Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14308–14313, November 1998 Immunology

Activation of human monocytes induces differential resistance to with rapid down regulation of -8͞FLICE

LIYANAGE P. PERERA* AND THOMAS A. WALDMANN

Metabolism Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Contributed by Thomas A. Waldmann, September 24, 1998

ABSTRACT Cells of the monocyte͞macrophage lineage species. Although it is widely recognized that peripheral blood play a central role in both innate and acquired immunity of the monocytes undergo apoptosis spontaneously upon culture host. However, the acquisition of functional competence and unless supplemented with serum, growth factors, bacterial the ability to respond to a variety of activating or modulating products, or inflammatory cytokines such as IL-1␤ or TNF-␣ signals require maturation and differentiation of circulating (7–15), at present, the molecular mechanisms of activation- monocytes and entail alterations in both biochemical and induced survival signals in monocytes remain obscure. In an phenotypic profiles of the cells. The process of activation also attempt to define the mechanistic basis of activation-induced confers survival signals essential for the functional integrity resistance to apoptosis in monocytes at the molecular level, we of monocytes enabling the cells to remain viable in microen- evaluated the modulation of expression profiles of vironments of immune or inflammatory lesions that are rich associated with the cellular pathways in cytotoxic inflammatory mediators and reactive free-radical after stimulation of human monocytes with bacterial products species. However, the molecular mechanisms of activation- including lipopolysaccharide (LPS). We demonstrate that the induced survival signals in monocytes remain obscure. To activation-induced resistance to apoptosis is selective and is define the mechanistic basis of activation-induced resistance dependent on the triggering stimulus and involves rapid down- to apoptosis in human monocytes at the molecular level, we regulation of the most apical cysteine protease, caspase-8͞ evaluated the modulation of expression profiles of genes FLICE that is critical for the Fas͞TNF receptor death- associated with the cellular apoptotic pathways upon activa- inducing signaling pathway (16–19) and also results in dra- tion and demonstrate the following: (i) activation results in matic induction of the Bfl-1 (20–22), an antiapoptotic selective resistance to apoptosis particularly to that induced member of the Bcl-2 family. by signaling via death receptors and DNA damage; (ii) con- current with activation, the most apical protease in the death MATERIALS AND METHODS receptor pathway, caspase-8͞FLICE is rapidly down- regulated at the mRNA level representing a novel regulatory Cell Culture. Monocytes were isolated from the blood of mechanism; and (iii) activation of monocytes also leads to normal healthy donors by a two-step procedure beginning with dramatic induction of the Bfl-1 gene, an anti apoptotic member an automated leukopheresis followed by counterflow elutria- of the Bcl-2 family. Our findings thus provide a potential tion. Cells were cultured in RPMI medium 1640 supplemented mechanistic basis for the activation-induced resistance to with penicillin, streptomycin, 2 mM glutamine, and 20% apoptosis in human monocytes. heat-inactivated pooled AB human serum. Reagents and Antibodies. Staurosporine, C2-ceramide, eto- Cells of the monocyte͞macrophage lineage play a central role poside, and the Dx2 mAb against the human were in both innate and acquired immunity of the host (1, 2). These purchased from CLONTECH. Staurosporine was used at a ␮ cells are crucial in the defense against invading pathogens and final concentration of 1.1 M. C2-ceramide was used at a final ␮ in addition, exert a wide variety of functions that include concentration of 50 M. Etoposide was used at a final con- ␮ regulation of the immune response, scavenging of senescent centration of 10.5 M. Dx2 antibody was used at a final ͞ cells, lysis of infected or malignant cells, wound healing, repair, concentration of 500 ng ml. Phenol-extracted LPS from Esch- and remodeling of tissues (3, 4). However, the acquisition of erichia coli serotype O128:B12 was purchased from Sigma. For functional competence and the ability to respond to a variety UV irradiation experiments, cells were irradiated at room of activating or modulating signals require maturation and temperature after aspirating the culture medium with a Blak- differentiation of circulating monocytes to macrophages that Ray UV lamp Model XX-15S from Ultraviolet Products. Cells ͞ 2 in turn entail alterations in both biochemical and phenotypic were exposed to a dose of 15 J M ; growth medium was then profiles of the cells (5, 6). Prominent manifestations of acti- replenished, and the cells were incubated at 37°C for the vation include the expression of adhesion molecules and indicated time. secretion of potent proinflammatory cytokines such as tumor Determination of Apoptosis by DNA Electrophoresis. Cells ␣ ␤ (2 ϫ 107) were washed once with PBS and resuspended in 350 necrosis factor (TNF)- , and IL-1 , enhanced metabolic ac- ␮ tivity with generation of free-radical metabolites, all of which l of lysis buffer containing 10 mM Tris, 10 mM EDTA, and 0.5% Triton X-100 and incubated on ice for 20 min before enable these cells to converge, attack, and eliminate the ϫ noxious stimulus. Importantly, the process of activation also centrifugation (13,000 g) at 4°C for 20 min to pellet chromosomal DNA. Supernatant was then collected and confers survival signals essential for the functional integrity of ͞ monocytes enabling the cells to remain viable in microenvi- RNase A was added to a final concentration of 0.1 mg ml before incubation at 37°C for 1 hr followed by the addition of ronments of immune or inflammatory lesions that are rich in ͞ cytotoxic inflammatory mediators and reactive free-radical proteinase K (1 mg ml final concentration) and SDS (1% final

The publication costs of this article were defrayed in part by page charge Abbreviations: TNF, tumor necrosis factor; LPS, lipopolysaccharide; DISC, death-inducing signaling complex; RPA, ribonuclease protec- payment. This article must therefore be hereby marked ‘‘advertisement’’ in tion assay; IAP, inhibitors of apoptosis; AFC, 7-amino-4-trifluoromethyl accordance with 18 U.S.C. §1734 solely to indicate this fact. coumarin. 0027-8424͞98͞9514308-6$0.00͞0 *To whom reprint requests should be addressed. e-mail: lperera@ PNAS is available online at www.pnas.org. atlas.niaid.nih.gov.

14308 Downloaded by guest on September 27, 2021 Immunology: Perera et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14309

concentration). Samples were then incubated at 50°C for 2 hr and extracted with phenol͞chloroform twice before precipi- tating the DNA by adding 35 ␮l of 3 M sodium acetate. Precipitated DNA was resuspended in 25 ␮l of water, loaded on a 1.2% agarose gel containing ethidium bromide (100 ng͞ml) and separated by electrophoresis. Caspase-8 Assay. Caspase-8 activity in whole cell lysates was determined by using the ApoAlert FLICE͞caspase-8 Fluores- cent Assay Kit from CLONTECH according to the manufac- turer’s instructions. Briefly, cell lysates from 106 cells were incubated with IETD tetrapeptide conjugated to 7-amino-4- trifluoromethyl coumarin (AFC) for 1 hr at 37°C. Free AFC accumulation that resulted from cleavage of the aspartic-AFC bond was monitored by using a fluorometer (Wallac, Gaith- ersburg, MD) equipped with a 400-nm excitation filter and 505-nm emission filter. Ribonuclease Protection Assay (RPA). Total cellular RNA was isolated from monocytes by using TRIzol (Life Technol- ogies, Gaithersburg, MD) according to the manufacturer’s instructions. The expression of various apoptosis-associated genes was measured by multi-probe RPAs (23). Template sets for the multi-probe RPA were purchased from PharMingen, and the assays were performed according to the manufactur- er’s instructions. Briefly, 50 ng of DNA from each multi-probe set were used to generate 32P-labeled riboprobes of defined FIG. 1. Activation of human monocytes leads to selective resis- length with T7 RNA polymerase in the presence of 150 ␮Ci of tance to apoptosis. Elutriated monocytes were cultured in RPMI [32P]UTP. Template DNA was then eliminated by digestion medium 1640 supplemented with 20% pooled human serum and ͞ with DNase free of RNase, followed by precipitation of labeled activated with E. coli LPS (25 ng ml) for 6 hr before the introduction of indicated apoptotic agents. Staurosporine, C2-ceramide, and eto- RNA. Fifteen micrograms of total cellular RNA were then ␮ ␮ ␮ ϫ 5 32 poside were added at 1.1 M, 50 M, and 10.5 M, respectively. mixed with 6 10 cpm of P-labeled riboprobe mixture in a Anti-Fas antibody was used at a concentration of 500 ng͞ml. A dose hybridization buffer containing 40 mM Pipes, 1 mM EDTA, of 15 J͞m2 was used in UV irradiation experiments. The cells were 0.4 M NaCl, and 80% formamide and incubated at 90°C for 5 then cultured for an additional 12 hr before harvesting to assess the min followed by 56°C for 12 hr. The hybridized RNA duplexes extent of oligonucleosomal DNA fragmentation as described in Ma- were then treated with an RNase mixture consisting of RNase terials and Methods. Similar results were obtained from monocytes A and RNase T1 followed by proteinase K digestion. RNase derived from three other donors. resistant duplex RNA was then extracted with phenol once and precipitated by the addition of an equal volume of 4 M evident in UV or staurosporine-induced apoptosis. The results ammonium acetate and 2 volumes of ethanol. Then, the RNA shown in Fig. 1 thus suggest that the activation of monocytes pellet was solubilized, resolved on a 6% sequencing gel, dried, imparts a selective desensitization to apoptotic signals medi- and subjected to autoradiography or phosphorimage analysis. ated via specific signaling pathways but does not exert a global protective effect from programmed cell death. This phenom- enon was not specific for LPS-mediated activation because RESULTS AND DISCUSSION another bacterial product, i.e., a soluble lysate of Mycobacte- It is well established that freshly isolated monocytes from rium leprae (kindly provided by P. Sieling, University of peripheral blood undergo apoptosis rapidly when cultured in California School of Medicine, Los Angeles, CA) when used the absence of serum (7). Nonetheless, in the presence of to activate the monocytes, yielded a similar pattern of selective serum or other activating factors such as IL-1␤, TNF-␣,or resistance (data not shown). Our observation that monocytes bacterial products such as LPS, the cells not only resist once activated resist induction of apoptosis by crosslinking the spontaneous loss of viability, but also resist apoptosis induced cell surface death receptor Fas confirms previous similar ␥ by FAS receptor crosslinking, -irradiation, H2O2, or NO (12, observations (12, 24). 24–27). However, at present, it is not clear whether the Death receptor-induced apoptosis is an important event in activation-induced resistance to apoptosis is a global effect or, tissue homeostasis. In addition to the Fas receptor (31), other rather, is limited to specific death-inducing signals via a members of the family of cell surface death receptors include particular signaling pathway(s). To explore this issue, we TNF R1 (32), DR3 [also known as TRAMP (33–36)], DR4 cultured freshly isolated human monocytes in the presence of (also known as TRAIL-R), and DR5 (37–40). The accumu- 20% pooled human serum, which as expected prevented lating evidence indicates that the signal transduction after the spontaneous apoptosis as assessed by both cell morphology as engagement of various cell surface death receptors may pro- well as the absence of oligonucleosomal DNA fragmentation. ceed via a common pathway requiring efficient recruitment As shown in Fig. 1, the cells cultured in the presence of serum and activation of the caspase-8͞FLICE enzyme, which is the remained refractory to spontaneous apoptosis but were still most apical caspase. The resistance of activated but not resting fully susceptible to apoptosis induced by staurosporine (28), a monocytes to induction of apoptosis by surface crosslinking of broad spectrum kinase inhibitor; C2-ceramide (28), a Fas receptors taken together with the observation that the water soluble ceramide analog able to penetrate cells; etopo- activated monocytes synthesize copious quantities of TNF-␣ side, a topoisomerase II inhibitor (29); ultraviolet light (UV); without any obvious self injury suggest that with activation the and crosslinking of surface Fas receptor (30). Intriguingly, assembly of the death-inducing signaling complex (DISC) is when cells were activated by E. coli LPS, the activated cells interrupted (41, 42). To gain insights as to how activation of displayed differential resistance to the same set of apoptotic human monocytes leads to disruption of the death receptor- inducers. Specifically, cells activated with LPS effectively mediated apoptosis, the effects of activation on the expression suppressed C2-ceramide, etoposide, and Fas antibody- of a panel of cellular genes associated with death receptor mediated apoptosis whereas no significant protection was pathway signaling were assessed by a sensitive RPA after Downloaded by guest on September 27, 2021 14310 Immunology: Perera et al. Proc. Natl. Acad. Sci. USA 95 (1998)

exposure of monocytes to either E. coli LPS or a soluble lysate of M. leprae. As shown in Fig. 2, when we examined the steady–state mRNA levels of Fas receptor after stimulation of monocytes with LPS for 6 hr, no significant quantitative differences were apparent in comparison to unstimulated cells. Our results are consistent with previous observations that the expression of Fas was not modulated during activation of monocytes (12, 24). The activation-induced resistance to apoptosis in the presence of an agonist anti-Fas antibody is then likely to operate downstream of receptor engagement. Moreover, the mRNA expression of TNF-R1 (p55), which was abundant in mono- cytes, remained unmodulated after stimulation with bacterial products. Similarly, activation of monocytes failed to modulate the mRNA expression of adaptor molecules TRADD (43), RIP (44), or FADD (45) that facilitate the recruitment of caspase-8͞FLICE to assemble the DISC. However, the ex- pression of caspase-8͞FLICE diminished sharply upon activa- tion (see Figs. 2 and 3). We also noted a slight reduction in TRAIL mRNA expression upon activation. The expression levels of FasL (46), DR3 death receptor (34–36), FAP (47), and FAF-1 (48) mRNA in human monocytes were minimal and required lengthy exposures of the autoradiograms to visualize signals from these genes and were not altered upon activation. To further explore whether the reduction in the ͞ FIG. 3. Effects of monocyte activation on the expression of cellular steady–state mRNA levels of caspase-8 FLICE as detected by . Elutriated monocytes were treated with E. coli LPS or a the RPA actually results in loss of intracellular caspase-8͞ soluble lysate of M. leprae for 6 hr. Total cellular RNA was extracted FLICE activity, we first attempted to determine the presence and RPAs were performed essentially as described in the Fig. 2 legend. of any detectable caspase-8͞FLICE activity in human mono- Granzyme B was included because of its ability to cleave and activate cytes. Concordant with the abundant levels of steady–state multiple caspase members. mRNA as evident in Fig. 2, detectable levels of specific ͞ possess inherent protease activity. Nonetheless, as shown in caspase-8 FLICE activity were present in lysates of freshly Ϸ ͞ isolated human monocytes. In the absence of any apoptotic Table 1, there was 90% reduction in the cellular caspase-8 FLICE activity after LPS-mediated activation. The reduction manifestations in these cells, the caspase-8 activity is presum- ͞ ably due to unprocessed proprotein, which has been shown to in caspase-8 FLICE activity was maintained up to 30 hr after LPS treatment and then gradually returned to normal levels over a period of 48 hr (data not shown). Having established that, upon activation of human mono- cytes, there was rapid down regulation of the most apical protease in the Fas death pathway, we next assessed the effects of monocyte activation, if any, on the expression of other cellular caspases (reviewed in refs. 49 and 50). As shown in Fig. 3, caspase-1 was most abundantly expressed although no modulation of its expression was evident upon activation. Similarly, there was detectable caspase-9 expression although that too was refractory to any alterations upon activation. Expression of caspase-2 and caspase-6 were minimal in human monocytes. Importantly, unlike caspase-8, the steady–state mRNA levels of caspase-5 and caspase-7 were consistently elevated although slightly upon activation. Granzyme B is an aspartate-directed serine protease that plays a dominant ef- fector function in cytotoxic T cell-mediated killing of target cells (51). It is the only non-caspase enzyme known to cleave and activate multiple caspase members involved in apoptosis including caspase-2, -3, -6, -7, -8, -9, and -10, but not the ICE subfamily proteases (ref. 52 and references therein). As can be seen in Fig. 3, there were relatively high levels of constitutively

Table 1. Activation of monocytes leads to down-regulation of caspase-8͞FLICE activity

FIG. 2. Effects of monocyte activation on the expression of cell Treatment % Inhibition of fluorescence* surface death receptors and death receptor-associated genes. Elutri- LPS 25 ng͞ml 86.0 Ϯ 0.4 ated monocytes were treated with E. coli LPS or a soluble lysate of M. LPS 250 ng͞ml 86.6 Ϯ 0.5 leprae for 6 hr. Total cellular RNA was extracted, and RPAs were performed as described in Materials and Methods. The expression *Cell lysates from LPS-treated cells were incubated with IETD- levels of ribosomal L32 and cellular glyceraldehyde-3-phosphate de- AFC. Free AFC accumulation was monitored by using a fluorometer hydrogenase (GAPDH) serve as internal controls as well as RPA with 400-nm excitation filter and 505-nm emmision filter. Percent performed with yeast tRNA and with RNA derived from monocytes inhibition of fluorescence was calculated in comparison to untreated cultured in medium alone (␾, control); similar results were obtained control samples and represents the mean Ϯ SD of samples done in from cells derived from two other donors. triplicate. Downloaded by guest on September 27, 2021 Immunology: Perera et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14311

expressed Granzyme B in monocytes, which up-regulated the IAP to the TNF receptors in monocytes (59). As shown in modestly upon activation. Fig. 5, XIAP (56), NAIP (57), MIHB (c-IAP1), MIHC (c- Considering the pivotal role of Bcl-2 family of in IAP2) (58, 59), TRPM2 (60), and CRAF (61), all showed modulating the cellular death program, we next assessed detectable levels of expression in human monocytes. However, whether during the activation of monocytes the mRNA ex- upon activation of monocytes, the expression of these genes pression profiles of Bcl-2 family members were altered (re- did not change meaningfully (less than 2-fold). The expression viewed in refs. 49, 53, 54). In Fig. 4, anti-apoptotic Bcl-2 and of TRAF2 was minimal whereas abundant expression of BclXL were constitutively expressed in monocytes although TRAF1 was evident in human monocytes although its respon- levels were consistently higher for the BclXL gene. Upon siveness to activation was minimal. activation, however, no significant enhancement of expression In Fig. 6, we examined the mRNA expression profile of an was noted for either of these genes. In addition, the expression additional set of genes including Granzyme A (62), Granzyme profiles of the proapoptotic members namely, Bik, Bak, and B (63), DAD-1 (64), FASTK (65), Granzyme H (66), RVP-1 Bax remained unchanged upon activation of monocytes. None- (67), Dr mm 23 (68), Granzyme 3 (69), Requiem (70), CAS theless, it should be noted that the basal constitutive expres- (71), and perforin (72). The expression of each one of these sion levels of proapototic Bak and particularly Bax were quite genes has been shown to influence the cellular death pathway high in human monocytes. Mcl-1 is also a member of the Bcl-2 in various systems. In addition to Granzyme B, only Granzyme family and encodes a 37 kDa polypeptide, which forms het- H and DAD-1 displayed any detectable levels of expression in erodimers with Bax to promote cell survival under various human monocytes although their expression remained un- conditions that cause apoptotic death (49). As evident from changed upon activation. Nonetheless, reaffirming the data Fig. 4, Mcl-1 was the most abundantly expressed member in previously shown in Fig. 3, Granzyme B expression levels were human monocytes. However, its expression was not affected by elevated modestly upon activation. activation and thus, is less likely to be relevant for activation After assessing the expression of an array of genes associated induced resistance to apoptosis. Bfl-1 is another member of the with the cellular death pathway, two potential mechanisms that Bcl-2 family and encodes a 175-amino acid, early response could confer an antiapoptotic state in activated monocytes seem to emerge from the present work: (i) rapid down polypeptide predominantly expressed in hematopoietic tissues ͞ (20–22). Bfl-1 has been shown to effectively suppress apoptosis regulation of caspase-8 FLICE expression upon activation of induced by p53 tumor suppressor polypeptide, TNF-␣, and human monocytes, and (ii) dramatic induction of the antiapo- IL-3 deprivation (49). Consistent with previous reports, there ptotic Bfl-1 gene that belongs to the Bcl-2 family. was a dramatic increase in the expression of Bfl-1 gene when Cell surface death receptors constitute a steadily growing monocytes were activated. In fact, Bfl-1 was the most inducible group of cell surface molecules belonging to the TNF receptor superfamily and include CD95 (Fas receptor), TNF-R1, DR3, Bcl-2 family member in human monocytes. DR4, and DR5. The death receptors share a cytoplasmic Proteins that can specifically inhibit the apoptotic death ‘‘death domain’’ responsible for recruiting adaptor molecules pathway were first identified in viruses, and the subsequent such as FADD and͞or TRADD proteins to the plasma mem- discovery of their cellular counterparts continues to expand brane through homotypic interactions. Receptor bound adap- our understanding of the cellular suicide machinery (55). To tor molecules then couple the death protease caspase-8͞ assess whether any of these inhibitors of apoptosis (IAP) could FLICE to the death receptor leading to its proteolytic pro- dictate the anti-apoptotic state seen in activated monocytes, cessing and activation. Thus FLICE represents the most apical we examined the mRNA expression profiles of an array of IAP caspase in the death receptor pathway, and activation of genes as well as TRAF-1 and TRAF-2, which recruit many of FLICE initiates the apoptotic cascade presumably by cleaving relevant downstream substrates (reviewed in ref. 49). Our data

FIG. 5. Effects of monocyte activation on the expression of cellular FIG. 4. Effects of monocyte activation on the expression of genes genes encoding IAP. Elutriated monocytes were treated with E. coli belonging to Bcl-2 family. Elutriated monocytes were treated with E. LPS for 6 hr. Total cellular RNA was extracted, and RPAs were coli LPS or a soluble lysate of M. leprae for 6 hr. Total cellular RNA performed essentially as described in the Fig. 2 legend. TRAF1 and was extracted, and RPAs were performed essentially as described in TRAF2 also were included because of their involvement in the the Fig. 2 legend. recruitment of IAP. Downloaded by guest on September 27, 2021 14312 Immunology: Perera et al. Proc. Natl. Acad. Sci. USA 95 (1998)

rapid generation of large quantities of free radical reactive species intracellularly upon activation of monocytes, it is conceivable that these cells have evolved effective strategies to prevent DNA damage and͞or to protect from DNA-damage induced cell death. The expression profiles of the bcl-2 family are relevant in this regard because of their importance in reactive oxygen͞DNA damage induced apoptosis (49, 53, 54). The ratio of death antagonists (Bcl-2, Bcl-XL, Mcl-1, and Bfl-1) to agonists (Bax, Bak, and Bik) regulates the compet- itive dimerization between selective pairs of antagonists and agonists of Bcl-2 family proteins. Induced expression of an- tagonists favor heterodimerization, which in turn leads to cell survival. Dramatic induction of Bfl-1 during activation of monocytes (see Fig. 4) thus could very well favor equilibrium toward heterodimerization. Thus, the ability of Bfl-1 to sup- press DNA-damage induced apoptosis largely regulated by p53 tumor suppressor protein and also TNF-␣ mediated apoptosis taken together with its rapid inductive profile makes a com- pelling argument for Bfl-1 to be important in activation- induced resistance to apoptosis in human monocytes. In conclusion, our data suggest that activation of monocytes results in selective resistance to apoptosis particularly to signaling via death receptors and DNA damage. Moreover, rapid induction of Bfl-1, an anti apoptotic member of the bcl-2 family and a marked reduction in the expression of the most FIG. 6. Effects of monocyte activation on the expression of apo- apical caspase, i.e., caspase-8͞FLICE provide a potential ptotic modulators. Elutriated monocytes were treated with E. coli LPS for 6 hr. Total cellular RNA was extracted, and RPAs were performed mechanistic basis for the activation-induced resistance to essentially as described in the Fig. 2 legend. apoptosis in human monocytes.

suggest that concurrent with monocyte activation there is rapid We thank Dr. Yutaka Tagaya for critical reading of the manuscript, down regulation of caspase-8͞FLICE expression at the level of Dr. Pin-Yu Perera for helpful suggestions, and Dr. Peter Seiling for mRNA thereby effectively preventing the formation of the providing M. leprae lysates. DISC that could be triggered by the engagement of cell surface 1. Van Furth, R. (1993) in Hematopoietic Growth Factors and death receptors. Mononuclear Phagocytes, ed. Van Furth, R. (Karger, Basel), pp. Control of the death receptor-induced suicide program at 14–38. the level of FLICE appears to be evolutionarily favored. For 2. Solbach, W., Moll, H. & Rollinghoff, M. (1991) Immunol. Today instance, complex large DNA viruses such as gamma herpes- 12, 4–6. viruses and molluscipoxviruses encode proteins, known as viral 3. Unanue, E. R. & Allen, P. M. (1987) Science 236, 551–557. FLICE inhibitory proteins (v-FLIPS) that efficiently interfere 4. Evans, R. & Alexander, P. (1972) Immunology 23, 615–626. with the recruitment of FLICE by interacting with the adaptor 5. Mayernik, D. G., Ul-Haq, A. & Rinehart, J. J. (1983) J. Immunol. molecule FADD thereby preventing the assembly of a receptor 130, 2156–2160. 6. Karnovsky, M. L. & Lazdins, J. K. (1978) J. Immunol. 121, associated DISC (55, 73). More recently, existence of a cellular 809–813. homologue of viral FLIPs has been established and its tem- 7. Musson, R. A. (1983) Am. J. Pathol. 111, 331–340. poral expression during the early phase of T cell activation 8. Becker, S., Warren, M. K. & Haskill, S. (1987) J. Immunol. 139, appear to rescue T cells from premature activation-induced 3703–3709. cell death and allow activated T cells to mediate help or 9. Pabst, M. J., Hedegaard, H. B. & Johnston, R. B. (1982) cytotoxicity (74). However, because of the transient nature of J. Immunol. 128, 123–128. c-FLIP expression, with its disappearance, Fas-mediated sig- 10. Mangan, D. F. & Wahl, S. M. (1991) J. Immunol. 147, 3408–3412. naling is reestablished and T cells undergo activation-induced 11. Mangan, D. F., Welch, G. R. & Wahl, S. M. (1991) J. Immunol. 146, 1541–1546. cell death to maintain homeostasis. 12. Kiener, P. A., Davis, P. M., Starling, G. C., Mehlin, C., Klebanoff, Our demonstration that, in monocytes during activation, S. J., Ledbetter, J. A. & Liles, W. C. (1997) J. Exp. Med. 185, there is down regulation of FLICE both at the level of mRNA 1511–1516. as well as protein represents yet another unique mechanism 13. Kiener, P. A., Moran-Davis, P., Rankin, B. M., Wahl, A. F., nature has exploited to control the death receptor-signaling Aruffo, A. & Hollenbaugh, D. (1995) J. Immunol. 155, 4917– pathway. Because FLICE is central in linking signals originat- 4925. ing from various cell surface death receptors to the cellular 14. Heidenreich, S., Schmidt, M., August, C., Cullen, P., Rademaek- 159, death pathway, rapid repression of its synthesis during the early ers, A. & Pauels, H. G. (1997) J. Immunol. 3178–3188. 15. Okada, S., Zhang, H., Hatano, M. & Tokuhisa, T. (1998) phase of activation ensures the survival of monocytes to J. Immunol. 160, 2590–2596. execute their functions especially in a micro-environment rich 16. Chinnaiyan, A. M. & Dixit, V. M. (1997) Semin. Immunol. 9, in cytotoxic proinflammatory cytokines such as TNF-␣ as well 69–76. as activated cells bearing FasL. It is important to note that the 17. Muzio, M., Salvesen, G. S. & Dixit, V. M. (1997) J. Biol. Chem. subsequent return of FLICE activity may still permit death 272, 2952–2956. receptor-mediated programmed cell death in elimination of 18. Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., Litwack, functionally redundant activated macrophages that could be G. & Alnemri, E. S. (1996) Proc. Natl. Acad. Sci. USA 93, detrimental to the host (27, 75). 14486–14491. 19. Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A., Activated monocytes in addition to being resistant to death Mann, M., Krammer, P. H. & Peter, M. E. (1997) EMBO J. 16, receptor mediated apoptosis are also resistant to apoptosis 2794–2804. triggered by ␥-irradiation as well as DNA damaging agents, for 20. Choi, S. S., Park, I. C., Yun, J. W., Sung, Y. C., Hong, S. I. & Shin, example, etoposide as illustrated in Fig. 1. Considering the H. S. (1995) Oncogene 9, 1693–1698. Downloaded by guest on September 27, 2021 Immunology: Perera et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14313

21. Karsan, A., Yee, E., Kaushansky, K. & Harlan, J. M. (1996) Blood 48. Chu, K., Niu, X. & Williams, L. T. (1995) Proc. Natl. Acad. Sci. 87, 3089–3096. USA 92, 11894–11898. 22. Kenny, J. J., Knobloch, T. J., Augustus, M., Carter, K. C., Rosen, 49. Allen, R. T., Cluck, M. W. & Agrawal, D. K. (1998) Cell. Mol. Life C. A. & Lang, J. C. (1997) Oncogene 14, 997–1001. Sci. 54, 427–445. 23. Naylor, M. S., Relf, M. & Balkwill, F. R. (1995) in Cytokines: A 50. Cohen, G. M. (1997) Biochem. J. 326, 1–16. Practical Approach, ed. Balkwill, F. R. (Oxford Univ. Press, 51. Berke, G. (1995) Cell 81, 9–12. Oxford), pp. 35–56. 52. Humke, E. W., Ni, J. & Dixit, V. M. (1998) J. Biol. Chem. 273, 24. Um, H.-D., Orenstein, J. M. & Wahl, S. M. (1996) J. Immunol. 15702–15707. 156, 3469–3477. 53. Chao, D. T. & Korsmeyer, S. J. (1998) Annu. Rev. Immunol. 16, 25. Kikuchi, H., Iizuka, R., Sugiyama, S., Gon, G., Mori, H., Arai, M., 395–419. Mizumoto, K. & Imajoh-Ohmi, S. (1996) J. Leukocyte Biol. 60, 54. Kroemer, G. (1997) Nat. Med. 3, 614–620. 778–783. 55. Tschopp, J., Thome, M., Hofmann, K. & Meinl, E. (1998) Curr. 26. Estaquier, J. & Ameisen, J. C. (1997) Blood 90, 1618–1625. Opin. Genet. Dev. 8, 82–87. 27. Munn, D. H., Beall, A. C., Song, D., Wrenn, R. W. & Throck- 56. Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., Cherton- morton, D. C. (1995) J. Exp. Med. 181, 127–136. Horvat, G., Farahani, R., McLean, M., Ikeda, J. E., MacKenzie, 28. Martin, S. J., Reutelingsperger, C. P., McGahon, A. J., Rader, A., et al. (1996) Nature (London) 379, 349–353. J. A., van Schie, R. C., LaFace, D. M. & Green, D. R. (1995) J. 57. Roy, N., Mahadevan, M. S., McLean, M., Shutler, G., Yaraghi, Z., Exp. Med. 182, 1545–1556. Farahani, R., Baird, S., Besner-Johnston, A., Lefebvre, C., Kang, 29. Onishi, Y., Azuma, Y., Sato, Y., Mizuno, Y., Tadakuma, T. & X., et al. (1995) Cell 81, 167–178. Kizaki, H. (1993) Biochim. Biophys. Acta 1175, 147–154. 58. Uren, A. G., Pakusch, M., Hawkins, C. J., Puls, K. L. & Vaux, 30. Yonehara, S., Ishii, A. & Yonehara, M. (1989) J. Exp. Med. 169, D. L. (1996) Proc. Natl. Acad. Sci. USA 93, 4974–4978. 1747–1756. 59. Rothe, M., Pan, M. G., Henzel, W. J., Ayres, T. M. & Goeddel, 31. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., D. V. (1995) Cell 83, 1243–1252. Sameshima, M., Hase, A., Seto, Y. & Nagata, S. (1991) Cell 66, 60. O’Bryan, M. K., Baker, H. W., Saunders, J. R., Kirszbaum, L., 233–243. Walker, I. D., Hudson, P., Liu, D. Y., Glew, M. D., d’Apice, A. J. 32. Tartaglia, L. A., Ayres, T. M., Wong, G. H. & Goeddel, D. V. & Murphy, B. F. (1990) J. Clin. Invest. 85, 1477–1486. 74, (1993) Cell 845–853. 61. Cheng, G., Cleary, A. M., Ye, Z. S., Hong, D. I., Lederman, S. 33. Bodmer, J. L., Burns, K., Schneider, P., Hofmann, K., Steiner, V., & Baltimore, D. (1995) Science 267, 1494–1498. Thome, M., Bornand, T., Hahne, M., Schroter, M., Becker, K., et 62. Gershenfeld, H. K., Hershberger, R. J., Shows, T. B. & Weiss- al. (1997) Immunity 6, 79–88. man, I. L. (1988) Proc. Natl. Acad. Sci. USA 85, 1184–1188. 34. Kitson, J., Raven, T., Jiang, Y. P., Goeddel, D. V., Giles, K. M., 63. Haddad, P., Clement, M. V., Bernard, O., Larsen, C. J., Degos, Pun, K. T., Grinham, C. J., Brown, R. & Farrow, S. N. (1996) L., Sasportes, M. & Mathieu-Mahul, D. (1990) Gene 87, 265–271. Nature (London) 384, 372–375. 64. Nakashima, T., Sekiguchi, T., Kuraoka, A., Fukushima, K., 35. Marsters, S. A., Sheridan, J. P., Donahue, C. J., Pitti, R. M., Gray, Shibata, Y., Komiyama, S. & Nishimoto, T. (1993) Mol. Cell. Biol. C. L., Goddard, A. D., Bauer, K. D. & Ashkenazi, A. (1996) Curr. 13, Biol. 6, 1669–1676. 6367–6374. 36. Yu, G. L., Lyons, R. H., Garg, M., Duan, D. R., Xing, L., Gentz, 65. Tian, Q., Taupin, J., Elledge, S., Robertson, M. & Anderson, P. R., Ni, J. & Dixit, V. M. (1996) Science 274, 990–992. (1995) J. Exp. Med. 182, 865–874. 37. Pan, G., O’Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., 66. Haddad, P., Jenne, D., Tschopp, J., Clement, M. V., Mathieu- Ni, J. & Dixit, V. M. (1997) Science 276, 111–113. Mahul, D. & Sasportes, M. (1991) Int. Immunol. 3, 57–66. 38. Pan, G., Ni, J., Wei, Y. F., Yu, G., Gentz, R. & Dixit, V. M. (1997) 67. Briehl, M. M. & Miesfeld, R. L. (1991) Mol. Endocrinol. 5, Science 277, 815–818. 1381–1388. 39. Schneider, P., Bodmer, J. L., Thome, M., Hofmann, K., Holler, 68. Venturelli, D., Martinez, R., Melotti, P., Casella, I., Peschle, C., N. & Tschopp, J. (1997) FEBS Lett. 416, 329–334. Cucco, C., Spampinato, G., Darzynkiewicz, Z. & Calabretta, B. 40. Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., (1995) Proc. Natl. Acad. Sci. USA 92, 7435–7439. Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, 69. Przetak, M. M., Yoast, S. & Schmidt, B. F. (1995) FEBS Lett. 364, K., Wood, W. I., et al. (1997) Science 277, 818–821. 268–271. 41. Kischkel, F. C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, 70. Gabig, T. G., Mantel, P. L., Rosli, R. & Crean, C. D. (1994) M., Krammer, P. H. & Peter, M. E. (1995) EMBO J. 14, J. Biol. Chem. 269, 29515–29519. 5579–5588. 71. Brinkmann, U., Brinkmann, E., Gallo, M. & Pastan, I. (1995) 42. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Proc. Natl. Acad. Sci. USA 92, 10427–10431. Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., 72. Shinkai, Y., Yoshida, M. C., Maeda, K., Kobata, T., Maruyama, Gentz, R., et al. (1996) Cell 85, 817–827. K., Yodoi, J., Yagita, H. & Okumura, K. (1989) Immunogenet. 30, 43. Hsu, H., Xiong, J. & Goeddel, D. V. (1995) Cell 81, 495–504. 452–457. 44. Stanger, B. Z., Leder, P., Lee, T. H., Kim, E. & Seed, B. (1995) 73. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, Cell 81, 513–523. E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J. L., Schroter, 45. Chinnaiyan, A. M., O’Rourke, K., Tewari, M. & Dixit, V. M. M., et al. (1997) Nature (London) 386, 517–521. (1995) Cell 81, 505–512. 74. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., 46. Alderson, M. R., Tough, T. W., Davis-Smith, T., Braddy, S., Falk, Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., B., Schooley, K. A., Goodwin, R. G., Smith, C. A., Ramsdell, F. et al. (1997) Nature (London) 388, 190–195. & Lynch, D. H. (1995) J. Exp. Med. 181, 71–77. 75. Ashany, D., Song, X., Lacy, E., Nikolic-Zugic, J., Friedman, S. M. 47. Sato, T., Irie, S., Kitada, S. & Reed, J. C. (1995) Science 268, & Elkon, K. B. (1995) Proc. Natl. Acad. Sci. USA 92, 11225– 411–415. 11229. Downloaded by guest on September 27, 2021