View metadata, citation and similar papers at core.ac.uk brought to you by CORE 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,² molecules to the cytoplasmic regions of the receptors Christine Mirtsos,² Michelle Ng,² (Nagata, 1997). One major class of proximal signal trans- Daniel E. Speiser,³ Arda Shahinian,² duction molecules is the family of TNFR-associated fac- § ² Michael W. Marino, Tak W. Mak,* k tors (TRAFs) (Rothe et al., 1994, 1995; Cheng et al., 1995; ² 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- ² 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., ³ 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-kB (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-kB) (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-kB 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 activa- tion of NF-kB 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 mac- rophages were isolated from wild-type and traf22/2 mice of the C57BL/6 3 BALB/c (F1) background. Macrophages were incubated for 24 hr with either mIFNg (50 U/ml) plus the indicated concentrations of exogenous mTNF (A) or with the indicated concentrations of LPS (B). Culture supernatants were as- sayed 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 traf22/2 inflammatory peritoneal macrophages were left untreated or treated with LPS (5 mg/ml) or mIFNg (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 macro- phages isolated by peritoneal lavage of wild- type and traf22/2 mice of the C57BL/6 3 CD1 (F3) background were counted and analyzed by flow cytometry. Equal numbers of Mac-1 and F4/80 double-positive macrophages were plated with mIFNg (50 U/ml) plus the indicated concentrations of exogenous mTNF for 72 hr, and nitrite production was quantitated. (E) traf22/2 resident peritoneal macrophages were treated with IFNg (50 U/ml) in the absence or presence of antagonistic antibodies (2 mg each per well) against TNFR1 and TNFR2 for 48 hr, and nitrite production was quantitated. (F) Wild- type and traf22/2 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 3 BALB/c [Yeh et al., 1997] or naling pathways in vivo. We demonstrate that the ab- C57BL/6 3 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 traf22/2 mice of both the C57BL/6 3 tion. Furthermore, the elimination of TNF signaling in BALB/c (Figure 1A) and C57BL/6 3 CD1 (data not vivo, achieved by generating double-knockout traf22/2 shown) genetic backgrounds produced greatly ele- tnf2/2 or traf22/2 tnfr12/2 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-g (IFNg). Overproduction of NO by abnormalities observed in TRAF2 single-knockout ani- traf22/2 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 traf22/2 macrophages re- of TRAF2, and CD40-mediated proliferation and NF-kB 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 IFNg. 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 traf22/2 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 traf22/2 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 traf22/2 macrophages to produce inflammatory To rule out the in vivo effect of thioglycollate on mediators in response to TNF stimulation.