TNF Receptor-Associated Factor 6 Is an Essential Mediator of CD40-Activated Proinflammatory Pathways in Monocytes and Macrophages This information is current as of September 26, 2021. Lata Mukundan, Gail A. Bishop, Kimberly Z. Head, Lihua Zhang, Larry M. Wahl and Jill Suttles J Immunol 2005; 174:1081-1090; ; doi: 10.4049/jimmunol.174.2.1081 http://www.jimmunol.org/content/174/2/1081 Downloaded from References This article cites 55 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/174/2/1081.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology TNF Receptor-Associated Factor 6 Is an Essential Mediator of CD40-Activated Proinflammatory Pathways in Monocytes and Macrophages1 Lata Mukundan,* Gail A. Bishop,† Kimberly Z. Head,* Lihua Zhang,* Larry M. Wahl,‡ and Jill Suttles2* The interaction between CD40 and its ligand, CD154, has been shown to play a role in the onset and maintenance of inflammatory disease. Contributing to this process is the ability of CD40 to signal monocyte and macrophage inflammatory cytokine production. We have shown that this event is dependent on Src family tyrosine kinase activity and the subsequent activation of ERK1/2. To address the role of TNFR-associated factor (TRAF) family members in facilitating this signaling pathway, we transfected a CD40-deficient macrophage cell line with wild-type human CD40, or with CD40 containing disrupted TRAF binding sites. Ligation Downloaded from of either wild-type CD40, or a CD40 mutant unable to bind TRAF2/3/5, resulted in the stimulation of inflammatory cytokine production. However, ligation of a CD40 mutant lacking a functional TRAF6 binding site did not initiate inflammatory cytokine production, and this mutant was found to be defective in CD40-mediated activation of ERK1/2, as well as I␬B kinase (IKK) and -NF-␬B. Likewise, introduction of a dominant-negative TRAF6 into a wild-type (CD40؉) macrophage cell line resulted in abro gation of CD40-mediated induction of inflammatory cytokine synthesis. Finally, treatment of monocytes with a cell-permeable peptide corresponding to the TRAF6-binding motif of CD40 inhibited CD40 activation of ERK1/2, IKK, and inflammatory http://www.jimmunol.org/ cytokine production. These data demonstrate that TRAF6 acts as a critical adapter of both the Src/ERK1/2 and IKK/NF-␬B proinflammatory signaling pathways in monocytes and macrophages. The Journal of Immunology, 2005, 174: 1081–1090. member of the TNFR superfamily, CD40 was initially signaling through CD40 on B and T lymphocytes, myeloid cells, characterized on B cells, where it was found to play a and nonprofessional APCs plays a central role in the initiation and A role in the stimulation of B cell proliferation, Ig isotype maintenance of immune responses, including those associated with switching, and the development of humoral memory (1). Subse- chronic inflammatory diseases. quently, CD40 was shown to be present on a variety of cell types, In monocytes and macrophages, CD40 signaling leads to secre- including other classical APCs such as macrophages/monocytes, tion of inflammatory cytokines, chemokines, matrix metallopro- by guest on September 26, 2021 and dendritic cells, as well as nonleukocyte, nonprofessional teinases, production of NO, enhanced survival, and induction of APCs, such as endothelial cells, vascular smooth muscle cells, and costimulatory molecules such as ICAM-1, LFA-3, B7-1, and B7-2 fibroblasts (2–6). Ligation of CD40 on these cell types leads to the (2, 6, 10, 11). T cells derived from CD154-deficient mice are im- expression of predominantly proinflammatory genes and the en- paired in their ability to induce macrophage effector function (12), hancement of costimulatory molecule and adhesion molecule ex- and, consequently, these mice are highly susceptible to intracellu- pression (7). Thus, CD40 signaling enhances inflammatory re- lar pathogens that would otherwise have been cleared by a pro- sponses by up-regulation of APC activity and by recruitment of ductive T cell-macrophage interaction (13). Blockade of CD154- nonleukocytes as players in inflammatory responses. Recently, CD40 signaling has been found to reduce the severity of disease in CD40 expression has also been demonstrated on subsets of T cells, murine models of collagen-induced arthritis, experimental allergic and data suggest that CD40 signaling in T cells may contribute to encephalomyelitis, and atherosclerosis (14–16). Despite the sig- T cell memory and autoimmune responsiveness (8, 9). Therefore, nificant contribution of monocyte/macrophage CD40 responses to inflammatory disease, the signaling pathway(s) engaged via CD40 ligation in these cell types has not been fully characterized. CD40 *Department of Microbiology and Immunology, University of Louisville School of does not contain cytoplasmic sequences with catalytic activity and Medicine, Louisville, KY 40292; †Departments of Microbiology and Internal Medi- has been shown to use adaptor proteins of the TNFR-associated cine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, IA 52242; 3 and ‡Immunopathology Section, National Institute of Dental and Craniofacial Re- factor (TRAF) family to mediate signaling events. Ligation of search, National Institutes of Health, Bethesda, MD 20892 CD40 causes its oligomerization, which leads to recruitment of Received for publication August 6, 2004. Accepted for publication November specific TRAF proteins. Six TRAF members have been identified 1, 2004. to date, all of which share a common stretch of amino acids at the The costs of publication of this article were defrayed in part by the payment of page C terminus designated as the TRAF domain. The TRAF domain is charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. further divided into two subregions. The C terminus of the TRAF domain, TRAF-C, mediates homo- and heterodimerization with 1 This work was supported by an Arthritis Foundation Biomedical Sciences Grant (to J.S.), an American Heart Association Predoctoral Fellowship (to L.M.), and, in part, by the Kentucky Research Challenge Trust Fund. G.A.B. received support from a Veterans Affairs Hospitals Career Award, and National Institutes of Health Grants 3 Abbreviations used in this paper: TRAF, TNFR-associated factor; CBA, cytometric AI28847, AI49993, and CA099997. bead array; CHO, Chinese hamster ovary; DNTRAF6, dominant-negative TRAF6 2 Address correspondence and reprint requests to Dr. Jill Suttles, Department of Mi- mutant; hCD40, human CD40; IKK, I␬B kinase; PP2, pyrazolopyrimidine 2; PTK, crobiology and Immunology, University of Louisville School of Medicine, 319 Abra- protein tyrosine kinase; TRAF6BP, TRAF6-binding peptide; TRANCE, TNF-related ham Flexner Way, Louisville, KY 40292. E-mail address: [email protected] activation-induced cytokine; TRANCE-R, TRANCE receptor. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 1082 ROLE OF TRAF6 IN MONOCYTE/MACROPHAGE CD40 SIGNALING other TRAF proteins and also to the receptor that recruits them. aspartic acid (D) mutation (shown in italics) that enhances affinity to The N-terminal region of this domain is a coiled coil structure that TRAF6 (24). is less conserved. With the exception of TRAF1, all TRAFs con- Cells and cell culture tain a ring finger and five to six zinc finger-like motifs N-terminal to the TRAF domain, which presumably play a role in NF-␬B and Primary human monocytes were isolated by counterflow elutriation from human PBMC, as described previously (25). Monocyte-derived macro- JNK activation (17). The cytoplasmic domain of human CD40 phages were generated, as previously described by Welsh et al. (26), with (hCD40) has a proximal TRAF6 binding site and a more distal slight modifications. Briefly, PBMC, obtained from healthy donors using TRAF2/3/5 binding site. Both the TRAF binding sites play distinct plasma-Percoll gradients, as described previously (27), were washed twice roles in the CD40 signaling of B lymphocytes. TRAF6 and its in physiological saline with final resuspension at 2 ϫ 106/ml in RPMI 1640 corresponding binding site on CD40 are required for CD40-medi- supplemented with 5% heat-inactivated FBS, 0.01 M HEPES, and 50 ␮g/ml gentamicin, henceforth referred to as R5. PBMCs were plated in ated IgM production, IL-6 secretion, and isotype switching (18), Costar 6 ultra-low attachment plates (Corning Costar) at a volume of 6–8 whereas the TRAF2/3/5 binding site on CD40 is required for ml/well and were allowed to mature for 4–7 days in a humidified 37°C CD40-mediated up-regulation of B7 and protection from B cell Ag incubator with 5% CO2. After the maturation period, cells were removed receptor-mediated growth arrest (reviewed in Ref. 19). More re- from low attachment plates, washed twice in Dulbecco’s PBS with 0.2% FBS, and resuspended at 5 ϫ 106/ml in R5. PBMC were plated at varying cently, TRAF2 has been implicated in the CD40-mediated IgM concentrations depending on experiment in 96- or 24-well plates (Nalge and JNK activation using TRAF2-deficient B cell lines (20). Al- Nunc International) for stimulation assays.