TRAF2 Deficiency Results in Hyperactivity of Certain TNFR1 Signals and Impairment of CD40-Mediated Responses

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provided by Elsevier - Publisher Connector Immunity, Vol. 11, 379–389, September, 1999, Copyright 1999 by Cell Press TRAF2 Deficiency Results in Hyperactivity of Certain TNFR1 Signals and Impairment of CD40-Mediated Responses

Linh T. Nguyen,* Gordon S. Duncan,† molecules to the cytoplasmic regions of the receptors Christine Mirtsos,† Michelle Ng,† (Nagata, 1997). One major class of proximal signal trans- Daniel E. Speiser,‡ Arda Shahinian,† duction molecules is the family of TNFR-associated fac- § † Michael W. Marino, Tak W. Mak,* k tors (TRAFs) (Rothe et al., 1994, 1995; Cheng et al., 1995; † Pamela S. Ohashi,*k and Wen-Chen Yeh k# Regnier et al., 1995; Cao et al., 1996; Ishida et al., 1996a, *Department of Immunology 1996b). TRAF proteins are characterized by a conserved University of Toronto TRAF-C domain, a coiled-coil TRAF-N domain and, ex- Toronto, Ontario cept for TRAF1, an N-terminal ring finger domain (Rothe Canada M5G 2M9 et al., 1994). The TRAF-C domain mediates TRAF associ- † Amgen Institute ation with members of the TNFR superfamily as well as Ontario Cancer Institute interactions between TRAF proteins, while the ring finger Toronto, Ontario domain is implicated in downstream signaling events Canada M5G 2C1 (Rothe et al., 1995; Cao et al., 1996; Takeuchi et al., ‡ Division of Clinical Onco-Immunology 1996). Ludwig Institute for Cancer Research TRAF1 and TRAF2 are prototypical members of the Lausanne Branch TRAF family proteins. They were originally purified from Centre Hospitalier Universitaire de Lausanne a protein complex associated with TNFR2. It has been CH-1011 Lausanne previously shown that TRAF2, but not TRAF1, can trans- Switzerland duce signals required for TNF-mediated activation of § Ludwig Institute for Cancer Research the transcription factor NF-␬B (Rothe et al., 1995) and New York Branch at Memorial the stress-activated protein kinase (SAPK/JNK) (Liu et Sloan-Kettering Cancer Center al., 1996; Natoli et al., 1997). Whereas TRAF2 interacts New York, New York 10021 directly with TNFR2 (Rothe et al., 1994), TRAF2 is re- Department of Medical Biophysics k cruited to TNFR1 through its interaction with an adaptor University of Toronto protein called TRADD (TNFR1-associated death domain Ontario Cancer Institute protein) (Hsu et al., 1995, 1996). Toronto, Ontario Unlike its complex and indirect interaction with Canada M5G 2M9 TNFR1, TRAF2 can interact directly with other members of the TNF receptor superfamily that mediate important immune functions, including CD40, CD30, CD27, 4-1BB, Summary and RANK (receptor activator of NF-␬B) (for review, see Tumor necrosis factor (TNF) receptor–associated fac- Arch et al., 1998). However, these receptors typically tor 2 (TRAF2) can interact with various members of recruit additional TRAF proteins also capable of mediat- the TNF receptor family. Previously, we reported that ing intracellular signaling. CD40, for example, can asso- TRAF2-deficient mice die prematurely and have ele- ciate with TRAF2, 3, 5, and 6 (Cheng et al., 1995; Rothe vated serum TNF levels. In this study, we demonstrate et al., 1995; Sato et al., 1995; Ishida et al., 1996a, 1996b; that TRAF2-deficient macrophages produce increased Tewari and Dixit, 1996). CD40 is expressed on all anti- amounts of nitric oxide (NO) and TNF in response to gen-presenting cell (APC) types, including B cells, den- TNF stimulation. Furthermore, we could enhance the dritic cells, and macrophages. Engagement of CD40 survival of TRAF2-deficient mice by eliminating either with CD40 ligand (CD40L) has been shown to be impor- TNF or TNFR1. Using these double-knockout mice, we tant for B and T cell interactions that contribute to B cell show that in the absence of TRAF2, the T helper– activation, differentiation, and immunoglobulin isotype dependent antibody response, CD40-mediated prolif- class switching (Noelle et al., 1992; Laman et al., 1996). eration, and NF-␬B activation are defective. These Recent data suggest that CD40-CD40L interactions are data demonstrate two important roles of TRAF2, one also essential for the association of dendritic cells and as a negative regulator of certain TNFR1 signals and T cells and thus the initiation of T-dependent immune the other as a positive mediator of CD40 signaling. responses (Noelle, 1996; Grewal and Flavell, 1998). Ex vivo experiments have shown that TRAF2, TRAF5, and Introduction TRAF6 all have positive effects on CD40-induced activation of NF-␬B and JNK/SAPK (Rothe et al., 1995; Sato Members of the tumor necrosis factor receptor (TNFR) et al., 1995; Ishida et al., 1996a, 1996b; Lee et al., 1999; superfamily play important roles in cell death, bone and Tsukamoto et al., 1999). However, the precise contribu- fat metabolism, and immune function. Stimulation of a tion of each TRAF protein to CD40 signaling in vivo TNFR-related receptor results in receptor aggregation remains to be resolved. and the recruitment of intracellular signal transduction In this study, we have extended our analysis of TRAF2- deficient animals to determine the physiological role # To whom correspondence should be addressed (e-mail: wen-chen. of TRAF2 in the signaling of two receptor complexes, [email protected]). TNFR1 and CD40. First, we investigated whether TRAF2 Immunity 380

Figure 1. TNF Stimulates the Overproduction of Inflammatory Mediators by TRAF2-Defi- cient Cells (A–B) Thioglycollate-elicited peritoneal macrophages were isolated from wild-type and traf2Ϫ/Ϫ mice of the C57BL/6 ϫ BALB/c (F1) background. Macrophages were incubated for 24 hr with either mIFN␥ (50 U/ml) plus the indicated concentrations of exogenous mTNF (A) or with the indicated concentrations of LPS (B). Culture supernatants were assayed for nitrite production. Data shown here are the representative of two to three mice per genotype, and at least three independent experiments were performed. (C) Northern blot analysis of iNOS and TNF mRNA. Wild- type and traf2Ϫ/Ϫ inflammatory peritoneal macrophages were left untreated or treated with LPS (5 ␮g/ml) or mIFN␥ (50 U/ml) plus mTNF (10 ng/ml) for 18 hr. RNA blots were hybridized with 32P-labeled iNOS- and TNF- specific cDNA probes. Equal loading of RNA was demonstrated by staining with ethidium bromide. (D) Unstimulated resident macrophages isolated by peritoneal lavage of wild- type and traf2Ϫ/Ϫ mice of the C57BL/6 ϫ CD1 (F3) background were counted and analyzed by flow cytometry. Equal numbers of Mac-1 and F4/80 double-positive macrophages were plated with mIFN␥ (50 U/ml) plus the indicated concentrations of exogenous mTNF for 72 hr, and nitrite production was quantitated. (E) traf2Ϫ/Ϫ resident peritoneal macrophages were treated with IFN␥ (50 U/ml) in the absence or presence of antagonistic antibodies (2 ␮g each per well) against TNFR1 and TNFR2 for 48 hr, and nitrite production was quantitated. (F) Wild- type and traf2Ϫ/Ϫ EF cells were treated with various concentrations of TNF for 18 hr. Culture supernatants were then assayed for nitrite production as described.

deficiency disrupts the delicate balance of TNFR1 sig- background (C57BL/6 ϫ BALB/c [Yeh et al., 1997] or naling pathways in vivo. We demonstrate that the ab- C57BL/6 ϫ CD1 [data not shown]). We therefore utilized sence of TRAF2 causes defective TNF signaling, by the healthier mutant animals of mixed genetic back- showing that TRAF2-deficient macrophages overpro- grounds and their littermate controls for these experi- duce TNF and its downstream inflammatory mediator, ments. Surprisingly, thioglycollate-elicited macrophages nitric oxide (NO), specifically in response to TNF stimula- isolated from traf2Ϫ/Ϫ mice of both the C57BL/6 ϫ tion. Furthermore, the elimination of TNF signaling in BALB/c (Figure 1A) and C57BL/6 ϫ CD1 (data not vivo, achieved by generating double-knockout traf2Ϫ/Ϫ shown) genetic backgrounds produced greatly ele- tnfϪ/Ϫ or traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ mice, partially rescues the lethal vated levels of nitrite, the stable end product of nitric phenotype of TRAF2-deficient animals. These double- oxide (NO) production, in response to stimulation with knockout mice survive long term, free of many of the TNF plus interferon-␥ (IFN␥). Overproduction of NO by abnormalities observed in TRAF2 single-knockout ani- traf2Ϫ/Ϫ macrophages was specific to TNF stimulation, mals. With respect to the physiological role of TRAF2 as NO levels increased proportionately with the concen- in CD40 signaling, immunoglobulin isotype switching in tration of exogenous TNF, while lipopolysaccharide response to virus infection is defective in the absence (LPS) stimulation of activated traf2Ϫ/Ϫ macrophages re- of TRAF2, and CD40-mediated proliferation and NF-␬B sulted in normal or slightly reduced levels of NO produc- activation are impaired in mutant cells. Our results define tion (Figure 1B). two distinct and apparently opposing physiological Increased NO production in the absence of TRAF2 roles for TRAF2: negative regulation of certain signals was confirmed by the levels of inducible NO synthase downstream of TNFR1 and positive regulation of signal- (iNOS) mRNA in macrophages 18 hr after treatment with ing downstream of CD40. TNF plus IFN␥. The expression of iNOS mRNA in wild- type macrophages had returned to the level of that in Results unstimulated cells, while it was still strongly elevated in traf2Ϫ/Ϫ macrophages, consistent with their NO overpro- TRAF2-Deficient Macrophages Overproduce NO duction (Figure 1C). We also measured TNF mRNA 18 and TNF in Response to TNF Stimulation hr after stimulation of wild-type or traf2Ϫ/Ϫ macrophages. TNF plays an important role in the initiation and progres- In comparison to the wild-type, TNF production in re- sion of inflammatory responses. To investigate the role sponse to TNF stimulation was greatly amplified in the of TRAF2 in TNF-mediated responses, we evaluated the absence of TRAF2 (Figure 1C). ability of traf2Ϫ/Ϫ macrophages to produce inflammatory To rule out the in vivo effect of thioglycollate on mediators in response to TNF stimulation. The frequency TRAF2-deficient cells, equalized numbers of unstimu- of viable traf2Ϫ/Ϫ animals increases with a mixed genetic lated, resident peritoneal macrophages isolated from Role of TRAF2 in TNFR1 and CD40 Signaling 381

Figure 2. Elimination of TNF or TNFR1 Res- cues Growth Defects in Survivor traf2Ϫ/Ϫ Mice day-old traf2Ϫ/Ϫ-9ف A) A representative runted) mouse (middle) is shown with two wild-type littermates (top and bottom). (B) Elimination of TNF restores growth of traf2Ϫ/Ϫ mice in a dose-dependent manner. The adult traf2Ϫ/Ϫ tnfϪ/Ϫ mouse (second from the left) is comparable in size to its littermates, while the traf2Ϫ/Ϫ tnfϩ/Ϫ mouse (second from the right) is runted. (C) Elimination of TNFR1 also restores growth of traf2Ϫ/Ϫ mice. traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ mice (left) are comparable in size to their littermates (right). traf2Ϫ/Ϫ animals and their littermate controls (C57BL/6 ϫ 2B). To increase the probability of obtaining traf2Ϫ/Ϫ tnfϪ/Ϫ CD1) were utilized for experiments. As shown in Figure double-knockout mice, traf2ϩ/Ϫ tnfϩ/Ϫ females were 1D, naive TRAF2-deficient cells produced increased mated to traf2ϩ/Ϫ tnfϪ/Ϫ males. As shown in Table 1, the amounts of NO proportional to TNF stimulation. In con- rescue of TRAF2-deficient mice by mutation of TNF was trast to elicited macrophages, both wild-type and mutant incomplete since the percentage of survivors was below resident macrophages produced negligible amounts of that expected by the Mendelian ratio (7.8% versus the NO in response to LPS alone (data not shown). In addi- expected 12.5%). The partial rescue of TRAF2-deficient tion, the significant amount of NO produced by traf2Ϫ/Ϫ mice from premature lethality by elimination of TNF sug- macrophages in response to IFN␥ alone is likely due to gests that the regulation of TNF levels is critical during a small amount of endogenous TNF secreted by these embryogenesis and postnatal survival. mutant cells. As shown in Figure 1E, the combination To further investigate the mechanism of TNF-medi- of antagonist antibodies against TNF receptors blocked ated toxicity in TRAF2-deficient mice, we generated the NO production in cultures treated with IFN␥. Indeed, TRAF2/TNFR1 double-knockout animals. Elimination of ELISA measurements of traf2Ϫ/Ϫ culture supernatants TNFR1 in TRAF2-deficient mice significantly enhanced treated with IFN␥ alone revealed levels of 0.2 to 0.5 ng/ the viability of these animals (Table 1). In addition to ml TNF. Wild-type macrophage cultures also produced increasing viability, complete elimination of either TNF a similar amount of endogenous TNF in the absence of or TNFR1 in TRAF2-deficient mice restored postnatal any exogenous TNF. growth close to normal levels (Figures 2B and 2C), in Finally, we examined NO production by TRAF2-defi- sharp contrast to the severe wasting observed in TRAF2- cient embryonic fibroblasts (EF). As shown in Figure 1F, deficient survivors from the C57BL/6 ϫ 129J back- traf2Ϫ/Ϫ EF cells produced high levels of NO in response ground (Figure 2A). The growth of TRAF2/TNF double- to TNF stimulation, while under our assay conditions knockout mice was rescued to a slightly greater extent wild-type EF cells showed very little or no NO produc- (90%–95% of wild-type body weight) than that of TRAF2/ tion. Taken together, these results further strengthened TNFR1 double-knockout mice (75%–90% of wild-type the notion that TRAF2-deficient cells are indeed hyper- body weight). This discrepancy suggests that, in TRAF2- responsive to TNF. deficient mice, TNF may be exerting minor cytotoxic effects through TNFR2 in addition to TNFR1. Interest- TRAF2-Deficient Mice Are Partially Rescued ingly, the survival of TRAF2/TNFR1 double-knockout from Premature Lethality by Deficiency pups was influenced by the maternal genotype for for TNF or TNFR1 TNFR1. A tnfr1Ϫ/Ϫ maternal genotype correlated with a The observation that the absence of TRAF2 resulted in higher percentage of surviving traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ pups an increase in the basal serum TNF level and significant (closer to the Mendelian ratio) than a tnfr1ϩ/Ϫ maternal overproduction of TNF by macrophages suggested that genotype (Table 1), suggesting that TNF signaling of the severe phenotype of TRAF2-deficient mice was due maternal origin might also have an effect on embryonic at least in part to TNF-mediated toxicity. This possibility survival. Together, these results indicate that the en- was examined by the generation of traf2Ϫ/Ϫ mice that hanced TNF production in TRAF2-deficient mice re- were also deficient in TNF. When the progeny of parent duces viability and survival and that these effects are mice that were both compound heterozygotes (traf2ϩ/Ϫ largely mediated through TNFR1. tnfϩ/Ϫ) were analyzed, no traf2Ϫ/Ϫ tnfϩ/ϩ progeny survived at the time of weaning (3 weeks old), a result reproduced for 10 litters (data not shown). However, a few traf2Ϫ/Ϫ TNF-Induced NO and IL-12 Production Require TNFR1 tnfϪ/Ϫ pups in these litters were rescued from premature and Are Negatively Modulated by TRAF2 lethality and survived beyond 3 weeks of age (compare We further examined the role of TRAF2 in macrophage Figures 2A and 2B). In addition, consistent with the gene production of inflammatory mediators using TRAF2/TNF dosage effect observed in TNF mutant animals (Marino and TRAF2/TNFR1 double-knockout animals. Elicited et al., 1997), a few traf2Ϫ/Ϫ tnfϩ/Ϫ mice in these litters peritoneal macrophages isolated from traf2Ϫ/Ϫ tnfϪ/Ϫ ani- were also viable. However, the traf2Ϫ/Ϫ tnfϩ/Ϫ mice were mals overproduced NO in response to increasing con- not as healthy as their traf2Ϫ/Ϫ tnfϪ/Ϫ littermates, as they centrations of exogenous murine TNF plus IFN␥ (Figure were severely runted and also died prematurely (Figure 3A), recapitulating the phenotype observed in cells from Immunity 382

Table 1. Elimination of TNF or TNFR1 Rescues Viability of traf2Ϫ/Ϫ Mice Parents: Male: traf2ϩ/Ϫ tnfϪ/Ϫ x Female: traf2ϩ/Ϫ tnfϩ/Ϫ traf2 genotypes ϩ/ϩϩ/ϪϪ/Ϫ Total % traf2Ϫ/Ϫ of Total tnf genotypes ϩ/Ϫ 11 22 7 6.8% 103 tnf genotypes Ϫ/Ϫ 18 37 8 7.8% Parents: Male: traf2ϩ/Ϫ tnfr1Ϫ/Ϫ x Female: traf2ϩ/Ϫ tnfr1ϩ/Ϫ traf2 genotypes ϩ/ϩϩ/ϪϪ/Ϫ Total % traf2Ϫ/Ϫ of Total tnfr1 genotypes ϩ/Ϫ 32 63 0 0% 196 tnfr1 genotypes Ϫ/Ϫ 36 56 9 4.6% Parents: Male: traf2ϩ/Ϫ tnfr1Ϫ/Ϫ x Female: traf2ϩ/Ϫ tnfr1Ϫ/Ϫ traf2 genotypes ϩ/ϩϩ/ϪϪ/Ϫ Total % traf2Ϫ/Ϫ of Total tnfr1 genotypes Ϫ/Ϫ 13 33 9 55 16.4% Data are grouped based on maternal TNFR1 genotype. Percentages indicate the proportion of traf2Ϫ/Ϫ pups from all litters. Only pups that were viable at the time of weaning (3 weeks old) were enumerated.

TRAF2 single-knockout mice (Figures 1A and 1D). Simi- dosage effect of TRAF2 or hypersensitivity of TNF-defi- lar results were observed when unstimulated resident cient cells to exogenous TNF. peritoneal macrophages from traf2Ϫ/Ϫ tnfϪ/Ϫ animals were utilized (data not shown). In contrast, cells from Absence of TNFR1 Rescues Phenotypic Abnormalities neither traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ nor traf2ϩ/Ϫ tnfr1Ϫ/Ϫ animals were Observed in TRAF2-Deficient Animals able to produce NO in response to exogenous TNF plus Following the observation that partial rescue of pre- IFN␥ stimulation (Figure 3B). Consistent with a previous mature lethality resulted from the elimination of TNF report (Endres et al., 1997), TNFR1 appears to be the or TNFR1 in TRAF2-deficient mice, we investigated primary mediator of TNF-induced NO production since whether the elimination of TNFR1 rescued other abnor- all cells lacking TNFR1 failed to respond to TNF. In addi- malities observed in TRAF2-deficient mice. Thymocytes tion, we measured IL-12 production by elicited macro- from traf2Ϫ/Ϫ tnfr1ϩ/ϩ mice exhibit exaggerated apo- phages and found that this cytokine was also secreted ptosis in response to TNF stimulation (Yeh et al., 1997). at increased levels by traf2Ϫ/Ϫ tnfϪ/Ϫ cells in response to In contrast, both traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ and traf2ϩ/ϩ tnfr1Ϫ/Ϫ TNF treatment. In contrast, IL-12 secretion by traf2Ϫ/Ϫ thymocytes were resistant to TNF treatment (Figure 4A). tnfr1Ϫ/Ϫ and wild-type elicited macrophages were not Cells of all genotypes underwent dexamethasone- increased by stimulation with TNF (Figure 3C). Macro- induced apoptosis to a similar degree (Figure 4B), dem- phages from traf2ϩ/Ϫ tnfϪ/Ϫ mice produced moderately onstrating that the hypersensitivity of TRAF2-deficient elevated levels of NO and IL-12 at high concentrations cells to TNF is specifically mediated through TNFR1. We of TNF (Figures 3A and 3C), possibly due to a gene previously reported that traf2Ϫ/Ϫ animals have elevated

Figure 3. TNF-Induced NO and IL-12 Pro- duction Requires TNFR1 and Is Negatively Modulated by TRAF2 (A and B) Peritoneal exudate macrophages were isolated from traf2Ϫ/Ϫ tnfϪ/Ϫ (A) or traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ (B) mice as well as appropriate littermate controls. Macrophages were incubated for 24 hr with mIFN␥ (50 U/ml) plus the indicated concentrations of mTNF, and culture supernatants were assayed for nitrite production. (C) Culture supernatants from the inflammatory macrophages were assayed for IL-12p70 by ELISA. Data shown here are representative of two to three mice per genotype. Role of TRAF2 in TNFR1 and CD40 Signaling 383

Figure 4. Elimination of TNFR1 Rescues Vari- ous Phenotypes Associated with TRAF2 Defi- ciency (A) Rescue of traf2Ϫ/Ϫ CD4ϩCD8ϩ (DP) thymocyte hypersensitivity to TNF-induced apoptosis. Thymocytes were plated at 5 ϫ 105 cells per well (24-well plates) and incubated for 20 hr with medium alone or medium containing the indicated concentrations of mTNF (A) or dexamethasone (B). Cells were triple- stained with anti-CD8␣, anti-CD4, and 7-AAD. Viable DP cells (7-AAD-negative) from indi- vidual treatments were plotted as percentages of the total viable DP cells in the treated cultures compared to the untreated controls of the respective genotypes. (C) Absence of TNFR1 in traf2Ϫ/Ϫ mice restores normal basal serum TNF levels. Sera from different animals of indicated genotypes were taken for analyses. (D) Bone marrow cells harvested from adult traf2ϩ/ϩ tnfr1Ϫ/Ϫ (TNFR1-KO control), traf2ϩ/ϩ tnfr1ϩ/Ϫ (WT control), and traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ mice were analyzed by flow cytometry. The numbers shown in each region represent the percentage of total cells.

basal serum TNF levels. Analysis of serum TNF levels TRAF2 Deficiency Results in Severely Impaired in TRAF2/TNFR1 double-knockout mice demonstrated VSV-Induced Isotype Switching a complete rescue of this phenotype, with a striking We next investigated the role of TRAF2 in regulating reduction in serum TNF to normal undetectable levels immunity to pathogens by evaluating the production of (Figure 4C). The partial rescue of the lethal phenotype neutralizing antibodies in response to vesicular stomati- of TRAF2-deficient mice by the elimination of TNF or tis virus (VSV) infection. Infection of wild-type mice with TNFR1, together with the restoration of normal serum VSV results in T-independent production of virus-spe- TNF levels, suggests that TNFR1 signals are involved in cific IgM, followed by T cell–dependent isotype switch- regulating TNF levels, and that TRAF2 plays a negative ing to IgG2a and IgG2b (Oxenius et al., 1996). Double- regulatory role in this pathway. knockout mice lacking TRAF2 and either TNFR1 or TNF We previously reported that the absence of TRAF2 were used as the experimental animals for these studies, also results in a reduction of B220ϩ IgMϪ CD43Ϫ and since these mutants, unlike TRAF2 single-knockouts, B220ϩ IgMϩ B cell precursors in the bone marrow. This are viable. Normal VSV-specific IgM and IgG responses depletion could either be due to impaired early B cell were mounted in traf2ϩ/Ϫ tnfϪ/Ϫ mice. In contrast, traf2Ϫ/Ϫ development or could occur as a secondary effect of tnfϪ/Ϫ mice mounted normal IgM responses but had se- TNF toxicity. Elimination of TNFR1 in TRAF2-deficient verely impaired IgG responses (about 64-fold lower) mice restored the number of B cell precursors to normal (Figure 5A). Similar results were obtained when traf2Ϫ/Ϫ levels (Figure 4D). This suggests that the decrease in B tnfr1Ϫ/Ϫ mice were compared to traf2ϩ/Ϫ tnfr1Ϫ/Ϫ mice cell precursors observed in traf2Ϫ/Ϫ animals is most likely (Figure 5B). Immunoglobulin (Ig) isotype switching has a secondary effect of TNF toxicity. Collectively, these been shown to depend on collaboration between type results demonstrate that abrogation of TNFR1 signaling 1 T helper cells and B cells (Zinkernagel et al., 1990). prevents the appearance of phenotypes seen in TRAF2- Defective isotype switching observed in the absence of deficient mice. TRAF2 therefore implicates this protein in the positive Immunity 384

Figure 6. TRAF2 Is Required for CD40-Induced Proliferation and NF-␬B Activation in Splenocytes (A) Splenocytes were plated at 1 ϫ 105 cells/well and left untreated or treated with anti-CD40 mAb (10 ␮g/ml) or LPS (10 ␮g/ml) for 2 days. Plates were then pulsed with [3H]-thymidine for 14 hr. Each bar represents the mean of quadruplicate samples. (B) Gel mobility Figure 5. TRAF2 Is Required for Virus-Induced Neutralizing Anti- shift assays were performed on splenocytes that were left untreated body Responses or treated with anti-CD40 mAb (10 ␮g/ml) for the indicated time- 5 (A and B) Mice were immunized with 2 ϫ 10 PFU VSV-IND intrave- points. Nuclear extracts were prepared as described in the Experi- nously. Sera were collected after 4 and 8 days and assayed for mental Procedures, incubated with an oligonucleotide probe spe- neutralizing IgM and IgG titers as described in Experimental Proce- cific for NF-␬B, fractionated on a 5% polyacrylamide gel, and dures. Each bar represents the mean of the sera from two mice. visualized by autoradiography. The position of activated NF-␬Bis The experiment in (A) using TRAF2/TNF double-knockout mice was indicated. done once. The experiment in (B) using TRAF2/TNFR1 double- knockout mice was repeated twice. these double-knockout mutant cells proliferated nor- regulation of signaling events that are important in either mally upon LPS stimulation. Furthermore, the prolifera- the activation of T helper cells and/or in the collaboration tion of traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ splenocytes in response to vari- between T and B cells. ous T cell stimuli was normal (data not shown). A critical event downstream of CD40 cross-linking Absence of CD40-Induced Proliferation and NF-␬B is the activation of the transcription factor NF-␬B. We Activation in TRAF2-Deficient Splenocytes examined the ability of traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ splenocytes to The failure to mount significant IgG responses to VSV activate NF-␬B in response to CD40 stimulation using infection has also been demonstrated in CD40L knock- a gel mobility shift assay. Elimination of TRAF2 resulted out mice (Oxenius et al., 1996). Recent biochemical stud- in complete abrogation of NF-␬B activation in response ies have shown that CD40 cross-linking recruits TRAF2, to CD40 cross-linking (Figure 6B). These data provide 3, and 5 directly to the C-terminal cytoplasmic domain of physiological evidence that TRAF2 is essential for trans- CD40 and TRAF6 to its N-terminal cytoplasmic domain ducing a positive signal triggered by CD40 ligation. (Pullen et al., 1998; Lee et al., 1999; Tsukamoto et al., 1999). To distinguish the function of TRAF2 from the Discussion roles played by other TRAF proteins in the CD40 signaling pathway, we investigated the responses of TRAF2- Biochemical studies have linked TRAF2 to the signaling deficient cells to CD40 cross-linking in vitro. Total spleno- complexes of many TNF receptor family members. cytes were isolated and evaluated for their responses These receptors include TNFR1, where a complex to CD40 cross-linking and LPS, agents known to stimu- signaling path involves interactions between TRADD, late B cells. In contrast to cells from traf2ϩ/ϩ tnfr1ϩ/Ϫ or TRAF2, and receptor interacting protein (RIP). In addi- traf2ϩ/ϩ tnfr1Ϫ/Ϫ mice, the B cell proliferative response tion, TRAF2, among other TRAFs, can bind directly to induced by CD40 cross-linking was completely abro- CD40. From studies utilizing in vitro or overexpression gated in traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ splenocytes (Figure 6A). This systems, TRAF2 has been reported to play a positive role defect was specific for CD40-induced proliferation, as in mediating signaling from both receptors. Specifically, Role of TRAF2 in TNFR1 and CD40 Signaling 385

TRAF2 was implicated as the principal transducer of of the many molecules downstream of TRAF2 that serve receptor-mediated signals leading to the activation of to execute its effects. NF-␬B and stress-activated kinases (JNK/SAPK) (Liu et al., 1996). In this report, we provide in vivo evidence that TRAF2 is indeed required for CD40 signaling. However, Regulation of the Production of Inflammatory the loss of TRAF2 results in not decreased but excessive Mediators by TRAF2 TNF signaling mediated primarily through TNFR1. This Macrophages produce various inflammatory cytokines sharp contrast illustrates not only the disparate nature and downstream mediators in response to activation by of CD40 and TNFR1 signaling but also suggests a novel a number of stimuli. Many of these stimuli, including molecular mechanism of how TRAF2 might modulate TNF and LPS, trigger signaling events resulting in the TNF responses. activation of the transcription factor NF-␬B, which is Several mechanisms have been proposed for main- required to transactivate the expression of many proin- taining a physiological equilibrium of TNF signals in vivo. flammatory cytokine genes as well as iNOS (Nussler and First, the action of TNF may be regulated by stimuli- Billiar, 1993; Baeuerle and Henkel, 1994). In this report, triggered metalloprotease-dependent cleavage of mem- we show that TRAF2-deficient macrophages produce brane-bound TNF receptors. This cleavage not only increased amounts of nitric oxide and TNF specifically clears TNF receptors from the membrane but also gener- in response to exogenous TNF treatment (Figures 1A, ates a pool of soluble receptors that competitively bind 1C, and 1D). Such increases seem to occur at the mRNA to and sequester the ligand (TNF). Failure of this receptor level, possibly due to either enhanced inos and tnf gene shedding would result in a reduced amount of soluble transcription or a reduced rate of mRNA degradation in TNF receptors in the circulation and the retention of cells lacking TRAF2. Since TNF-induced NF-␬B activity functional receptors on the cell membrane. Recently, it was previously found to be normal or only slightly de- was reported that mutations in the TNFR1 extracellular creased in TRAF2-deficient cells (Yeh et al., 1997), it is domain that impair receptor shedding are linked to in- likely that an as yet undefined TRAF2-dependent but herited autoinflammatory syndromes in humans (Mc- NF-␬B-independent pathway is responsible for the TNF- Dermott et al., 1999). While this phenomenon presents induced increase in inflammatory mediator production. a novel mechanism for dysregulated TNF signaling, it is Our results also show that the dysregulated produc- unlikely that TRAF2 deficiency results in impaired TNF tion of NO and TNF in traf2Ϫ/Ϫ macrophages is specifi- receptor shedding. Indeed, levels of soluble TNFR1 and cally dependent on stimulation by TNF. While endoge- TNFR2 in sera of TRAF2-deficient animals are not re- nous TNF plays a role in increased NO production by duced compared to wild-type animals (L. N. and W. C. Y., traf2Ϫ/Ϫ macrophages (Figures 1A, 1D, and 1E), traf2Ϫ/Ϫ unpublished data). tnfϪ/Ϫ macrophages also exhibit a hyperactivity in re- A second mechanism to downregulate TNF signaling sponse to exogenous TNF (Figure 3A). Moreover, elimi- involves the intracellular protein SODD (silencer of death nation of TNFR1 in macrophages completely abrogated domains), which can bind to the cytoplasmic domain their proinflammatory responses to TNF and IFN␥ (Fig- of TNFR1 and prevent the recruitment of TRADD and ure 3B). In addition, we have demonstrated that TRAF2 consequently other signaling proteins (Jiang et al., plays a negative regulatory role in TNF-induced NO pro- 1999). In this scenario, SODD could inhibit ligand-inde- duction not only in macrophages but also in another pendent self-association of TNFR1 molecules in resting cell type such as embryonic fibroblasts (Figure 1F). Stim- cells and could also participate in quenching TNFR1 ulation of TRAF2-deficient macrophages with LPS ap- signaling shortly after receptor stimulation. It remains peared to result in reduced NO production compared unclear how the absence of TRAF2 might alter the inter- to wild-type cells (Figure 1B), suggesting that optimal action between SODD and TNFR1 and thus the TNFR1 LPS signaling in macrophages requires TRAF2. How- signaling complex. ever, we also observed that TRAF2-deficient spleno- A third proposed mechanism postulates that stimula- cytes proliferated normally in response to LPS stimulation by TNF can induce a negative feedback loop in a tion. Further experiments are required to address the cell such that it downregulates endogenous TNF synthe- possibility of a tissue-specific dependence on TRAF2 sis. It was recently discovered that TNF treatment in- in LPS signaling. duces the expression of tristetraprolin (TTP), a molecule that can bind to AU-rich elements (ARE) in the 3Ј-untrans- lated region of TNF mRNA and induce its destabilization The Role of TRAF2 in TNFR1 Signaling (Carballo et al., 1998). Such a negative feedback loop We have discussed above experimental results that re- becomes very complicated, once one considers the veal an unconventional role for TRAF2 as a negative number of components that could potentially be regu- regulator of TNFR1 signaling. However, TRAF2 may lated. These would include elements in the signal trans- carry out this negative regulatory function by serving duction cascades for both TNF receptors, the transcrip- as a positive mediator in TNF-induced pathways that tion factor complex that binds to regulatory elements antagonize the proapoptotic and other detrimental ef- of the tnf gene, and the protein complex (including TTP) fects of TNFR1 signaling. As mentioned above, stimula- that affects the stability of TNF mRNA. The potential tion of TNFR1 can simultaneously activate pathways involvement of TRAF2 in a feedback loop controlling leading to apoptosis and antiapoptosis. Proximal signal- TNF synthesis is an intriguing possibility, particularly ing of TNF-mediated apoptosis involves the interaction since TRAF2 deficiency results in increased levels of of TRADD and FADD, while the cytoprotective pathway TNF mRNA and circulating TNF. Further support for this depends on a complex composed of TRADD, RIP, and model will require the identification and characterization TRAF2. In the absence of TRAF2, TNF signaling through Immunity 386

TNFR1 may be dominated by the TRADD-FADD interac- utilizes multiple TRAF proteins for signaling. While the tion, which leads to apoptosis. This suggests that there exact mechanism of signaling is unclear, CD40 appar- exists an important balance of TNF signaling in normal ently recruits TRAF2, 3, and 5 to the C-terminal cyto- cells and that TRAF2 may be essential for mediating a plasmic tail of the receptor and TRAF6 to an N-terminal signaling pathway that antagonizes TNF-induced apo- domain of the cytoplasmic tail. From biochemical and ptosis. NF-␬B activation has been implicated as a key protein overexpression studies, both parts of the CD40 event in mediating cell survival. However, we have shown cytoplasmic region as well as TRAF2, TRAF5, and that TRAF2 plays only a minor role in TNF-induced TRAF6 are thought to mediate CD40-induced NF-␬B NF-␬B activation (Yeh et al., 1997). More relevant may activation. Using human CD40 constructs with muta- be the observation (also reported by others) that TRAF2 tions at specific sites in the cytoplasmic domains, two is specifically required for TNF-induced SAPK/JNK acti- distinct pathways of CD40 signaling have been demon- vation (Lee et al., 1997; Yeh et al., 1997). TRAF2 can strated: one dependent on TRAF2 or TRAF5 and the also interact with cIAP (cellular inhibitor of apoptosis other dependent on TRAF6 (Tsukamoto et al., 1999). protein) family proteins, which have been recently pro- A report by Lee et al. (1999) implicated TRAF2 in the posed to bind directly to and inhibit terminal caspases upregulation of intercellular adhesion molecule-1 (ICAM-1) (Deveraux et al., 1997, 1998; Roy et al., 1997). Thus, induced by CD40 engagement. However, conflicting evi- there are several mediators downstream of TRAF2 with dence was reported by Lee et al. (1997), who used a the potential to contribute to TNF-induced, NF-␬B-inde- transgenic mouse strain expressing a dominant-nega- pendent cytoprotection. tive mutation of the TRAF2 protein to show increased It is also clear that TRAF2 can mediate pathways that proliferation of B cells and upregulation of ICAM-1, B7-1, play regulatory roles in nonapoptotic responses induced and CD23 in response to induction by CD40. via TNF and TNFR1. TRAF2-deficient mice exhibit a Using cells isolated from TRAF2-deficient mice, we cachexia-like phenotype possibly caused by elevated have demonstrated unequivocally and physiologically serum TNF levels. Strikingly, elimination of TNF or that TRAF2 is required for CD40-induced proliferation TNFR1 signaling in traf2Ϫ/Ϫ mice was able to rescue and NF-␬B activation. This result is further bolstered many of them from premature lethality. It has been dem- by our recent observation in TRAF6-deficient mice that onstrated that TNF can exert an anabolic effect through CD40 also requires TRAF6 for signaling (Lomaga et al., inhibition of insulin signaling and adipocyte develop- 1999). Gene-targeted disruption of TRAF6 completely ment (Hotamisligil et al., 1994, 1996; Argiles et al., 1997). abolished CD40-mediated NF-␬B activation. Taken to- However, a detailed molecular mechanism connecting gether, these data suggest that TRAF2 and TRAF6 are the TNF signal transduction machinery to the insulin both essential for CD40 signaling. It is possible that signaling cascade remains to be elucidated. It is possi- TRAF2 and TRAF6 reside in two separate pathways, ble that TRAF2 mediates signals keeping the cachectic neither of which is sufficient on its own to mediate CD40 as well as the proapoptotic effects of TNF in check, so signaling. Alternatively, TRAF2 might interact weakly that wild-type animals and cells are able to avoid TNF with TRAF6 via the TRAF domains, as has been pre- toxicity. viously suggested from studies using a yeast two-hybrid The rescue of traf2Ϫ/Ϫ animal viability by deletion of system (Cao et al., 1996). Intermolecular signals could TNF or TNFR1 was not complete. A number of traf2Ϫ/Ϫ conceivably be coordinated using the TRAF ring finger tnfϪ/Ϫ and traf2Ϫ/Ϫ tnfr1Ϫ/Ϫ animals died at the embryonic domains. Such a scheme might help reconcile the dis- stage. In contrast, almost all double-knockout mice that crepancy between our results and those obtained from survived embryogenesis achieved long-term survival the study of the TRAF2-dominant-negative transgenic (6–9 months). This finding suggests that signaling by animals. It is possible that, in the complete absence of molecules related to TNF or via receptors that can re- any form of TRAF2 (as in the TRAF2 knockout mouse), place TNFR1 may play a role in regulating embryonic the proposed TRAF2-TRAF6 complex required for CD40 survival in the absence of TRAF2. Lymphotoxin-␣ (LT␣) signal transduction simply cannot assemble. However, is a cytokine that can bind to TNFR1 (Ruddle, 1992), in the dominant-negative TRAF2 transgenic animal, while TNFR2 is the alternate receptor for TNF in traf2Ϫ/Ϫ wild-type TRAF2, TRAF6, and a mutant TRAF2 protein tnfr1Ϫ/Ϫ animals. Determination of the effects of these containing only the TRAF domain would all be present. molecules on embryonic survival awaits the generation A CD40 signaling complex could still assemble and, of triple-knockout mouse strains. Other candidates in- in fact, the TRAF2-TRAF6 interaction could be much clude death receptors such as death receptors 3 and 6 stronger than in wild-type cells due to locally increased (DR3 and DR6), which, like TNFR1, utilize TRADD to concentrations of the TRAF domain. Such a scenario initiate their signaling cascades (Ashkenazi and Dixit, could account for an increased CD40-induced response 1998; Pan et al., 1998). It is possible that some of the in the presence of dominant-negative TRAF2 mutant phenotypes of TRAF2-deficient mice result from disrup- molecules but a decreased response to CD40 in the tions of signaling mediated by these other receptors. complete absence of TRAF2. The phenotypes of the double-knockout mice empha- Our results also showed that TRAF2 has a role in the size the potential complexity of the signaling web sur- T-dependent B cell response, specifically in Ig-isotype rounding TRAF2. switching. TRAF2-deficient animals fail to mount IgG responses to VSV infection (even at time points up to The Role of TRAF2 in CD40 Signaling 18 days after infection; data not shown), and CD40 sig- CD40 belongs to the subset of TNF receptor superfamily naling was impaired in splenocytes in the absence of members that does not contain a death domain and TRAF2. These data are consistent with the phenotype Role of TRAF2 in TNFR1 and CD40 Signaling 387

of CD40L-deficient mice that mount normal T-indepen- Measurement of Serum TNF dent B cell responses to VSV but are defective in Ig- Whole blood was centrifuged in a Microtainer serum separator (Bec- isotype switching (Oxenius et al., 1996). If in fact the ton Dickinson) and the sera were stored at Ϫ70ЊC. Assay of serum TNF was performed in duplicate on serum samples diluted 1:2 or defect in Ig-isotype switching observed in TRAF2-defi- 1:4 using the mTNF␣ ELISA kit (Genzyme) according to the manufac- cient animals is due to an impairment of CD40 signaling, turer’s instructions. it implies that TRAF2 is involved in the activation of APC function (which in turn is required for activation of T VSV Infection and Neutralization Assay helper cells) and/or plays a direct role in Th-B collabora- Vesicular stomatitis virus, serotype Indiana (VSV-IND; Mudd-Sum- tion. Further studies are required to address the precise mers isolate), originally obtained from Lud Prevec (McMaster Univer- sity, Hamilton, Canada), was grown on BHK-21 cells (CRL 8544; role of TRAF2 in the T-dependent B cell response. Pres- ATCC) infected at low multiplicity and plaqued on Vero cells ently, we cannot rule out the involvement of other recep- (McCaren et al., 1959). For immunization of mice with VSV-IND, 2 ϫ tors utilizing TRAF2 signaling that might also contribute 105 PFU of live virus was injected i.v. in 200 ␮l of Balanced Salt to Ig-isotype switching. One such candidate is RANK, Solution. Neutralizing titers of sera were determined as described signaling through which has been implicated in dendritic (Roost et al., 1990). In brief, sera were prediluted 40-fold in MEM cell and activated T cell functions in addition to the supplemented with 2% FCS and then heat-inactivated for 30 min at 56ЊC. Serial 2-fold dilutions of heat-inactivated sera were mixed differentiation and activation of osteoclasts (Anderson with equal volumes of VSV preparations containing 500 PFU/ml. et al., 1997; Hsu et al., 1999; Kong et al., 1999). Further The mixture was incubated for 90 min at 37ЊC in an atmosphere examination of the RANK signaling pathway in TRAF2- containing 5% CO2. One hundred microliters of the serum-virus deficient animals will be required to clarify its involve- mixture was transferred onto Vero cell monolayers in 96-well plates, ment in the T-dependent B cell response. incubated for 60 min at 37ЊC, and then overlaid with 100 ml DMEM containing 1% methylcellulose. After incubation overnight at 37ЊC, the overlay was removed and the monolayer was fixed and stained Experimental Procedures with 0.5% crystal violet. The highest dilution of serum that reduced the number of plaques by 50% was taken as the neutralizing titer. Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Animal Husbandry and Generation of traf2 tnf and traf2 To determine IgG titers, undiluted serum was pretreated with an Ϫ/Ϫ tnfr1 Mice equal volume of 0.1 M 2-mercaptoethanol in saline as described Mice were housed in a specific-pathogen-free facility according to (Scott and Gershon, 1970). Titers represent log2 steps of 40-fold the ethical and institutional guidelines of the Ontario Cancer Insti- prediluted sera. tute. traf2Ϫ/Ϫ mice (Yeh et al., 1997) that had been backcrossed into the C57BL/6J background for at least four generations were Cellular Proliferation Assays Ϫ/Ϫ Ϫ/Ϫ crossbred to tnf (Marino et al., 1997) or tnfr1 (Pfeffer et al., 1 ϫ 105 viable nucleated splenocytes per well (96-well plates) were 1993) mice of similar genetic backgrounds. Genotypic analyses of cultured in quadruplicate in Iscove’s medium supplemented with the animals were performed as previously described (Pfeffer et al., 10% heat-inactivated fetal bovine serum (Sigma). Cells were un- 1993; Marino et al., 1997; Yeh et al., 1997). treated or treated with either rat IgG2a anti-mouse CD40 mAb (Phar- Mingen, 3/23, 10 ␮g/ml) or LPS (Difco, 10 ␮g/ml) for 2 days. Plates Nitric Oxide Production in Macrophage and Embryonic were pulsed with 1 ␮Ci [3H]-thymidine per well for 14 hr and har- Fibroblast Cultures vested onto glass fiber filters. [3H]-thymidine uptake was measured Elicited peritoneal macrophages (PEC MØ) were obtained from peri- using a scintillation counter (Topcount, Canberra Packard). toneal lavage of mice injected 5 days previously with 1.5 ml of 4% thioglycollate broth (Difco). Macrophages (2 ϫ 105) were incubated Gel Mobility Shift Assay with 1, 10, 100, or 1000 ng/ml LPS (Sigma) or 0.1, 1, 10, or 100 ng/ Splenocytes (2 ϫ 107), either untreated or stimulated with anti-mouse ml recombinant mTNF␣ plus 50 U/ml recombinant mIFN␥ (R&D CD40 mAb (10 ␮g/ml) for various times, were harvested for nuclear Systems) for 18 hr in 96-well flat-bottomed microtiter plates. In extracts that were then subjected to gel mobility shift assay for NF- experiments where unstimulated, resident macrophages were used, ␬B activity according to protocols previously described (Yeh et al., cells from peritoneal lavage were counted and analyzed by flow 1997). cytometry. Equal numbers (2.5 ϫ 104) of Mac-1 (PharMingen) and F4/80 (Caltag) double-positive macrophages were plated for treat- Acknowledgments ments for 72 hr. Antagonistic antibodies against TNFR1 (p55) and TNFR2 (p75) (2 ␮g each per well) were obtained from Genzyme. In This research is supported by the National Cancer Institute of Can- experiments where embryonic fibroblasts were used, 2 ϫ 105 cells ada with funds from the Terry Fox Run (#010028 [W.-C. Y.]), the were plated in 24-well plates for mTNF treatment for 18 hr. Superna- Medical Research Council (P. S. O.), and scholarships from Ontario tants were assayed for nitrite, the stable end product of NO, by the Graduate Scholarship and Natural Sciences and Engineering Re- Greiss method (Green et al., 1982). search Council of Canada (L. T. N.). We also thank Madeleine Bon- nard and Nobutaka Suzuki for helpful discussions, Betty Hum, Mi- Determination of IL-12p70 in Macrophage Cultures chael Bezuly, and Jennifer Tsang for technical support, Mary PEC MØ were obtained by peritoneal lavage 5 days after intraperito- Saunders for scientific editing, and Irene Ng for administrative neal injection of 1.5 ml 4% thioglycollate (Difco). 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