated and analyzed for the cytokines (61). Earlier studies have demonstrated that dexameth- Downloaded from expression of CD95L, TRAIL, RANKL, and TNF by Northern blotting asone also inhibits activation-induced apoptosis in T cell hybrid- analysis. omas and in the thymocytes (62–64), a ﬁnding that leads to the discovery of the role of steroid hormones in positive selection (63, 65, 66). Interestingly, the inhibitory effect of dexamethasone on mediated by Ca2ϩ mobilization alone. PKC activation is not re- activation-induced T cell hybridoma death has been revealed due 2ϩ quired, nor does it affect Ca mobilization-induced RANKL ex- to its effect on activation-induced CD95L expression (20, 67). We http://www.jimmunol.org/ pression. The expression of TNF in this experimental system is have examined the effect of dexamethasone on anti-CD3-induced induced by PKC activation. Ionomycin-induced Ca2ϩ mobilization expression of various TNF family genes. Dexamethasone inhibited has no effect on TNF production (Fig. 3). activation-induced expression of CD95L. It also blocked constitu- Mercep et al. (58) and we (48) have reported that blocking the tive expression of TRAIL. Dexamethasone only has moderate ef- calcineurin pathway with cyclosporin A completely inhibits AICD fect on anti-CD3-induced TNF production. Interestingly, it does in T cell hybridomas and immature T cells. It has recently been not have any effect on the activation-induced expression of reported that cyclosporine could inhibit activation-induced CD95L RANKL (Fig. 4). expression (21, 29). When the expression of other TNF family genes was examined, we found that cyclosporine also inhibited Differential requirement of protein synthesis for the expression by guest on September 25, 2021 RANKL. It, however, did not have effect on anti-CD3-induced of TNF family members expression of TNF-␣. Cyclosporin A also has no effect on consti- To determine whether the regulation of the TNF family members tutive expression of TRAIL (Fig. 3). is at the transcriptional level, it is of interest to test whether it Because cyclosporin A does not have any effect on cytosolic requires de novo protein synthesis. This possibility was examined ϩ Ca2 levels and thus does not interfere with the interaction of in aggregates incubated in the presence of a 5 ␮M concentration of ϩ Ca2 with other proteins, we tested the effect of a general inhibitor the protein synthesis inhibitor cycloheximide (Fig. 5). In the pres- ϩ of intracellular Ca2 mobilization, 8-(diethylamino)-octyl-3,4,5- ence of cycloheximide, induction of the CD95L, RANKL, and trimethoxybenzoate hydrochloride (TMB-8) (59). We have reported that TMB-8 completely blocked AICD and CD95L, but not

FIGURE 4. Dexamethasone has different effects on the expression of FIGURE 5. Differential requirement of protein synthesis for the expres- TNF family genes. A1.1 cells were activated with plastic-bound anti-CD3 sion of TNF family members. A1.1 cells were stimulated with anti-CD3 in in the presence or absence of dexamethasone (1 ␮M) for 4 h. Total RNA the presence or absence of cycloheximide (CHX) for 4 h. The levels of the was isolated and tested for the expression of CD95L, TRAIL, RANKL, and expression of CD95L, TRAIL, RANKL, and TNF were detected by North- TNF-␣ by Northern blotting hybridization. ern blotting. The Journal of Immunology 1987 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 6. Activation-induced TRAIL is cytotoxic to breast cancer. A1.1 cells were stimulated with plastic-bound anti-CD3 for 6 h. Breast cancer cells were treated with cycloheximide for 2 h and then incubated with supernatant of activated A1.1 cells at 1/1 dilution with or without DR5 or OPG. Cells were collected at 16 h after treatment and assessed for apoptosis by DNA content analysis.

TNF gene by anti-CD3 was blocked, providing evidence that de cells. We found that the supernatant of activated A1.1, but not novo protein synthesis was required for these two genes. Interest- unactivated A1.1 cells, exhibited cytotoxic activity to MDA-231 ingly, the constitutive expression of TRAIL was also blocked by cells (Fig. 6). To investigate whether the activity is due to activa- cycloheximide (Fig. 5). As a positive control, we show that tion-induced production of TRAIL, we employed soluble TRAIL OX40L expression is not affected by cycloheximide. The require- receptor DR5 (69) and a decoy receptor OPG. DR5 is specific to ment for ongoing protein synthesis for the expression of these TRAIL, and OPG blocks the activity of both TRAIL and RANKL genes suggests either that the key transcription factors are turned (70). We found that both fusion proteins effectively blocked the over rapidly or that anti-CD3 induces the de novo synthesis of a cytotoxic activity of supernatant from activated A1.1 cells, indi- missing regulatory protein (for CD95L, RANKL, and TNF). cating that the cytotoxic activity of the supernatant from activated A1.1 cells is due to the presence of TRAIL. Therefore, although Activation-induced TRAIL is cytotoxic to breast cancer cells not involved in AICD, activation-induced TRAIL expression is It has been shown that breast cancer cell line MDA-231 is sensitive functional. to TRAIL-induced apoptosis (68). We examined whether these cells are sensitive to the supernatant of anti-CD3-activated A1.1 Discussion Table I. Differential response of TNF family genes to the modulation of TCR signals Recent studies have revealed that various members of the TNF family are expressed on T cells (71). Since the discovery of TNF, there has been much interest in the elucidation of the molecular TNF-␣ CD95L RANKL TRAIL mechanisms controlling its expression. Along with the identifica- Anti-CD3 ϩϩ ϩ – tion of other members of the TNF family, a great amount of efforts ϩ ϩ Anti-CD3 cyclosporine A ––– have been focused on finding the mechanisms by which the ex- Anti-CD3 ϩ TNB-8 ϩ ––– Anti-CD3 ϩ dexamethasone Ϯ – ϩ – pression of these genes is regulated. It is now known that the PMA ϩ ––– expression of these genes is regulated by different pathways. Un- Ionomycine – ϮϩϮ fortunately, because the available information regarding the regu- PMA ϩ ionomycine ϩϩ ϩ – lation of these genes is drawn from various experimental systems, 1988 DIFFERENTIAL REGULATION OF GENES IN THE TNF SUPERFAMILY it has been difficult to establish a consensus concerning the regu- sion of CD95L is time dependent (data not shown). The expression lation of these genes. In this study, we used monoclonal T cell of TRAIL could also be detected on the cell surface at 5–6 h after hybridoma A1.1 cells (46, 48, 49) and examined the expression of activation (data not shown). Secreted TRAIL could kill breast can- TNF-␣, TRAIL, RANKL, and CD95L under identical conditions, cer cells (Fig. 6). In addition, we have found that activation-in- i.e., examined on the same blotting membranes. We show that the duced RANKL expression could be detected by Western blot anal- expression of these genes follows distinct kinetics in response to ysis (data not shown). Interestingly, RANKL expressed on A1.1 TCR stimulation. The expression of these genes requires continued cells could induce osteoclast formation. Therefore, the expression protein synthesis. Ca2ϩ mobilization is important for the induction of mRNA correlated with the protein expression. of RANKL and CD95, while PKC is important for modulating From our studies, it is clear that the expression of TNF family TRAIL and CD95L. Activation-induced RANKL and CD95L are molecules is regulated by different mechanisms. Although it has blocked by cyclosporin A. Dexamethasone only blocked CD95L, been reported that activation of T cells could result in the up- TRAIL, and to some extent TNF. However, it has no effect on regulation of both molecules, we have found that the initial acti- RANKL. Taken together, the expression of the genes of the TNF vation signals are actually inhibitory to the expression of TRAIL in superfamily is regulated by distinct signals. A summary of our data this T cell hybridoma system. Its expression only comes up at a is presented in Table I. later time. One explanation for this observation is that the TCR Our finding that different members of the TNF family are dif- signal does not directly mediate the expression of TRAIL. The ferentially regulated is important for the understanding of the di- signals for TRAIL expression may just well be secondary to the verse function of these molecules. It is important to point out that production of some factor(s). Nevertheless, it has been reported 2ϩ in in vivo situations, T cells at different stages of development, that mature NK cells use Ca -dependent granule exocytosis and Downloaded from differentiation, and activation occupy distinct milieus in various release of cytotoxic proteins, FasL, and membrane-bound or se- tissues. The particular environment provides specific adhesion and creted TNF-␣ to induce target cell death. In contrast, TRAIL is costimulation molecules, which could well be used to activate cer- expressed on immature NK cells (75). This is consistent with our tain members of this family of proteins. Detailed understanding of observation that TRAIL is constitutively expressed, while activa- their regulatory mechanisms could provide pivotal information tion leads to the reduction of TRAIL and simultaneously increases

concerning the role of these molecules in the modulation of the the expression of TNF and CD95L. http://www.jimmunol.org/ immune system. It is interesting to note that activation of this T cell hybridoma One important function of the members of the TNF family is to also induced expression of RANKL, a factor not only important for induce apoptosis in target cells in a receptor-specific manner (5). the formation of osteoclasts but that also plays an important role in The best-characterized proteins that execute apoptosis are CD95L the interaction between T cells and dendritic cells (39). Our study and TRAIL. It is well established that, upon activation, primary T is the first to characterize the signaling mechanisms leading to the cells express various members of the TNF/TNFR families. Among expression of RANKL in T cells. One question remaining is why them, CD95L is the best studied. It has been shown to be involved the immune system and the skeletal system share the same mole- in peripheral deletion of activated T cells and thus limiting the cule and how these two important functions are conserved on one extent of an immune response. Mice with mutations in CD95 (lpr) molecule during evolution. One possible explanation is that during by guest on September 25, 2021 or CD95L (gld) exhibit lymphoproliferative disorder and often de- an immune response, an increase in blood calcium level released velop autoimmune diseases (72). Human patients showing pheno- from the bone may be critical for full function of the immune types (Canale-Smith syndrome or autoimmune lymphoprolifera- system. Overall, the data reported in this study provide the first tive syndrome) similar to those in lpr or gld mice also carry direct evidence for the differential regulation of the expression of mutations in CD95 or CD95L (73). Therefore, activation-induced TNF family members. We hope that our studies begin to clarify the CD95L expression represents a key element in immune regulation. diverse biochemical events modulating the expression of genes of In our present study, we have found that CD95L is a key molecule the TNF superfamily. More detailed analysis of the expression in the modulation of AICD in T cell hybridomas. patterns of these ligands and receptors on lymphocyte subpopula- Among the TNF family members, TRAIL is another apoptosis- tions will be necessary to define their different roles in immune inducing molecule. A recent study has revealed that this molecule activation and suppression. on activated T cells is more important in causing tissue damage during autoimmune diseases (74) or in eliminating cancer cells Acknowledgment (68). In fact, upon activation, TRAIL receptor expression was de- We thank Drs. David Scott and Achsah Keegan for discussions, and Kristy creased, whereas TRAIL was up-regulated (74). 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