TNF Receptor-Associated Factor 6 Is an Essential Mediator of CD40

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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 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. Mature monocyte-derived mac- though the expression of the TRAF family members has been eval- rophages were selected by adherence after a further 1- to 2-day incubation uated in monocytes (21), the role of these proteins as mediators of of PBMC. monocyte and macrophage CD40 signaling pathways has not been Immortalized macrophage cell lines from CD40-deficient mice and Downloaded from wild-type C57BL/6J (B6J2 cell line) were established by transformation of explored. bone marrow with the murine recombinant J2 retrovirus, as previously We have previously demonstrated the requirement for protein described (28). For the generation of stable transfectants, the J2-trans- tyrosine kinase (PTK) activity and the subsequent activation of the formed lines were electroporated with 1 ␮g of DNA at 600 V, 20 ␮s, and MAPK family members ERK1/2 to be important components of 2 pulses. Twenty-four hours after transfection, Zeocin (100 ␮g/ml) was added to the medium. Cell lines used in this study also included Chinese the CD40 signaling pathway in monocytes (22). In the investiga- hamster ovary (CHO) and stable murine CD154 transfectants of CHO (29). tion of PTK(s) relevant to CD40 signaling, we have identified Src The cell lines were maintained in R5. http://www.jimmunol.org/ family kinase activity as an initiator of CD40 signaling in monocytes and macrophages. We report that CD40 associates with Src CD40 stimulation of monocytes and macrophages kinase activity upon stimulation, and that this kinase activity is Stimulation via CD40 was achieved by addition of either rCD154 or the required for ERK1/2 phosphorylation and the subsequent activa- CD154 CHO tranfectants. Primary elutriated human monocytes and the tion of inflammatory cytokine synthesis. An analysis of the role of murine macrophage cell lines transfected with hCD40 constructs were stimulated with rCD154 at a concentration of 1 ␮g/ml along with the ac- CD40-TRAF interactions in CD40-mediated activation of mono- companying cross-linking enhancer (Alexis Biochemicals), also at a con- cyte/macrophage inflammatory cytokine production revealed a centration of 1 ␮g/ml, for varying lengths of time, as indicated. In some critical requirement for TRAF6 in facilitating this pathway, as well experiments, murine macrophage lines were stimulated with control CHO as in mediating CD40 activation of I␬B kinase (IKK) and NF-␬B. or CHO-murine CD154 transfectants at a ratio of 2:1. by guest on September 26, 2021 Materials and Methods Flow cytometric analysis and selection of stable transfectants via cell sorting Reagents and Abs For the generation of stable lines expressing hCD40, Zeocin-resistant Sodium orthovanadate (Na VO ) was acquired from Fisher Scientific. Sol- 3 4 clones were labeled for surface expression of hCD40, and high hCD40 uble rCD154 with cross-linking enhancer was obtained from Alexis Bio- expressors were sorted using a FACSVantage SE (BD Biosciences). Cells chemicals. The pyrazolopyrimidine, PP2, a Src kinase inhibitor, was ob- were harvested by scraping and treated with Fc block (BD Pharmingen; tained from Calbiochem. Rabbit Abs recognizing the active, anti-mouse CD16/CD32 mAb) for 5 min on ice, and subsequently labeled phosphorylated (Thr183 and Tyr185) form of ERK1/2 were acquired from with PE-conjugated anti-human CD40 in Dulbecco’s PBS containing 5% Promega. HRP-conjugated F(abЈ) donkey anti-rabbit Ab was purchased 2 FBS and 0.01% azide serum for 30 min at room temperature, washed, and from Jackson ImmunoResearch Laboratories. Anti-IKK␣␤ and anti- analyzed on a FACSVantage SE (BD Biosciences). After establishment of ERK1/2 Abs were obtained from Santa Cruz Biotechnology, as were the stable transfectants, a comparative analysis of the level of CD40 expression Src kinase substrate Sam68 and the IKK substrate I␬B␣. The MAPK assay on the various lines was performed using the same protocol. kit for ERK1/2 and anti-Src and pSrc Abs was purchased from Cell Sig- naling Technology. PE-conjugated anti-human CD40 was from Beckman Western blot analysis Coulter. Anti-mouse CD16/CD32 mAb was from BD Pharmingen. The antibiotic Zeocin was purchased from InvivoGen. After stimulation in 24-well plates, cells were disrupted in lysis buffer (125

mM Tris, pH 6.8, 2% SDS, 20% glycerol, 1 mM Na3VO4), and total pro- DNA constructs tein content of the samples was assessed by micro-bicinchoninic acid assay The generation of wild-type hCD40 in the plasmid pRSV.5 (zeo), (Pierce). Equal amounts of protein per sample were analyzed by SDS- hCD40⌬32 in PCDNA (zeo), dominant-negative TRAF6 mutant PAGE on 10% Criterion gels (Bio-Rad). Protein transfer to nitrocellulose (DNTRAF6) in pRSV.5 (neo), and hCD40EEAA in pRSV.5 (neo) has been membranes (Hybond; Amersham) was performed using a Trans-Blot SD previously described (18, 23). DNTRAF6 and hCD40EEAA were sub- SemiDry Electrophoretic Transfer Cell (Bio-Rad). Ab-bound proteins were cloned into the vector pRSV.5 (zeo) to allow for selection based on Zeocin detected using an ECL Western blotting analysis system (Amersham), and resistance in macrophages. Plasmids containing the NF-␬B response ele- the membranes were exposed to Kodak Biomax XL x-ray film (Eastman ment upstream of a firefly luciferase gene (pNF-␬B-luc) were obtained Kodak). from Stratagene. Plasmids encoding the Renilla luciferase (pHRL-null) were obtained from Promega. Analysis of cytokine production TRAF6-binding peptide (TRAF6BP) Analysis of proinflammatory cytokine production by monocytes/macrophages stimulated through CD40 was performed by ELISA. Cells were Peptides corresponding to the TRAF6 binding domain of CD40 made cell plated in 96-well microtiter plates and stimulated with rCD154 or CHO- permeable by fusing the signal sequence of the Kaposi fibroblast growth CD154 transfectants. Controls included use of the CHO parent cell line. factor were manufactured by Biosynth International. The sequence of the Supernatants were harvested after an 18-h incubation and assayed by peptide is as follows: NH2-AAVALLPAVLLALLAPAPHPKQEPQEI- ELISA using OptEIA sets (BD Pharmingen), and analysis was performed DFPDD-OH. The underlined portion represents the Kaposi fibroblast using an E-max Precision microplate reader (Molecular Devices). For the growth factor signal sequence. The peptide contains an asparagine (N) to assay of murine TNF-␣ content, supernatants were analyzed by the mouse The Journal of Immunology 1083 inflammation cytometric bead array (CBA) kit (BD Pharmingen). Briefly, diluted supernatants or standards were incubated with the fluorescent beads and the PE-conjugated detection Abs. Cytokine-bound beads were analyzed with use of a FACSCalibur flow cytometer and the BD Biosciences CBA software. Analysis of cytokine mRNA synthesis Analysis of cytokine mRNA synthesis was performed by RNase protection assay. Cells were plated in six-well plates and stimulated via CD40 using CHO-CD154 or rCD154. RNA was extracted using TRIzol (Invitrogen Life Technologies), as per manufacturer’s instructions. RNA (5 ␮g) was hybridized with a radiolabeled probe generated from the human cytokine RNA template set, hCK-2b (BD Pharmingen), at 55°C overnight. RNase treatment was conducted at 37°C for 45 min, after which the protected probe was purified and resolved by electrophoresis using a 5% polyacryl- amide gel (Bio-Rad) in Tris-borate-EDTA buffer. The gel was dried and exposed to Kodak Biomax XL x-ray film (Eastman Kodak). With the un- digested probes as markers, a standard curve was plotted as migration distances vs nucleotide length on semilog paper. The identity of the RNase- protected bands in the samples was then extrapolated from the graph. Kinase assays Downloaded from Cells plated in six-well plates were stimulated via CD40 for 20 min, after which the samples were harvested in lysis buffer and protein levels were assessed using the bicinchoninic acid assay (Pierce). Lysate (100 ␮g protein per sample) was immunoprecipitated using the appropriate Ab for 18 h at 4°C, followed by incubation with recombinant protein A agarose for 1 h. The immunoprecipitates were subjected to an in vitro kinase assay using 1 ␮g of the appropriate substrate (Sam68, I␬B␣, or Elk1) in the presence or absence of 5 ␮Ci of [␥-32P]ATP for 20–30 min (for Src and IKK assays). http://www.jimmunol.org/ The samples were resolved by electrophoresis and visualized by autora- diography, or analyzed by Western blot using phosphospecific anti-Elk1 for the ERK1/2 kinase assay. NF-␬B luciferase reporter assay A total of 3 ϫ 106 macrophages was transfected with 20 ␮g of pNF-␬B-luc and 2 ␮g of pHRL-null by electroporation and plated in six-well plates. Two hours after transfection, cells were stimulated with CHO-CD154 or CHO control cells in triplicates. Sixteen hours after stimulation, cells were lysed in lysis buffer and luciferase assays were performed using the Pro- by guest on September 26, 2021 mega dual luciferase system, following the manufacturer’s instructions. Results CD40-mediated stimulation of monocyte and macrophage proinflammatory cytokine production requires Src kinase activity In previous work, we demonstrated a requirement for PTK activity for CD40-mediated monocyte cytokine synthesis and rescue of monocytes from apoptosis (10). CD40 stimulation of monocytes resulted in the phosphorylation of a number of proteins in the 52- FIGURE 1. CD40-mediated stimulation of monocyte proinflammatory to 65-kDa range, which corresponds to the molecular mass of most cytokine production requires Src kinase activity. A, Primary elutriated hu- Src family members (30). In addition, we have recently shown an man monocytes were pretreated for 1 h with the Src kinase inhibitor PP2 involvement of Src kinase activity in CD40-mediated activation of and subsequently stimulated via CD40 using rCD154, as described in Ma- vascular smooth muscle cell chemokine synthesis (31). Thus, Src terials and Methods, for 18 h. Supernatants were analyzed for TNF-␣, family kinases were considered as likely candidates for the PTK IL-6, and IL-1␤ production by ELISA. Values presented are means of activity induced via CD40 stimulation of monocytes. We stimu- triplicate determinations of monocyte supernatant IL-6, IL-1␤, or TNF-␣ lated primary elutriated human monocytes via CD40 using soluble content ϩ SD. B and C, Primary human elutriated monocytes were stim- rCD154 plus a cross-linking enhancer (as described in Materials ulated with rCD154 for3hintheabsence and presence of PP2 (30 ␮M) and Methods) in the presence of the specific Src kinase inhibitor and subjected to an RNase protection assay using the RiboQuant Multi- ␤ probe RNase system using the template hCK-2b. The histograms in C PP2 for 18 h. Culture supernatants were analyzed for IL-1 , IL-6, ␣ ␤ ␣ represent the density of the protected probes for IL-1 , IL-1 , and IL-6. and TNF- by ELISA. Ligation of CD40 using rCD154 resulted in These results are representative of data from two similar experiments. the robust production of the proinflammatory cytokines IL-1␤, IL-6, and TNF-␣ (Fig. 1A). A dose of 10 ␮M PP2 completely abrogated the CD40-mediated TNF-␣ and IL-1␤ production, ing that monocytes and macrophages share the requirement for Src whereas complete inhibition of IL-6 required higher concentra- family kinase activity for CD40 induction of TNF-␣ and IL-6 tions of PP2 (20–30 ␮M). The inhibitor did not have any adverse production. effects on cell viability at the concentrations used. The experimen- The reduction of CD40-induced cytokine production by PP2 tal protocol used in Fig. 1A for the analysis of CD40-induced pro- corresponded with reduced mRNA expression, as determined by tein expression was also performed using culture-generated mac- RNase protection assay (Fig. 1B). Stimulation via CD40 resulted rophages, prepared as described in Materials and Methods. These in the induction of IL-1␣, IL-1␤, and IL-6 mRNA expression. experiments yielded comparable results (data not shown), indicat- Pretreatment with PP2 (30 ␮M) inhibited the induction of IL-1␣ 1084 ROLE OF TRAF6 IN MONOCYTE/MACROPHAGE CD40 SIGNALING message by 70%, and that of IL-1␤ and IL-6 by 55%. Densito- strongly induced Src phosphorylation, observed at 5 min post- metric analysis is shown in Fig. 1C. The levels of the housekeep- stimulation, and Src phosphorylation above background was ap- ing genes L32 and GAPDH are equivalent in the different lanes, parent up to 60 min. To confirm the ability of PP2 to inhibit CD40- indicating that the differences in the levels of the proinflammatory activated Src activation, monocytes were preincubated with mRNA are not due to artifacts of gel loading. varying concentrations of PP2 before stimulation with rCD154. As shown in Fig. 2B, Src phosphorylation induced by rCD154 was CD40 ligation induces Src family kinase activity and CD40-Src blocked completely by PP2 in a dose-dependent manner. In the kinase association experiments presented in Fig. 2, A and B, there were no differences The data shown in Fig. 1 established a functional role for Src in the levels of total Src present in the samples tested (data not family kinase activity in monocyte and macrophage CD40 signal- shown). ing. To directly examine the effect of CD40 ligation on Src acti- We also analyzed Src family kinase activity of CD40-stimulated vation, monocytes were stimulated with rCD154 for different time monocyte cell lysates by in vitro kinase assay using the Src sub- intervals and cell lysates were analyzed using an anti-phospho-Src strate, Sam68 (Src-associated substrate during mitosis). Ligation Ab that recognizes Src family members when activated by phos- of CD40 resulted in the stimulation of Src kinase activity ϳ3-fold phorylation at Tyr416. As seen in Fig. 2A, treatment with rCD154 above background within 5 min. This activity remained constant over a 15-min stimulation (Fig. 2C). The possibility that CD40 physically associates with Src family kinase(s) was investigated by analysis of anti-CD40 immunoprecipitates for Src kinase activity.

We found detectable Src kinase activity in CD40 immunoprecipi- Downloaded from tates in the absence of stimulation, and CD40-associated Src activity increased ϳ2-fold upon treatment with rCD154 for 10 min, and returned to prestimulus levels by 15 min (Fig. 2D). These data conﬁrm that stimulation of monocyte CD40 results in activation of Src family kinase activity, and indicate that a CD40-Src kinase

physical association occurs under these conditions. http://www.jimmunol.org/ CD40 induction of Src kinase activity is essential for downstream ERK1/2 activation CD40 ligation of monocytes activates the MAPK family members ERK1/2, and in these studies we demonstrated that ERK1/2 activity is required for CD40 induction of inflammatory cytokine production (22). We had also observed that CD40 activation of ERK1/2 was regulated by an upstream PTK. With Src family kinase(s) now revealed as a candidate for the PTK activity observed by guest on September 26, 2021 previously, we tested the dependency of CD40-mediated ERK1/2 activation on Src. Primary human monocytes were preincubated with PP2 and subsequently stimulated with rCD154 for 20 min, and cell lysates were analyzed for ERK1/2 phosphorylation. Stim- ulation of monocytes with rCD154 for 20 min resulted in a 5-fold increase in the levels of phospho-ERK1/2, which was blocked by PP2 to background levels (Fig. 3). The histogram shows the density of the phosphorylated ERK1/2 bands. The blot was stripped and reprobed for total ERK1/2 (bottom panel), which demonstrated that the differences in the levels of phospho-ERK1/2 were not due to artifacts of gel loading or electroblotting. Because PP2 is an effective inhibitor of Src kinases and does not impact ERK1/2 activity (32, 33), these data indicate that Src family kinase activity acts upstream of ERK1/2 in this CD40-mediated proinflammatory pathway. CD40 induction of inflammatory cytokine synthesis requires an intact TRAF6 binding site FIGURE 2. CD40 ligation results in the phosphorylation and activation of Src kinase(s). A, Primary human monocytes were stimulated with It has been demonstrated that CD40 signaling pathways can be rCD154, as described in Materials and Methods, for the time periods in- relayed via both TRAF-dependent and independent means (34, dicated. Cell lysates were normalized and analyzed by Western blot using 35), and that engagement of specific TRAF family members anti-phospho-Src Ab. B, Monocytes were preincubated for 1 h with PP2 by CD40 varies considerably, dependent on cell type and func- (30 ␮M) and stimulated with rCD154 for 20 min. Cell lysates were nor- tional outcome (19, 36). To date, the role of CD40-TRAF inter- malized and analyzed by Western blot using anti-phospho-Src Ab. C, Hu- actions in monocyte and macrophage inflammatory pathways has man monocytes stimulated for varying intervals of time (as indicated) and not been explored. To investigate the role of TRAFs as potential cell lysates were subjected to an in vitro kinase assay using Sam68 as substrate. D, Human monocytes were stimulated at the times indicated, and mediators of the CD40-activated Src/ERK1/2 pathway in mono- cell lysates were immunoprecipitated with anti-CD40 Ab or isotype control cytes/macrophages, stable transfectants expressing either wild- (ISO) and subjected to an in vitro kinase assay, as in C. The histograms type hCD40 or hCD40 mutants missing specific TRAF binding represent the density of phospho-Sam 68. The results shown are represen- sites were generated in a CD40-deficient murine macrophage cell tative of three similar experiments. line. These included hCD40EEAA, deficient in TRAF6 binding, The Journal of Immunology 1085

FIGURE 5. CD40 signaling of macrophage proinflammatory activity requires an intact TRAF6 binding domain. The stable hCD40 transfectants, wild-type hCD40 (WT), hCD40⌬32 (⌬32), and hCD40EEAA (EEAA), were stimulated using CHO-CD154 or control CHO cells for 18 h. Super- natants were analyzed for TNF-␣ production by CBA (left panel) and IL-6 FIGURE 3. CD40-mediated ERK1/2 phosphorylation is downstream of production by ELISA (right panel). Values presented are means of tripli- Src kinase activity. Primary human monocytes were pretreated with indi- -p Ͻ 0.0005). The results shown are repre ,ء) cate determinations ϩ SD cated doses of PP2, followed by stimulation with rCD154, as described in sentative of four similar experiments. Materials and Methods, for 20 min. Cell lysates were analyzed by Western Downloaded from blot using Abs specific for the phosphorylated forms of ERK1/2 (top panel). Bottom panel, Shows the blot stripped and reprobed for total ERK1/2. The histogram represents ratio of phosphorylated ERK2 to total to LPS stimulation (data not shown); thus, deficiencies in CD40- ERK2 band density. The results shown are representative of three similar mediated cytokine production in the mutants were not due to an experiments. overall defect in responsiveness of these cell lines. These results ␣ indicate that CD40-mediated production of both IL-6 and TNF- http://www.jimmunol.org/ and hCD40⌬32, a truncated CD40 molecule that lacks the PVQET requires an intact TRAF6 binding site, and that the TRAF2/3/5 motif required for TRAF2/3/5 binding (18, 37). Stable transfec- binding site contributes to, but is not an essential requirement for tants, selected by flow cytometry and maintained as Zeocin-resis- CD40 stimulation of TNF-␣ production. tant lines, were analyzed to assure equivalent levels of CD40 ex- CD40 activation of monocyte/macrophage inflammatory cytokine pression for use in functional studies. As shown in Fig. 4, each of production is abrogated by a cell-permeable TRAF6BP the CD40-expressing clones (B–D) expressed comparable levels of CD40 receptor. The results of experiments using CD40 mutants indicated that in- The various CD40-expressing lines were stimulated using CHO- teraction of CD40 with TRAF6 is necessary for macrophage proin- CD154 transfectants or CHO control cells for 18 h. Supernatants flammatory signaling. To further evaluate the role of TRAF6, we by guest on September 26, 2021 were analyzed for IL-6 and TNF-␣ production by ELISA and addressed the requirement for an intact TRAF6 domain using cell- CBA, respectively. Ligation of CD40 resulted in a robust stimu- permeable decoy peptides corresponding to the TRAF6 binding lation of IL-6 and TNF-␣ production by the wild-type, but not by domain of hCD40. For these experiments, we returned to use of the transfectants containing the hCD40EEAA mutant (Fig. 5). In primary human monocytes and culture-generated macrophages. the analysis of the hCD40⌬32 mutant, we found that the level of The peptide was made cell permeable by fusing a hydrophobic CD40-stimulated TNF-␣ was 40% lower than that of wild-type sequence of Kaposi fibroblast growth factor signal peptide that has transfectants (Fig. 5, left panel). However, IL-6 levels produced by previously been used to deliver peptides into intact cells (38–40). the hCD40⌬32 mutant and wild-type cells were comparable (Fig. The TRAF6BP contains an asparagine (N) to aspartic acid (D) 5, right panel). All of the transfectants showed a similar response mutation that enhances the affinity to TRAF6. Thus, incubation of the cells with the peptide should act to block the recruitment of TRAF6 to CD40 and most likely block any responses that are TRAF6 dependent. In a recent study, cell-permeable peptides corresponding to the TRAF6 binding region of the TNF-related activation-induced cytokine (TRANCE) receptor were used to block TRANCE-induced osteoclast differentiation (40). Human monocytes were incubated with the TRAF6BP at a concentration range similar to that used in the TRANCE receptor (TRANCE-R) studies (40) before stimulation with rCD154. As shown in Fig. 6A, TRAF6BP inhibited CD40 stimulation of inflammatory cytokine in a dose-dependent manner. A concentration of 200 ␮M TRAF6BP blocked the CD40-mediated stimulation of IL-1␤, TNF-␣, and IL-6 to background levels. The peptide did not inhibit PMA-mediated cytokine production in monocytes (for which there is no evidence for TRAF6 involvement), thus demonstrating specificity of the peptide to TRAF6-mediated pathways (Fig. 6A, bot- FIGURE 4. Surface expression of CD40 by stable transfectants. The murine CD40-deficient macrophage cell line (mCD40KO), and the tom panel). The effects of the TRAF6BP on CD40-mediated proin- mCD40KO transfectants expressing wild-type hCD40, hCD40⌬3, and flammatory cytokine mRNA synthesis were evaluated by RNase hCD40EEAA were stained for surface expression of CD40, followed by protection assay. Preincubation of the TRAF6BP blocked CD40- flow cytometric analysis. Light lines represent background (unlabeled), mediated induction of IL-1␣, IL-1␤, IL-6 by 64, 61, and 71%, while dark lines represent the fluorescence intensity of labeled cells. respectively (Fig. 6, B and C). 1086 ROLE OF TRAF6 IN MONOCYTE/MACROPHAGE CD40 SIGNALING

A DNTRAF6 abrogates macrophage CD40-mediated inflammatory cytokine production The experiments described above indicate that a CD40-TRAF6 interaction is crucial to CD40-induced signal transduction, leading to inflammatory cytokine production. To further address the role of TRAF6 in this context, we tested the effects of introduction of a DNTRAF6 in murine macrophages expressing endogenous (wild- type) murine CD40. The B6/J2 macrophage line was stably transfected with DNTRAF6, which lacks the zinc and ring fingers, and therefore lacks catalytic activity, but contains the C-terminal region essential for binding to CD40 (18). B6J2 stably expressing DNTRAF6 and untransfected B6J2 cells were stimulated via CD40 using CHO-CD154 cells, with CHO cells serving as a control. Supernatants were analyzed for IL-6 and TNF-␣ content. Ex- pression of DNTRAF6 very effectively blocked CD40 stimulation of TNF-␣ and IL-6 production (Fig. 7), further supporting the role of TRAF6 as an essential component of the CD40-activated in-

ﬂammatory pathway in monocytes and macrophages. Downloaded from

CD40 signaling of monocyte/macrophage ERK1/2 kinase activation requires TRAF6-CD40 interactions and is not affected by disruption of the TRAF2/3/5 binding site

Because we have shown previously that ERK1/2 activity is re- http://www.jimmunol.org/ quired for CD40-induced cytokine synthesis in monocytes (22), the ﬁnding that CD40-mediated activation of cytokine synthesis is TRAF6 dependent suggested that TRAF6 may provide a link between CD40 ligation and ERK1/2 activation in monocytes/macrophages. Therefore, we explored the role of TRAFs in mediating ERK1/2 activation with use of the hCD40EEAA and hCD40⌬32 mutants. Ligation of CD40 on the wild-type and hCD40⌬32 mutant resulted in a 9-fold stimulation of ERK1/2 kinase activity by guest on September 26, 2021 above background, as measured by Elk1 phosphorylation, whereas stimulation of the hCD40EEAA mutant did not result in induction of ERK1/2 activity (Fig. 8A). We next addressed the requirement for TRAF6-CD40 interaction in the stimulation of ERK1/2 activation using TRAF6BP. Human monocytes were preincubated with the TRAF6BP for 3 h and subsequently stimulated via CD40 using rCD154 for 20 min. Cell lysates were analyzed by Western blot using anti-phospho-ERK1/2 Ab. Ligation of CD40 by rCD154 resulted in the phosphorylation of ERK1/2, which was reduced to background levels by TRAF6BP, indicating that TRAF6-CD40 interactions are required in the CD40 signaling of ERK1/2 activation (Fig. 8B). We also addressed whether TRAF6BP-CD40 interactions are required for CD40 induction of Src activity. Human monocytes were stimulated with rCD154 following preincubation with TRAF6BP, as described above. Although PP2 effectively inhibited CD40-induced Src activity, as also shown in Fig. 2B, pretreatment of monocytes with TRAF6BP did not impair CD40 induction of Src phosphorylation (Fig. 8C). In addition, CD40 induction of Src activity was intact in the hCD40EEAA transfec- tant (data not shown), suggesting that Src activity is upstream of TRAF6-mediated signaling.

FIGURE 6. CD40 activation of monocyte/macrophage inﬂammatory cytokine production is abrogated by TRAF6BP. A, Human monocytes were TRAF6BP (300 ␮M) for 3 h, then stimulated with rCD154 for 3 h. Samples preincubated with different doses of TRAF6BP for 3 h and stimulated with were analyzed by RNase protection assay using the template hCK-2b. The rCD154, as described in Materials and Methods, or PMA (as indicated) for histograms shown in C represent the density of the protected probes for 18 h. Supernatants were analyzed for TNF-␣, IL-6, and IL-1␤ (as indi- IL-1␣ (total pixels ϫ 104), IL-1␤ (total pixels ϫ 105), and IL-6 (total cated) by ELISA. Values presented are means of triplicate determinations pixels ϫ 104). The results shown are representative of two similar .p Ͻ 0.002). B, Human monocytes were preincubated with experiments ,ء) ϩ SD The Journal of Immunology 1087

FIGURE 7. CD40 signaling is disrupted by a TRAF6 dominant-negative mutant. B6J2 or B6J2 expressing DNTRAF6 were stimulated with CHO-murine CD154 or control CHO cells for 18 h. Supernatants were analyzed for IL-6 or TNF-␣ production by ELISA. Values presented are p Ͻ ,ء ,means of triplicate determinations of supernatant IL-6 (left panel .p Ͻ 0.0005) content ϩ SD ,ء ,or TNF-␣ (right panel (0.003

TRAF6 mediates CD40 signaling of monocyte/macrophage IKK and NF-␬B activation

In addition to ERK1/2, CD40 ligation activates NF-␬B, which con- Downloaded from tributes to the transcriptional regulation of genes encoding inﬂam- matory proteins. Studies done on TRAF-deﬁcient mice have implicated both TRAF2 and TRAF6 in CD40-mediated NF-␬B activation (41, 42). Similarly, both the TRAF binding sites prob- ably contribute to CD40 signaling of NF-␬B in B lymphocytes (20,

34). To address this issue in macrophages, we transiently trans- http://www.jimmunol.org/ fected the wild-type and CD40 cytoplasmic domain mutants with a plasmid containing the NF-␬B response element upstream of a luciferase gene (pNF-␬B-luc) and 2 ␮g of a Renilla luciferase (pHRL-luc) plasmid to normalize for transfection efﬁciency. The cells were subsequently stimulated via CD40 using CHO-CD154 for 16 h. Stimulation of both the wild-type and hCD⌬32 mutant resulted in a ϳ3-fold induction of NF-␬B luciferase activity above background, whereas stimulation of the hCD40EEAA mutant did

␬ by guest on September 26, 2021 not result in detectable stimulation of NF- B reporter activity (Fig. FIGURE 8. CD40 signaling of monocyte and macrophage proinflam- ␬ 9A). Thus, optimal NF- B activation requires an intact TRAF6 matory ERK1/2 phosphorylation requires TRAF6. A, The stable hCD40 binding domain of CD40. Interestingly, the hCD40⌬32, but not the transfectants, wild-type hCD40 (WT), hCD40⌬32 (⌬32), and hCD40EEAA mutant was found to be defective in CD40-mediated hCD40EEAA (EEAA), were stimulated with rCD154, as described in Ma- NF-␬B activation in B lymphocytes (23), which further demon- terials and Methods, for 20 min. Cell lysates were immunoprecipitated strates that CD40 signaling pathways vary between different cell using immobilized anti-ERK1/2 Ab, and the immunoprecipitates were types. NF-␬B is sequestered in cells via association with proteins washed and subjected to an in vitro kinase assay using Elk1 as substrate, of the I␬B family. Stimulus induces the serine phosphorylation of as described in Materials and Methods. Phosphorylated Elk1 was detected I␬B by the kinases IKK␣␤, which leads to its dissociation from by Western blot using anti-phospho-Elk1 Ab. The histograms represent the ␬ ␬ density of the phospho-Elk1 bands. B, Primary elutriated human mono- NF- B, liberating NF- B to activate gene transcription (43). Be- ␮ ⌬ cytes, preincubated with TRAF6BP (200 M) for 3 h, were stimulated with cause the hCD40EEAA, but not the hCD40 32 mutant was found rCD154 for 20 min. Lysates were analyzed by Western blot using anti- ␬ to be refractory to CD40-mediated NF- B activation, we antici- phospho-ERK1/2 Ab. Bottom panel, Shows the blot stripped and reprobed pated that this mutant would be deficient in CD40-mediated IKK for total ERK content. The histogram represents ratio of phosphorylated activation. Indeed, CD40 stimulation using rCD154 for 20 min ERK2 to total ERK2 band density. C, Cells were stimulated as in B, with resulted in a 5-fold induction of IKK activation measured by phos- preincubation with either TRAF6BP (200 ␮M) or 30 ␮M PP2. Lysates phorylation of I␬B␣ in the wild-type, but not the hCD40EEAA were analyzed by Western blot using anti-phospho-Src Abs. The results mutant (Fig. 9B). Similarly, preincubation of the TRAF6BP presented are representative of three similar experiments. blocked CD40-mediated I␬B␣ phosphorylation in human monocytes (Fig. 9C). Interestingly, the hCD40⌬32 mutant was also deficient in CD40-mediated IKK activation (Fig. 9B), despite having identify the components of the signaling pathways that culmi- shown levels of NF-␬B activation equivalent to the wild type (Fig. nate in CD40-mediated inflammatory activity. It is clear from 9A). Thus, it possible that CD40-mediated NF-␬B activation may published work that CD40 signaling differs from cell type to involve IKK-dependent and IKK-independent mechanisms, as has cell type, in particular with regard to and activation of MAPKs been observed in other systems (44, 45). and employment of TRAF family members. For example, CD40 activation of monocytes results in activation of ERK1/2, but Discussion does not induce JNK activation, nor increase levels of p38 Although it has been well established that CD40 induction of phosphorylation above background (22). In contrast, CD40 ac- proinflammatory activity in monocytes and macrophages con- tivation of vascular smooth muscle cells results in effective ac- tributes to chronic inflammation in autoimmune and vascular tivation/phosphorylation of both ERK1/2 and p38 (31). Like- disease, the signaling pathways mediating this activity have wise, studies of the role of TRAF family members in CD40 been only partially elucidated. A goal of our studies has been to signaling have revealed cell type differences (19, 36). Early 1088 ROLE OF TRAF6 IN MONOCYTE/MACROPHAGE CD40 SIGNALING

do not represent what occurs in cell types that naturally express CD40 (18, 23). The accumulating data demonstrating the divergence of signaling outcomes of CD40 ligation on various cell types underscore the importance of examining CD40 signaling as a cell-specific event. In this work, we have identified Src family kinase activity as the initiator of downstream ERK1/2 activation and subsequent cytokine production in monocytes/macrophages (Figs. 1 and 3), and we present evidence for a CD40-Src interaction (Fig. 2). At present, it is unclear which Src family member(s) is a participant in these pathways. Based on the protein phosphorylation pattern observed post-CD40 ligation, as well as on previous reports suggesting a potential role of the Src family kinase, Lyn, in CD40 signaling in dendritic cells (49), we considered Lyn as a likely candidate for CD40-induced Src activity. However, experiments performed in our laboratory using macrophages derived from Lyn- deficient mice indicated that Lyn is not involved in CD40-induced proinflammatory activity (our unpublished data). Thus, the identity

of the Src family kinase(s) involved in monocyte/macrophage Downloaded from CD40 proinﬂammatory signaling is still unresolved and is pres- ently under investigation. We have also shown in this work that the Src/ERK1/2 pathway, as well as CD40-mediated activation of the IKK/NF-␬B pathway in monocytes/macrophages, is dependent on the interaction of

CD40 with TRAF6. TRAF6 dependency was revealed using three http://www.jimmunol.org/ different strategies. First, we used a CD40-deficient murine macrophage cell line expressing either wild-type or mutants of CD40 missing either the TRAF2/3/5 or TRAF6 binding sites, and found that, upon CD40 ligation, the transfectants expressing the TRAF6 binding region mutant (hCD40EEAA) were profoundly impaired in production of TNF-␣ and IL-6 (Fig. 5), activation of ERK1/2 (Fig. 8A), and activation of NF-␬B and IKK activities (Fig. 9, A and B). In addition, we treated human monocytes and macrophages with TRAF6BP, which proved to be an effective inhibitor of by guest on September 26, 2021 monocyte and macrophage CD40-induced inflammatory cytokine mRNA synthesis and protein production (Fig. 6). Finally, we found that expression of a DNTRAF6 molecule into a wild-type murine CD40-expressing line proved to potently inhibit CD40- activated TNF-␣ and IL-6 production in wild-type murine macrophages (Fig. 8). Thus, using each of these approaches, we identified TRAF6 as an essential signal transducer for CD40-activated proinflammatory activity in monocytes and macrophages. In monocytes and macrophages, activation of CD40 signal transduction requires extensive multimerization of the receptor and FIGURE 9. The TRAF6-binding motif of CD40 is required for optimal NF-␬B activation. A, The stable hCD40 transfectants, wild-type hCD40 is not efficiently induced by monomeric CD154 or anti-CD154 IgG (WT), hCD40⌬32 (⌬32), and hCD40EEAA (EEAA), were transfected (6). Interestingly, surface plasmon resonance studies have demon- with 20 ␮gofpNF␬B-luc plasmid and 2 ␮g of pRL-luc plasmid. Two strated that TRAF6 has a low affinity for CD40 and that it requires hours after transfection, cells were stimulated with CHO control or CHO- extensive multimerization of CD40 to mediate signaling events CD154 for 17 h. Cell lysates were analyzed for luciferase activity. Relative (50). Therefore, our results identifying TRAF6 as a signal trans- promoter activities are reported as a ratio of firefly to Renilla luciferase ducer for CD40 in monocytes/macrophages may explain the re- activities to normalize for transfection efficiency. The transfectants (B)or quirement for extensive cross-linking to initiate CD40 signaling. primary elutriated human monocytes (C) were preincubated with Previous work supports the possibility that TRAF6 may act as an TRAF6BP, then stimulated via CD40 with rCD154 for 20 min. The sam- adapter linking CD40 to Src family activity. For example, stimu- ples were immunoprecipitated with anti-IKK Ab, and the immunoprecipi- lation of osetoclasts with TRANCE, a TNF family member, re- tates were subjected to an in vitro kinase assay using pI␬B as substrate. The histogram represents the density of the phosphorylated I␬B bands. The sulted in an association between TRAF6, Src, and TRANCE-R. results shown are representative of two similar experiments. The N-terminal ring/zinc finger domains of TRAF6 were found to enhance the activity of c-Src upon stimulation via the TRANCE-R (51). As yet, we have been unable to detect a direct Src-TRAF6 studies of CD40 signaling involved overexpressing exogenous interaction in monocytes/macrophages. Thus, the potential role of CD40 and the various TRAF molecules in 293 cells (24, 46– Src in recruitment of TRAF6 to CD40 remains to be determined. 48). These studies helped to identify the TRAF binding domains However, an interaction between CD40 and Src is evident (Fig. in CD40 and to establish affinities between CD40 and the dif- 2C), and the finding that inhibition of TRAF6-CD40 interactions ferent TRAF proteins. However, it is now apparent that the with TRAF6BP does not impair CD40-initiated Src activity (Fig. results of CD40 signaling studies conducted in 293 cells often 8C) suggests that Src acts upstream of TRAF6 in the ERK1/2 The Journal of Immunology 1089 pathway. Interestingly, IKK and NF-␬B activities induced via 6. Wagner, D. H. J., R. D. Stout, and J. Suttles. 1994. Role of the CD40-CD40 ϩ CD40 stimulation were not impaired by treatment of cells with ligand interaction in CD4 T cell contact-dependent activation of monocyte interleukin-1 synthesis. Eur. J. Immunol. 24:3148. PP2 (data not shown), suggesting that, despite the shared depen- 7. Stout, R. D., and J. Suttles. 1996. The many roles of CD40 in cell-mediated dency of TRAF6, CD40 activation of the ERK1/2 and IKK/NF-␬B inflammatory responses. Immunol. Today 17:487. 8. Bourgeois, C., B. Rocha, and C. Tanchot. 2002. A role for CD40 expression on pathways involves different initiating signals. ϩ ϩ CD8 T cells in the generation of CD8 T cell memory. Science 297:2060. In recent years, TRAF6 has been identified as a key adaptor 9. Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, and molecule regulating signal transduction pathways mediated by K. Haskins. 2002. Expression of CD40 identifies a unique pathogenic T cell members of the IL-1/TLR family (52, 53). Although both the up- population in type 1 diabetes. Proc. Natl. Acad. Sci. USA 99:3782. 10. Suttles, J., M. Evans, R. W. Miller, J. C. Poe, R. D. Stout, and L. M. Wahl. 1996. stream and downstream kinases and adapter proteins used by the T cell rescue of monocytes from apoptosis: role of the CD40-CD40L interaction various members of this receptor family are variable, a common and requirement for CD40-mediated induction of protein tyrosine kinase activity. J. Leukocyte Biol. 60:651. outcome is the induction of proinflammatory activity, in part me- 11. Van Kooten, C., and J. Banchereau. 2000. CD40-CD40 ligand. J. Leukocyte Biol. diated via activation of NF-␬B (51, 54, 55). The demonstration that 67:2. TRAF6 mediates multiple CD40-activated proinflammatory path- 12. Stout, R. D., J. Suttles, J. Xu, I. S. Grewal, and R. A. Flavell. 1996. Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice. J. Immunol. ways in monocytes/macrophages contributes to the evidence sug- 156:8. gesting that TRAF6 pushes cellular responses toward a proinflam- 13. Soong, L., J. C. Xu, I. S. Grewal, P. Kima, J. Sun, B. J. Longley, N. H. Ruddle, matory phenotype. D. McMahon-Pratt, and R. A. Flavell. 1996. Disruption of CD40-CD40 ligand interactions results in an enhanced susceptibility to Leishmania amazonensis in- The role of the TRAF2/3/5 binding site in the CD40 signaling of fection. Immunity 4:263. monocytes/macrophages requires further investigation. Our results 14. Durie, F. H., R. A. Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, and R. J. Noelle. demonstrate that the TRAF2/3/5 binding site contributes to, but is 1993. Prevention of collagen-induced arthritis with an antibody to gp39, the Downloaded from ␣ ligand for CD40. Science 261:1328. not an essential requirement in CD40-induced mediated TNF- 15. Carayanniotis, G., S. R. Masters, and R. J. Noelle. 1997. Suppression of murine production. It is possible that deficiency of the TRAF2/3/5 binding thyroiditis via blockade of the CD40-CD40L interaction. Immunology 90:421. site may impact CD40-mediated activation of the other transcrip- 16. Gerritse, K., J. D. Laman, R. J. Noelle, A. Aruffo, J. A. Ledbetter, W. J. Boersma, ␤ and E. Claassen. 1996. CD40-CD40 ligand interactions in experimental allergic tion factors (e.g., AP-1 and/or C/EBP ) that contribute to the syn- encephalomyelitis and multiple sclerosis. Proc. Natl. Acad. Sci. USA 93:2499. thesis of TNF-␣. Paradoxically, our results identify a requirement 17. Inoue, J., T. Ishida, N. Tsukamoto, N. Kobayashi, A. Naito, S. Azuma, and T. Yamamoto. 2000. Tumor necrosis factor receptor-associated factor (TRAF) for the TRAF2/3/5 binding site in CD40-mediated activation of http://www.jimmunol.org/ ␬ family: adapter proteins that mediate cytokine signaling. Exp. Cell Res. 254:14. IKK (Fig. 9B), but not NF- B transcriptional activity (Fig. 9A). 18. Jalukar, S. V., B. S. Hostager, and G. A. Bishop. 2000. Characterization of the However, it should be noted that IKK-independent activation of roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte ef- NF-␬B has been reported in other systems (44, 45). For example, fector functions. J. Immunol. 164:623. 19. Bishop, G. A., and B. S. Hostager. 2001. Molecular mechanisms of CD40 sig- the NS5A protein of hepatitis C virus was found to activate NF-␬B naling. Arch. Immunol. Ther. Exp. 49:129. via a mechanism requiring the tyrosine, but not serine, phosphor- 20. Hostager, B. S., S. A. Haxhinasto, S. L. Rowland, and G. A. Bishop. 2003. Tumor ylation of I␬B␣. This event was blocked by the PTK inhibitor necrosis factor receptor-associated factor 2 (TRAF2)-deficient B lymphocytes reveal novel roles for TRAF2 in CD40 signaling. J. Biol. Chem. 278:45382. genestein and the Syk kinase inhibitor piceatannol, but not the IKK 21. Pearson, L. L., B. E. Castle, and M. R. Kehry. 2001. CD40-mediated signaling in inhibitor BAY11-7085 (44). Further studies are required to explore monocytic cells: up-regulation of tumor necrosis factor receptor-associated factor the mechanisms involved in CD40-mediated NF-␬B activation of mRNAs and activation of mitogen-activated protein kinase signaling pathways. by guest on September 26, 2021 Int. Immunol. 13:273. monocytes/macrophages. 22. Suttles, J., D. M. Milhorn, R. W. Miller, J. C. Poe, L. M. Wahl, and R. D. Stout. Blockade of the CD40-CD154 interaction has been found to be 1999. CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway: a target of interleukin (IL)-4 and IL-10 anti- beneficial in several murine models of inflammatory diseases (14– inflammatory action. J. Biol. Chem. 274:5835. 16). Thus, components of the CD40 signaling pathway(s) are po- 23. Hsing, Y., B. S. Hostager, and G. A. Bishop. 1997. Characterization of CD40 tential therapeutic targets. Our results establish an important role signaling determinants regulating nuclear factor-␬B activation in B lymphocytes. J. Immunol. 159:4898. for TRAF6 in the CD40 signaling of monocytes/macrophages, and 24. Pullen, S. S., T. T. Dang, J. J. Crute, and M. R. Kehry. 1999. CD40 signaling our studies using TRAF6BP demonstrate that blockade of the through tumor necrosis factor receptor-associated factors (TRAFs): binding site CD40-TRAF6 interaction may be a potential strategy in the treat- specificity and activation of downstream pathways by distinct TRAFs. J. Biol. Chem. 274:14246. ment of inflammatory diseases. 25. Wahl, L. M., I. M. Katona, R. L. Wilder, C. C. Winter, B. Haraoui, I. Scher, and S. M. Wahl. 1984. Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B-lymphocyte-, T-lympho- Acknowledgments cyte-, and monocyte-enriched fractions by flow cytometric analysis. Cell. Immu- nol. 85:373. 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