Tumor necrosis factor receptor 1 associates with ... - The FASEB Journal

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Tumor necrosis factor receptor 1 associates with CD137 ligand and mediates its reverse signaling Mei Chung Moh,*,† Paolo Alberto Lorenzini,*,† Charles Gullo,‡,§ and Herbert Schwarz*,†,储,1 *Department of Physiology, †Immunology Program, ‡Department of Medical Education, Research, and Evaluation, Duke–National University of Singapore Graduate Medical School, §Department of Microbiology, and 储Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore Reverse signaling through CD137 ligand (CD137L) potently activates monocytes. However, the underlying mechanism is not well elucidated. This study provides evidence that tumor necrosis factor receptor 1 (TNFR1) acts as a coreceptor for CD137L and mediates CD137L signaling. CD137L colocalizes with TNFR1 on the plasma membrane and binds directly to TNFR1 via its extracellular domain. Using the human monocytic THP-1 cell line, we demonstrate that engagement of CD137L by recombinant CD137 protein promotes cell adhesion, apoptosis, expression of CD14, and production of IL-8 and tumor necrosis factor (TNF). Concomitantly, the expression of TNFR1 protein is down-regulated in response to CD137L activation, due to enhanced extracellular release and internalization of TNFR1. Activation of TNFR1 by TNF protein additively augments CD137L-induced IL-8 expression. Conversely, inhibition of TNFR1 activity by a TNFR1-neutralizing antibody inhibits CD137L-mediated cell adhesion, cell death, CD14 expression, and IL-8 production. Taken together, these data show that TNFR1 associates with CD137L and is required for CD137L reverse signaling.—Moh, M. C., Lorenzini, P. A., Gullo, C., Schwarz, H. Tumor necrosis factor receptor 1 associates with CD137 ligand and mediates its reverse signaling. FASEB J. 27, 2957–2966 (2013). www.fasebj.org

ABSTRACT

Key Words: TNFR1 䡠 THP-1 cells 䡠 monocytes CD137 ligand (CD137L; 4-1BBL, TNFSF9), a member of the tumor necrosis factor (TNF) family, is expressed as a type II transmembrane glycoprotein on antigenpresenting cells (APCs) consisting of monocytes, macrophages, B cells, and dendritic cells (1–3). Binding of Abbreviations: APC, antigen-presenting cell; CD137L, CD137 ligand; EGFR, epidermal growth factor receptor; FcR, Fc receptor; HRP, horseradish peroxidase; IgG, immunoglobulin G; JNK, c-Jun-N-terminal kinase; MDC, monodansylcadaverine; NF-␬B, nuclear factor ␬B; TLR, Toll-like receptor; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TRADD, TNFR1-associated death domain; WT, wild type 0892-6638/13/0027-2957 © FASEB

CD137L to its receptor CD137 on T cells provides costimulatory signals important for proliferation, survival and the cytolytic activity of T cells (2, 4 – 6). Besides acting as a T-cell costimulatory ligand, CD137L is able to receive and transmit signals back into the cells on which it is expressed, a process termed reverse signaling (3, 7). Reverse signaling and bidirectional communication are not restricted to CD137L but are frequent features of members of the TNF and TNF receptor (TNFR) families (8). Other TNF family members capable of transducing reverse signals include TNF, TRAIL, Fas ligand, CD40 ligand, OX40 ligand, and CD30 ligand (8 –13). The roles of CD137L-mediated reverse signal transduction have been extensively studied in APCs, particularly monocytes. CD137L signaling participates in monocyte activation. Cross-linking of surface CD137L by immobilized recombinant CD137 protein promotes adhesion and spreading of human monocytes, accompanied by the production of proinflammatory cytokines IL-6, IL-8, and TNF, and the inhibition of anti-inflammatory cytokine IL-10. Moreover, the expression of the monocyte activation marker ICAM-1 is increased (14). Stimulation of CD137L also regulates apoptosis, survival, and proliferation in primary monocytes (15, 16). Furthermore, CD137L reverse signaling induces differentiation of monocytes into dendritic cells. These mature monocyte-derived dendritic cells are more potent in promoting T-cell proliferation and Th1 responses than classical dendritic cells (17, 18). Engagement of CD137L enhances monocyte migration in vitro and in vivo. Mimicking in vivo extravasation of monocytes into inflamed tissues, Drenkard et al. (19) demonstrated that spheroids expressing CD137 recruit 6 times more monocytes than control spheroids. In addition, introduction of CD137-coated Matrigel into mice re1 Correspondence: Department of Physiology, National University of Singapore, Center for Life Sciences, #03– 05, 28 Medical Dr., Singapore 117456, Singapore. E-mail: phssh@ nus.edu.sg. doi: 10.1096/fj.12-225250 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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sults in an increased accumulation and infiltration of monocytes (19). The underlying mechanisms driving CD137L reverse signaling are not well elucidated. Studies have shown that CD137L is capable of activating signaling pathways that elicit different immune responses. These pathways primarily include nuclear factor ␬B (NF-␬B), phosphatidylinositol-3-kinase/Akt, and mitogen-activated protein kinase involving c-Jun-N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (20 –22). Except for a casein kinase recognition site, CD137L seems to lack conserved motifs for signal transduction in its short cytoplasmic domain. Moreover, no CD137Linteracting adaptor protein has been identified (13, 21, 23). Therefore, CD137L by itself may not be sufficient to initiate signaling cascades when activated by CD137 protein. Instead, it is conceivable that on binding to CD137, CD137L interacts with and signals through other cell surface proteins that perhaps provide binding sites for adaptor proteins and/or enzymatic activity to achieve distinct CD137L-mediated cellular responses. In a seminal discovery, Kang et al. (24) found that CD137L associates with Toll-like receptor (TLR)-4 and possibly other TLRs in murine macrophages and that CD137L is essential to sustain TNF secretion induced by LPS and several other TLR agonists, independently of CD137. However, the effects of CD137L signaling are not identical to that of TLR signaling, which indicates the involvement of additional signaling pathways (25). This study provides evidence of a physical interaction between CD137L and TNFR1. Using the human monocytic cell line THP-1, we show that cross-linking of CD137L by recombinant CD137 protein promotes cell adhesion, apoptosis, and surface CD14 expression and increases the production of IL-8 and TNF. Although CD137L activation down-regulates TNFR1 protein expression, functional studies show that TNFR1 is required for CD137- and CD137L-induced activities. Activation of TNFR1 by TNF further enhances IL-8 release triggered by CD137L engagement. Conversely, neutralization of TNFR1 activity with a TNFR1 blocking antibody suppresses CD137L-induced cell adhesion, apoptosis, CD14 expression, and IL-8 production.

MATERIALS AND METHODS Cell culture and treatments THP-1 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI 1640 medium (Gibco BRL; Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Biowest, Logan, UT, USA). MCF7 cells (ATCC) were grown in DMEM (SigmaAldrich, St. Louis, MO, USA) containing 10% FBS. THP-1 cells (106 cells/ml) were cultured on tissue culture plates coated with 10 ␮g/ml human immunoglobulin G1 (IgG1) Fc (Accurate Chemical & Scientific Corp., Westbury, NY, USA) or CD137-Fc (R&D Systems, Minneapolis, MN, USA) protein. The endotoxin concentration in the CD137-Fc protein is 0.01 2958

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EU/␮g protein by the Limulus amebocyte lysate (LAL) method. TNFR1 activity was blocked by 10 ␮g/ml TNFR1neutralizing antibody (H398; Enzo Life Sciences, Plymouth, PA, USA). Mouse IgG1 antibody (MOPC21; Sigma-Aldrich) was included as negative isotype control. Human TNF (Peprotech, Rocky Hill, NJ, USA) was used at 10 ng/ml. Endogenous TNF was neutralized by 1 ␮g/ml recombinant human TNFR1-Fc (R&D Systems). Where indicated, cells were preincubated with Fc receptor (FcR) blocking reagent (Miltenyi Biotec, Gladbach, Germany) for 30 min at 4°C. Endocytosis was inhibited by pretreating THP-1 cells with 250 ␮M of monodansylcadaverine (MDC; Sigma-Aldrich) for 2 h. Subsequently, the cells were cultured on Fc or CD137-Fc protein for a further 16 h in the presence of 50 ␮M MDC. Plasmids and transfection The complete coding sequences of CD137L and its truncated mutants were generated by PCR amplification. Wild-type (WT)_CD137L (residues 1–254), CD137L_⌬CT (residues 25– 254) and CD137L_⌬EX (residues 1– 48) were cloned into pcDNA6/V5-His vector (Life Technologies) at the HindIII/ XbaI restriction sites. The plasmids were transiently transfected into MCF7 cells using Lipofectamine Plus reagent (Life Technologies), according to the manufacturer’s instructions. RNA isolation and RT-PCR Total RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany) and treated with RNase-free DNase set (Qiagen). Semiquantitative RT-PCR was performed using the OneStep RT-PCR Kit (Qiagen). A forward primer (5=-CAGAAAACCACCTCAGACAC-3=) and a reverse primer (5=GTGTTCTGTTTCTCCTGGCA-3=) were used to amplify a TNFR1 fragment of 217 bp from 1 ␮g of RNA. GAPDH was included as loading control. RT-PCR products were analyzed by agarose gel electrophoresis. Immunofluorescence Cells in suspension or grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton-X 100. Nonspecific sites were blocked with 1% BSA (SigmaAldrich). Subcellular localization of CD137L and TNFR1 was detected by mouse anti-CD137L (41B-436; AdipoGen, Seoul, South Korea) and rabbit anti-TNFR1 (ab19139; Abcam, Cambridge, UK) antibodies, followed by Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG (Life Technologies) secondary antibodies. Background staining was controlled by mouse IgG1 (MOPC21) antibody. In MCF7 cells overexpressing CD137L, the extracellular CD137L domain was detected with mouse anti-CD137L (41B-436) antibody, while the cytoplasmic CD137L domain was detected with a custom-made anti-CD137L monoclonal antibody (GenScript, Piscataway, NJ, USA). Suspension cells were centrifuged onto glass slides using a cytospin. Fluorescence was visualized by the confocal microscope Olympus FV1000 (Olympus, Tokyo, Japan). Flow cytometry For detection of surface expression of CD137L and TNFR1, cells were blocked in 1% BSA and incubated with mouse anti-CD137L (41B-436) and rabbit anti-TNFR1 (ab19139) antibodies for 2 h at 4°C. The primary antibodies were detected by goat anti-mouse IgG eFluor 660 (eBioscience, San

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Diego, CA, USA) and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) antibodies. Nonspecific staining was controlled by mouse IgG1 antibody (MOPC21). Annexin V-PE/ 7-AAD staining was carried out according to the manufacturer’s recommendations (eBioscience). Anti-human CD14APC, CD44-APC, CD83-PE, and CD86-PE antibodies were purchased from eBioscience. Flow cytometry was performed on a CyAn ADP analyzer (Beckman Coulter, Brea, CA, USA). Data were analyzed and compensated using the Summit software (Beckman Coulter). ELISA Concentrations of human IL-6, IL-8, TNF, and TNFR1 in cell culture supernatants were determined using the appropriate DuoSet ELISA development reagents from R&D Systems as per the manufacturer’s instructions. Western blot and coimmunoprecipitation Cells were lysed in radioimmunoprecipitation assay buffer supplemented with protease inhibitors. For coimmunoprecipitation experiments, cells were lysed in 1% Nonidet P-40 lysis buffer containing protease inhibitors. The precleared cell lysates were incubated with rabbit anti-TNFR1 (C25C1; Cell Signaling, Beverly, CA, USA) or rabbit mAb IgG (clone DAIE; Cell Signaling) antibody and protein G agarose beads (Thermo Scientific, Rockford, IL, USA) overnight at 4°C. Mouse anti-epidermal growth factor receptor (EGFR) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was included as a negative control. To determine the direct interaction between CD137L and TNFR1, recombinant human CD137L (R&D) and TNFR1 (obtained from human sTNF RI/TNFRSF1A DuoSet; R&D) proteins in 1% Nonidet P-40 lysis buffer were agitated with rabbit anti-TNFR1 (ab19139) or normal rabbit IgG (Santa Cruz Biotechnology) antibody overnight at 4°C, followed by 1 h incubation with protein G agarose beads. The immunoprecipitates were washed 4 times with 1% Nonidet P-40 lysis buffer, resuspended in 2⫻ Laemmli sample buffer, and boiled for 10 min. Protein samples were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Richmond, CA, USA). CD137L and TNFR1 proteins were detected by mouse anti-CD137L (41B-436) and rabbit antiTNFR1 (C25C1) antibodies, respectively. Mouse anti-GAPDH antibody was purchased from Santa Cruz Biotechnology. Expression of exogenously expressed CD137L was detected by mouse anti-V5-horseradish peroxidase (HRP) antibody (Life Technologies). Bound unconjugated primary antibodies were detected with appropriate HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence (Thermo Scientific).

RESULTS Expression of CD137L and TNFR1 in THP-1 cells The total protein expression of TNFR1 and CD137L was examined in the human immune cell line THP-1 by Western blot analysis (Fig. 1A). The result shows that both TNFR1 and CD137L are expressed in THP-1 cells. These data were confirmed by flow cytometry analysis. About 80% of cells express both TNFR1 and CD137L on the cell surface (Fig. 1B). Functional effects of CD137L reverse signaling on THP-1 cells CD137L signaling can be induced in CD137L-expressing cells by a CD137-Fc fusion protein when it is immobilized onto the cell culture plates (14). THP-1 cells were cultured on plates with immobilized CD137-Fc or control Fc protein or on uncoated (PBS) plates for 24 h. After incubation, the floating cells were removed, and the attached cells were photographed. Cross-linking of CD137L by immobilized CD137-Fc protein increased the adhesion of THP-1 cells on the culture plates (Fig. 2A), while the majority of the cells cultured on plates that were either not coated or coated with the Fc control protein remained in suspension, with only a few attaching to the plates. In a separate experiment, the cell viability was assessed by annexin V/7-AAD staining after 48 h of stimulation. CD137L-activated cells had a lower percentage of viable cells (32.9%) compared to untreated (81.4%) or Fc-treated (78.7%) cells (Fig. 2B). Concomitantly, the percentage of early apoptotic (annexin V⫹/7-AAD⫺) cells

Cell adhesion assay Cells were treated for the indicated times. After treatment, suspended cells were harvested. Attached cells were photographed with a Zeiss Axiovert 40C/CFL inverted microscope (Carl Zeiss, Munich, Germany), and then harvested. The absolute number of suspended and attached cells was quantified by flow cytometry using CountBright absolute counting beads (Life Technologies) following the manufacturer’s instructions. Statistical analysis Nonparametric ANOVA was performed using the GraphPad InStat 3.0 (GraphPad, San Diego, CA, USA) software to calculate P values. Values of P ⬍ 0.05 were considered significant. TNFR1 IN CD137L SIGNALING

Figure 1. Expression of TNFR1 and CD137L in THP-1 cells. A) Western blot analysis of TNFR1 and CD137L using anti-TNFR1 (C25C1) and anti-CD137L (41B-436) antibodies. GAPDH was included as loading control. B) Flow cytometry analysis of the cell surface expression of TNFR1 and CD137L. Left dot plot displays isotype control. Right dot plot shows staining of TNFR1 and CD137L using anti-TNFR1 (ab19139) and anti-CD137L (41B-436) antibodies. 2959

Figure 2. Effects of CD137L activation on THP-1 cells. Cells were cultured on plates either uncoated (PBS) or coated with 10 ␮g/ml Fc or CD137-Fc. A) After 24 h of incubation, suspended cells were removed, and the attached cells were photographed under the inverted microscope (⫻10 magnification). B) Dot plots of annexin V/7-AAD staining to determine cell viability after 48 h incubation. C) Flow cytometry histograms showing surface CD14 expression. Values in histograms indicate percentages of positive cells and mean fluorescence intensities. D, E) Concentrations of IL-8 (D) and TNF (E) in cell culture supernatants as quantified by ELISA. Data represent means ⫾ sd. Representative data from 1 of 3 experiments are presented. **P ⬍ 0.001; ANOVA.

was higher in CD137-Fc-treated cells (61.5%) than in untreated (16.1%) or Fc-treated (17.6%) cells. Moreover, stimulation with CD137-Fc protein induced CD14 expression (Fig. 2C). Analysis of cell culture supernatants for cytokines revealed that CD137L engagement triggered IL-8 (Fig. 2D) and TNF (Fig. 2E) release from THP-1 cells. IL-6 was almost undetectable (data not shown). Therefore, these data indicate that reverse CD137L signaling induces activation and apoptosis of THP-1 cells. To rule out the possibility that FcRs on THP-1 cells contribute to the responses induced by CD137L, the cells were preincubated with FcR blocking reagent prior to CD137L stimulation. Inhibition of FcR did not cause significant changes in cell adhesion and apoptosis mediated by CD137L signaling (Supplemental Fig. S1), confirming the specificity of CD137L-induced activities. Since TNF is produced on CD137L activation (Fig. 2E), a possible role of TNF in CD137L-mediated activities was evaluated. As shown in Supplemental Fig. S2, neutralization of endogenous TNF by TNFR1-Fc had little or no effect on the CD137-Fc-mediated increase of CD14 or decrease of TNFR1 expression.

were stimulated with immobilized Fc or CD137-Fc protein. Western blot analysis clearly shows a reduced expression of total TNFR1 in cells treated with CD137-Fc protein (Fig. 3A). This observation is supported by flow cytometry and confocal microscopy analyses. An ⬃70% reduction in surface TNFR1 protein was detected in CD137-Fc-treated cells compared to unstimulated cells (Fig. 3B, C) This down-regulation seemed to be specific for TNFR1, since the expression levels of total (Fig. 3A) and surface CD137L (Fig. 3B) were unchanged by CD137L signaling, while expression of CD83, CD86, and CD44 was increased (Fig. 3B). To determine whether TNFR1 was suppressed at the transcriptional or translational level, RT-PCR was performed. The TNFR1 mRNA expression level in CD137Fc-stimulated cells was comparable to that of the controls (Fig. 3D), signifying that TNFR1 is regulated by CD137L signaling at the protein level.

Down-regulation of TNFR1 protein expression by CD137L cross-linking

To investigate whether TNFR1 protein was down-regulated through shedding of the receptor or through endocytic degradation, we measured soluble TNFR1 by ELISA in the cell supernatants and inhibited clathrinmediated endocytosis with MDC. Cells treated with

The expression of TNFR1 and CD137L was examined in unstimulated (PBS) THP-1 cells and in cells that 2960

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Increased processing of TNFR1 protein by CD137L activation


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Figure 3. Down-regulation of TNFR1 protein expression in CD137Lactivated THP-1 cells. Cells were cultured on plates either uncoated (PBS) or coated with 10 ␮g/ml Fc or CD137-Fc for 48 h. A) Western blot analysis of TNFR1 and CD137L using anti-TNFR1 (C25C1) and antiCD137L (41B-436) antibodies. B) Flow cytometric analysis of TNFR1 and CD137L. Analysis of CD83, CD86, and CD44 was included as a control. Values in histograms indicate percentages of positive cells and mean fluorescence intensities. C) Confocal images of surface TNFR1 expression on cells from B. Scale bar ⫽ 10 ␮m. D) Evaluation of TNFR1 mRNA expression by semiquantitative RT-PCR. GAPDH was included as loading control.

CD137-Fc secreted higher levels of TNFR1 than untreated or Fc-treated cells (Fig. 4A). In addition, MDC treatment resulted in an 18% increase in surface TNFR1 in CD137-Fc-stimulated cells compared to treatment with the solvent DMSO (control; Fig. 4B). Furthermore, the surface expression of CD137L was increased by 9% in CD137L-activated cells, while no increase was detected in Fc-treated cells on treatment with MDC. These data suggest that the loss of TNFR1 protein expression during CD137L-mediated reverse signaling can be attributed to an increased TNFR1 shedding, as well as to internalization of TNFR1.

croscopy. THP-1 cells either untreated (PBS) or treated with Fc or CD137-Fc protein were fixed, permeabilized, and stained with antibodies specific for CD137L and TNFR1. CD137L was expressed predominantly on the plasma membrane of cells in all treatment conditions. TNFR1, besides locating on the plasma membrane, displayed a strong intracellular staining, particularly in untreated cells (Fig. 5). Consistent with the results in Fig. 3, TNFR1, but not CD137L, had an overall reduced staining in Fc- and CD137-Fc-treated cells as compared to untreated cells. Partial colocalization of CD137L and TNFR1 was detected on the plasma membrane of THP-1 cells.

Colocalization of CD137L and TNFR1 on the cell surface of THP-1 cells

Physical association of CD137L and TNFR1

The subcellular localization and expression levels of CD137L and TNFR1 were visualized by confocal mi-

Since CD137L and TNFR1 colocalize on the plasma membrane, we investigated whether CD137L and TNFR1

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Figure 4. Increased TNFR1 processing in CD137L-activated THP-1 cells. A) Cells were either untreated (PBS) or treated with Fc or CD137-Fc for 48 h. Cell culture supernatants were harvested, and the concentration of soluble TNFR1 was measured by ELISA. Data represent means ⫾ sd. **P ⬍ 0.001; ANOVA. B) Cells were pretreated with 250 ␮M of MDC or the solvent DMSO for 2 h prior to culturing in the presence of Fc or CD137-Fc. Overlay histograms showing surface expression of TNFR1 and CD137L as determined by flow cytometry analysis. Data represent 1 of 3 experiments.

can interact. Coimmunoprecipitation was performed on MCF7 cells transiently transfected with CD137L plasmid. The cell lysates were immunoprecipitated with an anti-

TNFR1 antibody, and the immunoprecipitates were tested for CD137L by Western blot analysis using an anti-V5 antibody. The result demonstrates that CD137L binds TNFR1 but not EGFR, which was included as a negative control (Fig. 6A), thereby demonstrating the specificity of the CD137L-TNFR1 interaction. To identify the CD137L domain responsible for binding to TNFR1, plasmids encoding deletion mutants of CD137L were constructed (Fig. 6B) and transiently transfected into MCF7 cells. Confocal microscopy confirmed the expected expression pattern of the mutant CD137L constructs (Fig. 6C). As shown in the total cell lysate lanes of Fig. 6D, WT and truncated CD137L migrated as multiple bands possibly because of post-translational modifications. Moreover, the protein level of ⌬EX_CD137L was consistently lower than those of WT_CD137L and ⌬CT_CD137L, which is most likely due to a reduced stability of the protein. The predicted molecular masses of WT_CD137L, ⌬CT_CD137L, and ⌬EX_CD137L of ⬃27, 24, and 5 kDa, respectively, were used as references. Coimmunoprecipitation with the deletion mutants revealed that ⌬CT_CD137L, which lacks the cytoplasmic domain, bound to TNFR1, implying that binding to TNFR1 is mediated by the extracellular domain of CD137L. Indeed, no binding of ⌬EX_CD137L (lacking the extracellular domain) to TNFR1 was observed. Hence, the extracellular domain of CD137L is required for binding to TNFR1. The direct interaction of CD137L and TNFR1 was confirmed by coimmunoprecipitation of recombinant human CD137L and TNFR1 proteins (Fig. 6E). We have also attempted to capture the physical interaction of endogenously-expressed CD137L and TNFR1 in THP-1 cells. However, it is technically challenging because both proteins are ex-

Figure 5. Colocalization analysis of TNFR1 and CD137L. Fixed and permeabilized THP-1 cells were stained for TNFR1 and CD137L using antiCD137L (41B-436) and anti-TNFR1 (ab19139) antibodies. Fluorescence was visualized by confocal microscopy. Arrows indicate sites of colocalization of TNFR1 and CD137L. Scale bar ⫽ 10 ␮m.

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Figure 6. Coimmunoprecipitation of TNFR1 and CD137L. A) MCF7 cells were transiently transfected with V5-tagged, WT CD137L plasmid for 48 h before cell lysis. Equal amounts of precleared cell lysates were immunoprecipitated (IP) with anti-TNFR1 antibody (clone C25C1; left panel) or anti-EGFR antibody as negative control (right panel). Isotype-matched IgG was included as negative controls. Immunoprecipitates were analyzed for CD137L expression by immunoblotting (IB) using an anti-V5-HRP antibody. Total cell lysate (TL) was included as a positive control. IgG heavy chain detected with appropriate HRP-conjugated secondary antibody is shown as loading control. B) Schematic diagram of WT and truncated mutants of CD137L. EX, extracellular domain; TM, transmembrane domain; CT, cytoplasmic tail. C) MCF7 cells were transiently transfected with WT or truncated CD137L plasmids. Cells were stained with antibodies against the extracellular (extCD137L) or the cytoplasmic (cytCD137L) CD137L domain and visualized by confocal microscopy. D) Precleared cell lysates were immunoprecipitated with anti-TNFR1 antibody (clone C25C1) or rabbit mAb IgG (clone DAIE) as a control. Anti-V5-HRP antibody was used to detect CD137L in the immunoprecipitates. Arrows indicate the predicted molecular masses of WT and mutated CD137L. E) A mixture of recombinant human CD137L and TNFR1 proteins was immunoprecipitated with anti-TNFR1 antibody (clone ab19139) or normal rabbit IgG. CD137L expression was detected with anti-CD137L (clone 41B-436) antibody.

pressed at low levels in THP-1 cells. In addition, detergent solubilization is known to alter the native conformational state of transmembrane proteins, thus interfering with their ligand binding capacity (26).

ipates in CD137L-induced apoptosis. Moreover, inhibition of TNFR1 in CD137-Fc-stimulated cells significantly reduced CD14-expressing cells (Fig. 8D) and IL-8 production (Fig. 8E). Together, the data demonstrate that TNFR1 participates in the signaling of CD137L.

Functional significance of CD137L-TNFR1 interaction in THP-1 cells Soluble TNF is known to activate TNFR1. The role of TNFR1 in CD137L-mediated signaling was first evaluated by activating TNFR1 on THP-1 cells with recombinant human TNF and culturing the cells in the absence or presence of immobilized Fc or CD137-Fc. IL-8 concentrations were measured by ELISA after 48 h of stimulation. Although TNF induced IL-8 production in all treatments, an additive effect was observed in CD137L-activated cells (Fig. 7). This result was verified by neutralizing TNFR1 with a TNFR1-blocking antibody. THP-1 cells treated with either TNFR1-blocking antibody (H398) or control antibody (MOPC21) were grown in Fc- or CD137-Fc-coated plates for 24 or 48 h. At both time points, adhesion of CD137L-activated cells was impeded by TNFR1-blocking antibody (Fig. 8A, B). Analysis of cell viability revealed that neutralization of TNFR1 activity had little effect on Fc-treated cells, but resulted in a near 2-fold increase in survival of CD137Lactivated cells (Fig. 8C), indicating that TNFR1 particTNFR1 IN CD137L SIGNALING

Figure 7. Enhanced IL-8 production in TNFR1 and CD137L coactivated THP-1 cells. Cells untreated or treated with 10 ng/ml TNF were seeded onto plates either uncoated (PBS) or coated with Fc or CD137-Fc. After 48 h of incubation, the concentration of IL-8 in the harvested cell culture supernatants was measured by ELISA. Data represent means ⫾ sd. **P ⬍ 0.001; ANOVA. 2963

Figure 8. Effect of TNFR1 inhibition on CD137L-mediated reverse signaling. THP-1 cells either untreated or treated with 10 ␮g/ml mouse IgG1 isotype antibody or anti-TNFR1 blocking antibody were cultured in the presence of Fc or CD137Fc. A) Attached cells were photographed under the inverted microscope (⫻10 view). B) Absolute number of suspended and attached cells. Suspended and attached cells were harvested separately at the indicated time points and quantified using CountBright absolute counting beads. C) Dot plots of annexin V/7-AAD staining of cells 48 h post-treatment. D) Flow cytometry histograms showing surface CD14 expression. Values in histograms indicate percentages of positive cells and mean fluorescence intensities. E) ELISA assay of IL-8 concentration in the cell culture supernatants. Data represent means ⫾ sd. Representative data from 1 of 3 experiments are presented. **P ⬍ 0.001; ANOVA.

DISCUSSION CD137L reverse signaling plays important roles in immune responses (7). The lack of binding sites for signaling molecules and enzymatic activity in the cytoplasmic domain of CD137L made it challenging to delineate the signaling pathways responsible for the induced cellular activities. In this study, we initially found through colocalization and coimmunoprecipitation analyses that CD137L colocalizes with TNFR1 on the cell surface and directly binds to TNFR1 via its extracellular domain. Therefore, it is attractive to speculate that CD137L reverse signals via the TNFR1 pathway. Activation of TNFR1 by TNF mediates both proapoptotic and proinflammatory effects. On binding to TNF, TNFR1 recruits the adaptor protein TNFR1-associated death domain (TRADD) to its cytoplasmic death domain. TRADD, in turn, serves as an assembly platform for binding receptor interacting protein-1 and TNF-receptor-associated factor 2, leading to the activation of proinflammatory signaling pathways of NF-␬B and JNK (27–30). TRADD can also form a complex with Fas-associated death domain protein to mediate caspase activation and apoptosis (31). CD137L-activated THP-1 cells undergo both activation and apoptosis as displayed by the enhanced cell adhesion, CD14 expression, and production of proinflammatory cytokines (TNF and IL-8), as well as the increased cell death. Since TNFR1 is able to elicit similar responses, we investigated whether TNFR1 is involved in CD137L-mediated signaling. Costimulation 2964

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of TNFR1 and CD137L with recombinant TNF and CD137 proteins results in a greater increase in IL-8 release. Moreover, inhibition of TNFR1 activity with a TNFR1-neutralizing antibody inhibits cell adhesion and suppresses CD137L-mediated apoptosis and IL-8 production. The number of CD137-Fc-activated CD14expressing cells is also reduced. These data suggest that TNFR1 participates in CD137L reverse signaling in THP-1 cells. However, the downstream signaling cascade of CD137L-TNFR1 requires a detailed characterization. Our findings also show that the expression of TNFR1 can be regulated by CD137L. Ligation of CD137L by recombinant CD137 reduces total and membrane TNFR1 protein levels without affecting the mRNA expression. The loss of TNFR1 protein could be attributed to an accelerated turnover of TNFR1 in the presence of CD137L. Indeed, CD137L has been demonstrated to be internalized when engaged by CD137 (32) and could, therefore, lead to cointernalization of TNFR1 when the two proteins are associated. Several mechanisms for TNFR1 turnover have been described. TNF-TNFR1 complexes can be internalized via clathrin-dependent and caveolae-mediated endocytosis (33, 34). Endocytosis was initially regarded as a mechanism to terminate receptor signaling. However, it has been shown that TNFR1 internalization is required for TNF-induced apoptosis (35). Other turnover mechanisms that play a role in preventing excessive immune or inflammatory responses include proteolytic cleavage and


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shedding of the TNFR1 ectodomain (36), and exosomelike vesicle release of full-length TNFR1 (37). Experimentally, we have shown that inhibition of endocytosis by MDC partially restores surface expression of TNFR1 in THP-1 cells that are stimulated with CD137 protein. In addition, an increased level of soluble TNFR1 is detected in the cell culture supernatant of CD137L-activated cells. Taken together, the results indicate that the CD137L-mediated down-regulation of TNFR1 occurs through TNFR1 internalization and release into the extracellular environment. MDC also increases the surface expression of CD137L in CD137-stimulated cells. However, it is still unclear whether TNFR1 and CD137L internalize as a complex into the cells. CD137L reverse signaling induces TNF production (14). Consistently, an increased level of TNF is secreted from THP-1 cells stimulated with recombinant CD137 protein. The high concentration of soluble TNF could be sustained, at least in part, by the increased turnover of TNFR1. The reduction of membrane-bound TNFR1 might result in a decline in TNF uptake. In addition, the increase in soluble TNFR1 might delay the spontaneous degradation of TNF by binding and stabilizing the trimeric form of TNF (38). CD137L not only uses other molecules, i.e., TNFR1 as adaptor molecule in signaling, but it serves itself as an adaptor molecule for TLR4. CD137L and TLR4 associate via their cytoplasmic domains, which prolongs LPS-induced TNF secretion. Whether CD137L acts as a bridging molecule between TLR4 and TNFRI, thereby leading to the formation of a larger signaling complex remains to be determined by future research. If that were the case the proinflammatory activities of TNF and LPS could have their basis in an at least partly common signaling pathway. In summary, our findings show that CD137L binds directly to TNFR1 via its extracellular domain. Using THP-1 cells, we have demonstrated that engagement of CD137L by CD137 protein down-modulates TNFR1 expression by increasing TNFR1 internalization and shedding. Once activated, sCD137L utilizes TNFR1 to trigger cell activation and apoptosis in response to CD137L stimulation. This study was supported by a Singapore Agency for Science, Technology, and Research (A*STAR)–Hungary National Office for Research and Technology (NKTH) joint grant (10/1/21/24/637).

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Received for publication December 2, 2012. Accepted for publication April 1, 2013.

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