TLR2-Mediated B Cell Activation by Decoy Receptor 3 Suppresses

Decoy Receptor 3 Suppresses TLR2-Mediated B Cell Activation by Targeting NF-κB

This information is current as Zi-Ming Huang, Jhi-Kai Kang, Chih-Yu Chen, Tz-Hau of September 24, 2021. Tseng, Chien-Wen Chang, Yung-Chi Chang, Shyh-Kuan Tai, Shie-Liang Hsieh and Chuen-Miin Leu J Immunol published online 11 May 2012 http://www.jimmunol.org/content/early/2012/05/11/jimmun

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 11, 2012, doi:10.4049/jimmunol.1102516 The Journal of Immunology

Decoy Receptor 3 Suppresses TLR2-Mediated B Cell Activation by Targeting NF-kB

Zi-Ming Huang,* Jhi-Kai Kang,* Chih-Yu Chen,* Tz-Hau Tseng,* Chien-Wen Chang,* Yung-Chi Chang,*,1 Shyh-Kuan Tai,†,‡ Shie-Liang Hsieh,*,†,x,{ and Chuen-Miin Leu*

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of

B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam3CSK4-, and LPS-mediated B cell proliferation but did not affect

cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 Downloaded from may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-bR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-kB p65 activation and NF-kB–driven

luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by http://www.jimmunol.org/ DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-kB. The Journal of Immunology, 2012, 188: 000–000.

ecoy receptor 3 (DcR3, also known as TR6 or M68) is a receptor expressed by T lymphocytes (LIGHT), and TNF-like a soluble receptor that belongs to the TNFR superfamily. molecule 1A (TL1A) (1, 7, 8). DcR3 prevents FasL- and LIGHT- The gene encoding DcR3 produces a 300-aa polypeptide, induced tumor cell apoptosis by neutralizing the interactions of

D by guest on September 24, 2021 and it is found in humans but not in mice. DcR3 is barely detectable FasL and LIGHT with Fas and lymphotoxin b-receptor (LT-bR), in normal tissues; however, it is overexpressed in a variety of tumor respectively (1, 6, 7). DcR3 also promotes angiogenesis to help cells such as adenocarcinomas of the esophagus, stomach, colon, tumor growth by binding to TL1A and subsequently stimulating rectums, and pancreas (1–4) and in lymphomas and gliomas (5, 6). the proliferation of endothelial cells (9). Earlier reports showed that DcR3 binds to Fas ligand (FasL), ho- Accumulating evidence has demonstrated a potent immuno- mologous to lymphotoxin, showing inducible expression, and com- modulatory function of DcR3. DcR3 can neutralize T cell-mediated peting with HSV glycoprotein D for herpes virus entry mediator, apoptosis by binding FasL and LIGHT (1, 7). Furthermore, DcR3 may regulate T cell differentiation. The T cell response is biased to a Th2 phenotype with an increased IL-4/IFN-g ratio in a DcR3- *Institute of Microbiology and Immunology, National Yang-Ming University, Taipei transgenic mouse model (10). Another study reported that the 11221, Taiwan; †Department of Medicine, Institute of Clinical Medicine, National Yang-Ming University, Taipei 11221, Taiwan; ‡Department of Otolaryngology, Tai- intrathecal injection of the DcR3.Fc protein into mice with ex- x pei Veterans General Hospital, Taipei 11217, Taiwan; Infection and Immunity Cen- perimental autoimmune encephalomyelitis signiﬁcantly reduced ter, National Yang-Ming University, Taipei Veterans General Hospital, Taipei 11217, Taiwan; and {Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan the number of Th17 cells and the production of IL-17 (11). These observations suggest that DcR3 may favor the differentiation of 1Current address: Department of Pediatrics, University of California, San Diego, La Jolla, CA. Th2 cells and suppress Th1 and Th17 cells, although the mecha- Received for publication August 31, 2011. Accepted for publication April 9, 2012. nism by which DcR3 controls T cell differentiation remains to be This work was supported by National Science Council Grants NSC96-2320-B-010- elucidated. In addition to its neutralization activity, DcR3 has been 011-MY3 and NSC99-2320-B-010-005-MY3 and Taipei Veterans General Hospital demonstrated to have direct modulatory activities (also called Grants V95S5-008, V96S5-005, V97S5-001, and V98S5-005. reverse signaling) on various types of immune cells. Through its Address correspondence and reprint requests to Dr. Chuen-Miin Leu, Institute of binding to LIGHT, DcR3 induces signals and inhibits T cell Microbiology and Immunology, National Yang-Ming University, 155 Sec. 2, Li- Nong Street, Taipei 11221, Taiwan, Republic of China. E-mail address: cmleu@ym. chemotaxis (12). DcR3 has been reported to transduce reverse edu.tw signaling to modulate the differentiation and maturation of den- Abbreviations used in this article: DC, dendritic cell; DcR3, decoy receptor 3; FasL, dritic cells (DCs) and macrophages; intriguingly, these effects are Fas ligand; hIgG1, human IgG1; HSPG, heparan sulfate proteoglycan; LIGHT, independent of FasL, LIGHT, and TL1A (13–15). The DcR3 homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by ligands in myeloid lineage that mediate the regulation of DcR3 T lymphocytes; LT-bR, lymphotoxin b receptor; PI, propidium iodide; RA, rheuma- have been identiﬁed recently as heparan sulfate proteoglycans toid arthritis; SAC, Staphylococcus aureus Cowan strain; SAW, Staphylococcus au- (HSPGs), such as CD44v3 and sydecan-2 (16). Although DcR3 is reus Wood strain; SLE, systemic lupus erythematosus; TL1A, TNF-like molecule 1A. reported to modulate the functions of T cells, DCs, and macro- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 phages, whether it regulates B cell remains to be elucidated.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102516 2 DcR3 ATTENUATES B CELL ACTIVATION VIA SUPPRESSING NF-kB

B cell activation is the first step of the humoral immune response. and BALB/c mouse strains were used in this study, and similar results were After encountering T-independent or T-dependent Ags, B cells obtained. Ly294002 and PD98059 were purchased from Calbiochem. Bay undergo activation, proliferation, and differentiation to become 11-7082, Pam3CSK4, and CpG ODN1826 were obtained from InvivoGen (San Diego, CA). LPS and human IgG1 (hIgG1) were purchased from memory or plasma cells. Several microbial products, including Sigma-Aldrich (St. Louis, MO). Anti-human CD40 Ab and recombinant peptidoglycans, LPS, and unmethylated CpG oligonucleotides human IL-10 were purchased from R&D Systems (Minneapolis, MN). stimulate polyclonal B cell proliferation and may drive limited Anti-human IgM F(ab9)2 fragment was from Jackson ImmunoResearch plasma cell differentiation without the help of T cells (17–19). Laboratories (West Grove, PA). Recombinant human IL-2 was from Roche Applied Science (Mannheim, Germany). In the past decades, extensive studies have revealed the receptors responsible for the response to these microbial B cell mitogens, The production of recombinant fusion proteins namely the TLRs. The recombinant fusion proteins DcR3.Fc, DcR3.Fcmut, Fas.Fc, LT-bR.Fc, TLRs are pattern recognition receptors that play a critical role and DR3.Fc were expressed by the FreeStyle 293 expression system as in the regulation of innate and adaptive responses. There are 10 described previously (10, 14). Briefly, the expression vector containing the TLRs in humans and 12 TLRs in mice (20). Peptidoglycan and fusion protein was transfected into 293F cells, and the supernatant was the synthetic agonist Pam CSK are recognized by TLR1/2, LPS collected on days 2, 4, and 6. The purification of the fusion proteins was 3 4 performed on a Sepharose-protein A column (Amersham Biosciences), and binds to TLR4, and CpG binds to TLR9. TLR ligands stimulate the purity of fusion proteins was determined by silver staining. The bio- B cell activation, the increased expression of activation markers activity of DcR3.Fc was checked by THP-1 cell adhesion assay (14) before (such as CD25, CD69, CD80, CD86 and MHC class II), and cell it was applied to B cells. proliferation. The production of cytokines, including IL-12p40, The DcR3.Fc binding assay IL-12p70, IL-6, IL-10, and TNF-a, is simultaneously induced Downloaded from (21). Upon TLR activation, all TLRs except TLR3 recruit the Before staining, peripheral blood cells were incubated with 1 mg/ml human adaptor molecule MyD88 to the plasma membrane and evoke IgG to block Fc receptors, and mouse B220 cells were preblocked with 100 mg/ml anti-mouse CD16/32 mAb (clone 2.4G2). To assess DcR3.Fc a series of reactions that lead to the activation to NF-kB and binding, the cells were incubated with biotinylated DcR3.Fc followed by AP-1, two transcription factors that regulate gene expression. TLR streptavidin-allophycocyanin with the PE-labeled lineage-specific markers signaling also activates MAPK pathways and the PI3K pathway and then analyzed by flow cytometry. To test if DcR3.Fc binds to HSPGs, 6

(22–25). Genetic approaches have shown the importance of the 10 cells were incubated with 10 mg/ml biotinylated DcR3.Fc in the ab- http://www.jimmunol.org/ NF-kB and PI3K pathways in TLR4-mediated B cell proliferation sence or presence of various concentrations of heparin or heparan sulfate on ice for 10 min. After washing with PBS, the cells were incubated with (26–28); however, the biochemical mechanisms by which other streptavidin-PE for an additional 10 min. The cells were washed twice with TLRs regulate B cell activation remain to be elucidated. PBS and analyzed by flow cytometry. In the current study, we found an unexpected effect of DcR3.Fc on B cell activation. DcR3.Fc binds to human and mouse B cells, The analysis of B cell proliferation and activation and it is able to suppress the activation and proliferation of B cells. The purified human B cells were incubated in 96-well plates (105/well in We found that resting B cells do not express FasL, LIGHT, or 200 ml RPMI 1640 supplemented with 10% FCS) for 72 h in the presence or absence of the fixed Staphylococcus aureus Cowan (SAC) or Wood (SAW) TL1A. Furthermore, the biological function of DcR3.Fc is not ∼

strains (Sigma-Aldrich). SAC produces 10-fold more protein A than does by guest on September 24, 2021 blocked by heparin. The administration of Fas.Fc, LT-bR.Fc SAW. The cells were pulsed for an additional 24 h with [3H]thymidine (LIGHT receptor), and DR3.Fc (TL1A receptor) does not suppress (1 mCi/well; PerkinElmer), harvested, and the [3H]thymidine incorporation B cell proliferation, suggesting that its effects are mediated via was assessed with a liquid scintillation counter. For the mouse splenic + 5 unknown ligand(s) on the B cells. We discovered that DcR3.Fc B cells, the purified B220 B cells (1 3 10 ) were plated and incubated with Pam3CSK4, LPS, or CpG in the presence or absence of hIgG1 attenuates the TLR2 agonist-induced cytokine expression. Finally, 3 (10 mg/ml) or DcR3.Fc (10 mg/ml). After 48 h, [ H]thymidine was added we found that the TLR2-stimulated NF-kB activation was sup- (1 mCi/well), and the cells were incubated for another 24 h. These cells pressed by DcR3. Taken together, our results reveal a regulatory were harvested with a cell harvester, and the incorporated radioactivity was function of DcR3 on B cells. measured using a b counter (PerkinElmer Wallac 1450 Microbeta Trilux Scintillation Counter; PerkinElmer). The stimulation percentage of test samples was calculated as follows: % stimulation = 100% 3 (cpm of the Materials and Methods test sample 2 cpm of medium control)/(cpm of the hIgG1 control 2 cpm of medium control). For the cell division assay, the purified B cells were Cells, Abs, and chemicals resuspended in PBS/5% FCS, and CFSE (Invitrogen, Carlsbad, CA) was Human and mouse cells were cultured in RPMI 1640 medium containing added to a final concentration of 5 mM and incubated for 10 min at room 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 10% temperature. The cells were then washed once with PBS/5% FCS. The FCS (Life Technologies, Grand Island, NY). The Ramos, Daudi, and cells were counted and stimulated as described. The data from 10,000 total IIA1.6 B cell lines were from Dr. Max D. Cooper (Emory University, events were acquired and analyzed using FACSCanto (BD Biosciences, Atlanta, GA). The Raji B cell line was from Dr. Cheng-po Hu (Tunghai Mountain View, CA). The cell-surface expression of CD69 was assessed University, Taichung, Taiwan). The THP-1 cell line was provided by 18–24 h after the cells were treated as described above. The percentage 3 Dr. Shie-Liang Hsieh. The BCL-1 cell line was generously provided by of inhibition is calculated as follows: 100% (percentage of the hIgG1 2 2 Dr. Kuo-I Lin (Academia Sinica, Taipei, Taiwan). The human tonsils and control percentage of the test sample)/(percentage of the hIgG1 control blood were obtained in accordance with the policies established by the percentage of medium control). Taipei Veterans General Hospital Institutional Review Board. The mono- nuclear cells in the tonsils or the buffy coat were isolated by Ficoll- The apoptosis assay Hypaque gradient centrifugation. The human CD19+ B cells and mouse + To quantify the apoptotic cells, the cells were incubated with FITC-Annexin B220 splenic B cells were purified using Ab-conjugated microbeads V and propidium iodide (PI) according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The cell purity was determined by staining (BD Pharmingen). The apoptotic cells were monitored by flow cytometry with either anti-human CD20 or anti-mouse B220 Abs. The PE-conjugated (FACSCanto; BD Biosciences). anti-human CD19, CD20, CD69, CD38, IgM, and IgD Abs were purchased from BD Pharmingen (San Diego, CA). The Alexa 488-conjugated anti- RT-PCR and quantitative RT-PCR mouse B220 and PE-conjugated anti-mouse CD69 Abs were from Bio- Legend (San Diego, CA). Anti-mouse CD16/32 Ab (clone 2.4G2) was PCR primers for detection of human FasL, LIGHT, and TL1A are human from BD Pharmingen. The plasma-derived human IgG was purchased FasL: sense, 59-CAGCTCTTCCACCTACAGAAGG-39 and antisense, 59- from Calbiochem (La Jolla, CA). All the animal experiments were con- CTCTTAGAGCTTATATAAGCCG-39; human LIGHT: sense, 59-AGA- ducted in accordance with the guidelines approved by the Animal Care and TCTTGACGGACCTGCAGGCTCC-39 and antisense, 59-CTTCACACC- Usage Committee of National Yang-Ming University. Both the C57B/L6 ATGAAAGCCC-39; human TL1A: sense, 59-GGAATTCCATGGCCGAG- The Journal of Immunology 3

GATCTGG-39 and antisense, 59-GTCTTCCGACTCTGGGATCAG-39; were detected by the incubation with specific anti–phospho-ERK or anti– mouse FasL: sense, 59-ACCACTACCACCGCCATCACAA-39 and anti- phospho-Akt Abs conjugated with HRP. The concentrations of phospho- sense, 59-CCAGAGATCAGAGCGGTTCCATA-39; mouse LIGHT: sense, ERK or phospho-Akt were evaluated by OD proportional to standards. 59-ATGGAGAGTGTGGTACAGCCTTC-39 and antisense, 59-GACCAT- GAAAGCTCCGAAATAGG-39; and mouse TL1A: sense, 59-AGTCCCA- Statistical analysis GTGGAAGTGCTG-39 and antisense, 59-GTGCTAAGTCCTGCGAGG- AT-39. To perform quantitative RT-PCR, the B220+ cells cultured for 8 h The two-tailed Student t test was used for the statistical analysis of the , with Pam CSK in the presence or absence of 10 mg/ml hIgG1 or DcR3.Fc results. The differences were considered to be significant when p 0.05. 3 4 , , , were harvested and lysed by TRIzol reagent (Invitrogen Life Technolo- *p 0.05, **p 0.01, ***p 0.005. gies). The RNA was purified, and the cDNA was generated by the Su- perScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s instructions. The PCR was performed on the LightCycler Results thermal cycler system (Roche Diagnositics, Mannhein, Germany) using DcR3.Fc binds to human and mouse B cells the LightCycler FirstStart DNA Master SYBR Green I system (Roche). The following primers were used in quantitative RT-PCR: 18S rRNA (59- In addition to DCs and macrophages, the DcR3.Fc fusion protein ACAATACAGGACTCTTTCGAG-39 and 59-GAGCTTTTTAACTGCAG- also bound to the B cells in the peripheral blood (Fig. 1A) and to CAAC-39); IL-6 (59-GCCTTCCCTACTTCACAAGT-39 and 59-GAATTG- mature human B cell lines (Raji and Daudi) (Fig. 1B). To test if CCATTGCACAACTCT-39); TNF-a (59-CCTCACACTCAGATCATCTT- DcR3.Fc could bind to all the differentiation stages of B cells, we C-39 and 59-CGGCTGGCACCACTAGTTG-39); IL-12p40 (59-ACATG- + GTGAAGACGGCCAG-39 and 59-GAAGTCTCTCTAGTAGCCAG-39); purified the CD19 B cells from the tonsils and performed the IL-10 (59-ATGCAGGACTTTAAGGGTTAC-39 and 59-CCTGAGGGTC- binding assay. Biotinylated hIgG1 was used as negative control. TTCAGCTTC-39); and IkB(59-AACCTGCAGCAGACTCCAC-39 and 59- Based on the expression of IgD and CD38, tonsillar B cells can GCTACACTGGCCAGGCAG-39). be divided into five populations: naive cells, pregerminal center Downloaded from The DNA-binding assay for NF-kB cells, germinal center cells, plasma cells, and memory cells (29). We found that all tonsillar B cells were able to bind DcR3.Fc to The purified splenic B cells were treated with 0.3 mg/ml Pam3CSK4 with 10 various degrees (Fig. 1C). The binding of DcR3.Fc to naive cells mg/ml hIgG1 or DcR3.Fc and incubated at 37˚C for 60 min. The cells were was strong and the highest in the pregerminal center cells. Fur- then harvested, and the nuclear extracts were collected by the Nuclear + Extract kit (Active Motif, Carlsbad, CA) according to the manufacturer’s thermore, DcR3.Fc also bound to the mouse spleen B cells (B220 ) instructions. The DNA binding activity of the NF-kB subunit p65 was and two mouse B cell lines (BCL-1 and IIA1.6) (Fig. 1B). Al- http://www.jimmunol.org/ assessed by the TransAM NF-kB kit (Active Motif). Briefly, 4 mg nuclear though the DcR3.Fc binding to splenic B cells was weak, it was extract was incubated in wells coated with oligonucleotides with the consistently observed in all of the experiments (n =6).The NF-kB consensus sequence for 1 h. After washing, an anti-p65–specific Ab was added to each well and incubated for 1 h. The secondary HRP- binding of DcR3.Fc was specific because DcR3.Fc did not bind conjugated Ab was then added and incubated for another hour. After the to the peripheral blood T cells (Fig. 1A). Based on these results, colorimetric reaction with the substrate, the absorbance was measured we speculated that DcR3 might regulate the function of human at 450 nm. and mouse B cells. The preparation of the lentiviral vector and the luciferase DcR3.Fc suppresses human B cell proliferation and activation

reporter assay by guest on September 24, 2021 To test if DcR3 could regulate the function of B cells, we first To generate a lentiviral reporter vector, a DNA fragment containing an examined its effects on B cell activation and proliferation. CD19+ NF-kB response element that drives the luciferase reporter gene luc2P was B cells were purified from the buffy coat of human peripheral blood amplified by PCR (forward primer, 59-CATAGGATCCTACCTGAGCT- CGCTAG-39; reverse primer, 59-GAAACTGCAGTTAGACGTTGATCCT- and stimulated with fixed S. aureus in the presence or absence of 39) using pGL4.32[luc2P/NF-kB-RE/Hygro] as a template. The PCR DcR3.Fc ex vivo. The purified human IgG1 was used as the neg- product was cleaved and cloned into a BamHI- and PstI-digested lentiviral ative control. S. aureus can stimulate B cell division and activation vector, pLKOAS3.puro (generously provided by Dr. Lih-Hwa Hwang, in a T cell-independent manner. Two strains of S. aureus were used Institute of Microbiology and Immunology, National Yang-Ming Univer- in this study: SAW and SAC. We found that DcR3.Fc inhibited sity, Taipei, Taiwan). This lentiviral reporter vector was named pLKOAS3- 3 NF-kB-RE-luc2P.puro. To prepare the lentiviruses, the pLKOAS3-NF-kB- SAW- or SAC-induced proliferation as measured by the [ H]thy- RE-luc2P.puro, pCMV-D R8.91, and pMD.G vectors (gifts from Dr. midine incorporation assay (Fig. 2A and data not shown) in human Lih-Hwa Hwang) were cotransfected into 293T cells. Fresh medium was B cells. DcR3.Fc suppressed B cell proliferation in 12 out of added after 6 h, and the supernatant containing the viral particles was 13 donors in .10 independent experiments with an average inhi- collected 40 and 64 h posttransfection. The viral supernatant was filtered 6 through a 0.22-mm filter and kept at 270˚C until use. The splenic B220+ bition of 50.7 11.9% (Fig. 2B, right panel). The effect of DcR3 cells were purified and stimulated with 10 mg/ml LPS for 24 h before was not limited to mitogen-stimulated proliferation. When we used transduction. The virions were added to the stimulated B cells in the anti-CD40 plus anti-IgM Abs to mimic T-dependent stimulation, presence of 10 mg/ml LPS and 8 mg/ml polybrene and centrifuged for 50 DcR3.Fc was also able to reduce B cell proliferation (Fig. 2B; 3 g min at 450 . The viral supernatant was removed after an overnight average inhibition 37.3 6 14.1%; n = 4). The inhibitory effect of incubation, and fresh medium containing 10 mg/ml LPS was added for another 24 h. The cells were moved to a medium without LPS for 2 h and DcR3 on SAW- or BCR-induced proliferation was specific because then plated and stimulated under various conditions for 6 h. The cells were DcR3.Fc did not affect IL-2– plus IL-10–mediated human B cell lysed, and the firefly luciferase activity was measured with the reagents proliferation (Fig. 2B; average inhibition 22.7 6 19.7%; n =3). from the Luciferase Assay System (Promega) according to the manu- The DcR3.Fcmut fusion protein cannot bind FcgR and showed facturer’s instructions. a similar inhibitory effect on human B cell proliferation, indicating The detection of the active forms of Erk and Akt that this suppression was attributed to the DcR3 portion (Fig. 2B, left panel). The purified splenic B cells were cultured for 30 min with Pam3CSK4 in the presence or absence of hIgG1 (10 mg/ml) or DcR3.Fc (10 mg/ml), Similarly, the SAC-induced B cell activation was suppressed by harvested, and lysed with RIPA buffer containing protease inhibitors and DcR3.Fc as evaluated by the upregulation of activation marker phosphatase inhibitors. The phosphorylation of Erk and Akt were detected CD69 (Fig. 2C). The B cells from four out of six donors responded with either the phospho-ERK1/2 Enzyme Immunometric Assay Kit (Assay + 473 to DcR3.Fc, and the percentage of CD69 cells was reduced by Designs, Ann Arbor, MI) or the phospho-Akt (Ser ) STAR ELISA Kit 6 6 (Upstate, Millipore) according to the manufacturer’s instructions. Briefly, 27.5% (hIgG1, 29.1 10.5% versus DcR3.Fc, 21.1 10.3%; the cell lysate was added to microtiter plates precoated with monoclonal p , 0.005). To confirm the antiproliferation activity of DcR3, we anti-ERK or anti-Akt Abs. After washing, the active forms of ERK or Akt used the CFSE assay to address the same question. Consistent 4 DcR3 ATTENUATES B CELL ACTIVATION VIA SUPPRESSING NF-kB

spleen B cells from C57BL/6 mice were puriﬁed and incubated with various stimuli. In accordance with its effect on human B cells, DcR3.Fc suppressed the SAW- or SAC-induced proliferation of mouse spleen B cells (data not shown). A similar inhibitory effect of DcR3 on the splenic B cells from BALB/c mice was also observed (data not shown). To study how DcR3 reduces B cell proliferation and activation, we examined its activity on TLR- mediated stimulation because the signaling pathways transduced by TLRs are relatively deﬁned. TLR2, TLR4, and TLR9 agonists were used to stimulate mouse B cells because all of them have been reported to induce B cell proliferation and activation (17, 19). We found that DcR3.Fc reduced the cell proliferation mediated by Pam3CSK4 and LPS but not by CpG (Fig. 3A). At a concentration of 10 mg/ml, DcR3.Fc suppressed the TLR2- induced B cell proliferation in 9 out of 10 mice with an average inhibition of 38 6 6% (Fig. 3A, bottom right panel). Similarly, DcR3.Fc reduced the LPS-stimulated [3H]thymidine uptake in seven out of eight mice by an average of 49.2 6 13.1%. The

antiproliferative activity of DcR3.Fc on the TLR2 and TLR4 Downloaded from ligands was further evaluated by the CFSE assay, and similar inhibitory results were observed (Fig. 3C). The percentage of Pam3CSK4-stimulated cell division was reduced from 36 6 9.4% to 23.2 6 5.7% (36% decrease; n =3,p , 0.05) by DcR3.Fc. Similarly, LPS-induced cell division was also suppressed by

DcR3.Fc (59.1 6 8.6% to 46.2 6 9.4%; n =3,p , 0.005) http://www.jimmunol.org/ Consistent with the cell proliferation, the CD69 upregulation by Pam3CSK4 and LPS was also substantially suppressed by DcR3 (Fig. 3B). DcR3.Fc treatment led to a 30.9% decrease of the + Pam3CSK4- stimulated CD69 cells (n =5;p , 0.005) and a 37% FIGURE 1. DcR3.Fc binds to B cells in humans and mice. (A) DcR3.Fc decrease of the LPS-stimulated cells (n =3;p , 0.005). Together, binds to blood B cells. PBMCs were incubated with 1 mg/ml human IgGs our observations indicate that DcR3.Fc selectively attenuates the to block Fc receptors. The cells were then incubated with biotin-labeled TLR2- and TLR4-mediated cell proliferation and activation in DcR3.Fc or hIgG1 followed by streptavidin-conjugated allophycocyanin. mouse B cells.

The Abs to the lineage-specific markers (CD3, CD20, and CD14) were by guest on September 24, 2021 added to identify each cell type. Filled peak, hIgG1-biotin; thin line, DcR3. DcR3.Fc does not affect B cell apoptosis in vitro Fc-biotin. One representative result of four independent experiments is shown. (B) DcR3.Fc binds to B cell lines. Raji and Daudi are human B cell Recently, DcR3 has been reported to induce the apoptosis of human lines; THP-1 is a human monocytic cell line; B220+ indicates the purified DCs (30). This raises the possibility that the suppression of pro- mouse splenic B220+ B cells. BCL-1 is a surface IgM+ murine B cell line. liferation by DcR3.Fc may be due to the induction of B cell death. + IIA1.6 is a surface IgG+ mouse B cell line. Filled peak, hIgG1-biotin; To test this, we treated human CD19 B cells with or without thin line, DcR3.Fc-biotin. One representative result of at least three in- DcR3.Fc in the presence of SAC and measured the percentage of dependent experiments is shown. (C) DcR3.Fc binds to all stages of B apoptotic cells. We found that the SAC treatment slightly reduced + cells. CD19 tonsillar B cells were purified by magnetic beads and used to the Annexin V-positive cells compared with the medium control A perform the DcR3.Fc binding assay as described in ( ), except that anti- (Fig. 4A; the dot plots show one representative result, and the bar IgD and CD38 Abs were used to dissect the five stages of B cells. One graph is a summary of three donors). In the presence of SAC, representative result of five independent experiments is shown. GC, ger- DcR3.Fc marginally augmented the percentage of apoptotic cells minal center cells. (by an average of ∼5% at 24, 48, or 72 h) when compared with hIgG1. To further examine the direct effect of DcR3 on human with the [3H]thymidine uptake results, we observed that DcR3.Fc B cell apoptosis, we cultured CD19+ B cells with DcR3.Fc or reduced the SAC- or BCR-induced cell division (Fig. 2D). DcR3. hIgG1 and measured cell apoptosis. Compared to the control, Fc decreased cell division stimulated by SAC by 38% (hIgG1, DcR3.Fc did not increase human B cell apoptosis at 24, 48, or 72 h 22 6 8.6% versus DcR3.Fc, 13.6 6 9%; p , 0.005, n = 5) and by (Fig. 4C, left panel), although 10 mg/ml DcR3.Fc was reported to the anti-IgM plus anti-CD40 Abs by 20% (hIgG1, 32.4 6 18.1% dramatically induce DC apoptosis (30). Because DcR3.Fc only versus DcR3.Fc, 25.8 6 16.3%; p , 0.05, n = 3). In contrast, showed a marginal effect on human B cell apoptosis in the pres- DcR3.Fc slightly increased cell division induced by IL-2 plus IL-10 ence of SAC, and because it did not induce apoptosis alone, we by 8% (hIgG1, 40.8 6 10.9% versus DcR3.Fc, 44.4 6 12.7%; conclude that DcR3.Fc does not affect B cell apoptosis in humans. n = 4). Together, these results demonstrate that DcR3 specifically We have also examined the effect of DcR3.Fc on mouse B cell suppresses the SAW- and BCR-induced cell proliferation and apoptosis. As shown in Fig. 4B, .60% of the purified splenic activation in human B cells. B cells died when they were cultured in vitro for 24 or 48 h, and Pam3CSK4 treatment significantly reduced the number of apo- DcR3.Fc suppresses the TLR2- and TLR4-mediated mouse ptotic cells (medium, 70.5 6 13.5% versus Pam3CSK4, 49.2 6 B cell activation and proliferation 14% at 24 h and 83.5 6 8.1% versus 40.1 6 16.4% at 48 h; n = 7). In addition to human B cells, DcR3.Fc bound to mouse splenic In the presence of Pam3CSK4, DcR3.Fc did not increase mouse B cells and B cell lines (Fig. 1B). Therefore, we tested the effect of B cell apoptosis at 24 h. At 48 h, the DcR3.Fc treatment slightly DcR3.Fc on mouse B cell proliferation and activation. The B220+ increased the number of Annexin V+ cells (Fig. 4B, bar graph; The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ FIGURE 2. DcR3.Fc suppresses the proliferation and activation of human B cells. (A) DcR3.Fc attenuates S. aureus (Wood strain, marked as SAW)- induced cell proliferation. The CD19+ B cells were purified to ∼99% purity from the peripheral blood using magnetic beads. The cells (2 3 105) were seeded in 96-well plates and cultured with the indicated reagents for 3 d. After adding 1 mCi [3H]thymidine to each well, the cells were cultured for additional 24 h and harvested to detect the incorporated isotope. The results are presented as the means 6 SD of triplicate samples. The experiments were performed .10 times, and a set of representative data is presented. (B) DcR3.Fc inhibits anti-IgM and anti-CD40 Ab-stimulated cell proliferation. The B cells were stimulated either with anti-IgM (5 mg/ml) and anti-CD40 (1 mg/ml) or with anti-CD40 (1 mg/ml) plus IL-2 (100 U/ml) and IL-10 (25 ng/ml) in the presence of 10 mg/ml hIgG1, DcR3.Fc, or DcR3.Fcmut. Left panel, One representative of three independent experiments is shown. Right panel is a summary of all proliferation results in human B cells. Percentage of stimulation was calculated as described in Materials and Methods.(C) DcR3.Fc suppresses SAC-stimulated cell activation. The peripheral B cells were purified and stimulated as described in (A), and the cells were harvested 24 h later to stain CD69 and analyzed by flow cytometry. Dashed line, isotype control; filled peak and thin line, anti-CD69-PE. A set of representative data of four by guest on September 24, 2021 independent experiments is shown. (D) DcR3.Fc decreases the SAC- and BCR-induced B cell division. Filled peak, labeled cells without stimulation; thin line, cells after in vitro culture for 4 d. One representative of three to four independent experiments is shown. *p , 0.05, ***p , 0.005. hIgG1, 42.2 6 17.3% versus DcR3.Fc, 48.2 6 19.4%; n = 7). To the inhibition reached ∼100% at the concentration of 100 mg/ml further assess the role of DcR3.Fc on mouse B cell apoptosis, we (Fig. 5B). However, heparin could not block the DcR3.Fc binding incubated splenic B cells with hIgG1 or DcR3.Fc and measured to the human blood CD19+ B cells (Fig. 5B). Similarly, the cell apoptosis. We did not observe an increase of apoptotic cells binding of DcR3.Fc to the mouse splenic B220+ B cells was not by the DcR3.Fc treatment at 6, 18, 24, and 48 h (Fig. 4C, right changed by the addition of heparin, heparan sulfate, chondroitin panel and data not shown; n = 2–4). Taken together, these data sulfate, or dermatin sulfate (data not shown). Consistent with these show that DcR3.Fc seems to have little effect on the apoptosis of results, heparin did not neutralize the inhibitory activity of DcR3. mouse B cells. Fc in human B cell (Fig. 5C). Although we could not detect the expression of FasL, LIGHT, or The suppression of B cell proliferation by DcR3 is not TL1A in the resting B cells from humans and mice, these DcR3 correlated with FasL, LIGHT, TL1A, and HSPGs ligands might be upregulated upon B cell activation and subse- Next, we examined what receptor mediates the suppressive effect quently mediate the suppressive effect. To further test the in- of DcR3 in B cells. DcR3 has been reported to bind FasL, LIGHT, volvement of FasL, LIGHT, and TL1A in the suppression of B cell TL1A, and HSPGs (such as CD44v3 and sydecan-2). Using RT- proliferation, we used the recombinant fusion proteins Fas.Fc, PCR, we were unable to detect the expression of FasL, LIGHT, LT-bR.Fc, and DR3.Fc, which are receptors for FasL, LIGHT, or TL1A in the human peripheral blood CD19+ cells, the naive or TL1A, respectively, to test if they have the same suppressive tonsillar B cells, and the mouse splenic B220+ B cells (Fig. 5A and effects. Our results showed that neither the addition of each one of data not shown). these receptor fusion proteins nor the combination of all three To test if DcR3 can bind to B cells via HSPGs, we used heparin, receptors could reproduce the antiproliferative activity of DcR3.Fc heparan sulfate, and other polysaccharides to determine if they (Fig. 5D and data not shown). Based on these observations, we could prevent the DcR3 binding to human and mouse B cells. Our assume that the attenuation of DcR3 on B cells is mainly mediated results show that none of the tested carbohydrates could block by unknown ligand(s) and not by any of these identified ligands. the DcR3.Fc binding to B cells (Fig. 5B and data not shown), k including heparin and heparan sulfate, two HSPGs reported to DcR3.Fc suppresses the TLR2-induced NF- B activation and dramatically inhibit the binding and function of DcR3.Fc in cytokine expression THP-1 cells (16). We found that heparin significantly reduced the To elucidate the signaling pathway affected by DcR3, we first- DcR3.Fc binding to THP-1 cells in a dose-dependent manner, and dissected the signaling pathway critical for the TLR2- and 6 DcR3 ATTENUATES B CELL ACTIVATION VIA SUPPRESSING NF-kB Downloaded from

FIGURE 3. DcR3.Fc suppresses the TLR-mediated proliferation and activation of mouse B cells. (A) DcR3.Fc suppresses the Pam3CSK4- and LPS- induced B cell proliferation. Spleen B220+ cells (105) were seeded in 96-well plates and treated with the indicated reagents (concentration unit is mg/ml). A representative dataset of three to seven independent experiments is shown. The bottom right panel is a summary of proliferation results in mouse B cells.

Pam3CSK4 (n = 9), LPS (n = 7), and CpG (n = 3). (B) DcR3.Fc suppresses the Pam3CSK4- and LPS-induced B cell activation. CD69 expression in the cells http://www.jimmunol.org/ treated with 0.1 mg/ml Pam3CSK4 or 0.1 mg/ml LPS in the presence of 10 mg/ml hIgG1 or 10 mg/ml DcR3.Fc was measured 24 h after incubation. A representative dataset of three to ﬁve independent experiments is shown. (C) DcR3.Fc attenuates the Pam3CSK4- or LPS-induced cell division. Filled peak, labeled cells without stimulation; thin line, cells after in vitro culture for 3 d. One representative experiment of three is shown. **p , 0.01, ***p , 0.005.

TLR4-mediated B cell proliferation. In addition to the activation of The puriﬁed splenic B220+ cells were transduced with a lentiviral NF-kB and IFN regulatory factors, TLRs also activate ERK1/2 NF-kB reporter and stimulated with Pam3CSK4 in the presence of and PI3K/Akt pathways (22–25). Because the major function of hIgG1 or DcR3.Fc. As shown in Fig. 6D, Pam3CSK4 increased NF- IFN regulatory factors is to regulate the expression of type I IFNs, kB promoter activity in the presence of hIgG1 by ∼5-fold; however, we focused on the role of the NF-kB, ERK, or PI3K pathways in DcR3.Fc suppressed the induction of NF-kB activity by 2.8-fold by guest on September 24, 2021 the TLR-mediated B cell proliferation. As shown in Fig. 6A, the (10 mg/ml) or 1.9-fold (30 mg/ml). The average inhibition of the cell proliferation induced either by Pam3CSK4 or LPS was dra- NF-kB reporter activity by 10 mg/ml DcR3.Fc was 58.5 6 9.7% matically suppressed by NF-kB or PI3K inhibitors (BAY11-7082 (n = 3). A similar inhibitory effect of DcR3 was also observed in and LY294002, respectively) in a dose-dependent manner. At a the LPS-stimulated reporter assay (data not shown). These results concentration of 0.5 mM, the NF-kB inhibitor blocked the TLR2- indicate that DcR3 suppressed the TLR2- and TLR4-induced acti- or TLR4-stimulated cell growth almost completely. The PI3K vation of NF-kB. To further support this idea, we examined the inhibitor Ly294002 was less potent than the NF-kB inhibitor; expression of proinﬂammatory cytokines, such as IL-6, TNF-a,and however, it also completely suppressed cell proliferation at the p40 subunit of IL-12, following TLR2 stimulation. We found 10 mM. By contrast, the MEK inhibitor PD98059 had very little that DcR3.Fc reduced the TLR2-induced cytokine expressions effect on the TLR2 or TLR4-mediated B lymphocyte prolifera- by 50% (Fig. 6E, compare lane 4 to lane 3). Two other NK-kB tion. At a concentration of 20 mM, PD98059 did not suppress the target genes, IL-10 and IkB, were also suppressed by DcR3.Fc. The Pam3CSK4- or LPS-induced cell growth (data not shown). These levels of TLR2 and PTEN mRNA remained unchanged by the observations indicate that the NF-kB and PI3K pathways play treatment of DcR3.Fc (data not shown). Taken together, our results major roles in the TLR2- and TLR4-mediated B cell proliferation. demonstrate that DcR3.Fc partially suppresses the activation of the We hypothesized that these two pathways may be the targets for NF-kB pathway and subsequently reduces B cell activation and DcR3. proliferation. To test our hypothesis, we measured the activation of Akt and NF-kB upon TLR2 stimulation with or without DcR3.Fc. An Discussion ELISA-based analysis was used to detect the active forms of Akt DcR3 has been reported to regulate the differentiation and func- (pAkt), ERKs (pERKs), and NF-kB after the B220+ B cells were tions of T cells, macrophages, and DCs; however, its effect on stimulated in vitro by Pam3CSK4. The TLR2 agonist induced a B cells remains largely unknown. In this study, we demonstrated 2.5-fold increase of pERKs and a 3–5-fold increase of pAkt (Fig. that DcR3.Fc suppressed B cell activation and proliferation, and 6B); however, DcR3.Fc did not attenuate the activation of ERKs or this effect was not due to the induction of cell death. Furthermore, Akt. In contrast, the DNA-binding activity of NF-kB p65 subunit the NF-kB activation and cytokine expression stimulated by TLR was substantially reduced by DcR3.Fc upon Pam3CSK4 treatment agonists were substantially reduced by DcR3. The inhibition of in a dose-dependent manner (Fig. 6C). In three independent B cells by DcR3 might be mediated by a novel ligand(s) of DcR3. experiments, DcR3.Fc suppressed the NF-kB DNA-binding activity In summary, our results indicate a very complex role for DcR3 in by 28.3 6 5.5% or 54.3 6 16.8% at a concentration of 10 or the regulation of the immune system. In addition to neutralizing 30 mg/ml, respectively. To ascertain that DcR3 suppressed NF-kB or inducing reverse signaling in T cells and myeloid cells, DcR3 activation, an NF-kB–driven luciferase reporter system was used. may also modulate B cell activation. When this article was under The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. DcR3.Fc does not induce B cell apoptosis. The human peripheral blood CD19+ B cells (A) and mouse splenic B220+ B cells (B) were purified and incubated with the indicated reagents in culture for various time periods. The cells were harvested at indicated time point and stained with Annexin V and PI and then analyzed by flow cytometry. A total of 0.3 mg/ml Pam3CSK4, 10 mg/ml hIgG1, and 10 mg/ml DcR3.Fc was used. Dot plots are repre- sentatives of three (A) or five (B) independent experiments. Bar graphs represent the mean Annexin V+ percentages 6 SD of data. n =3(A); n =7(B). (C) DcR3.Fc does not induce B cell apoptosis by itself. Human blood B cells (left panel, n = 3) and mouse splenic B220+ B cells (right panel, n = 4) were purified and incubated with 1, 3, and 10 mg/ml DcR3.Fc, and the cells were stained with Annexin V and PI at indicated time points. The data are shown as the mean percentages 6 SD of all the independent experiments. review, Cheng et al. (31) reported that DcR3.Fc suppresses (30). However, we did not observe any significant anti- or pro- Pam3CKS4-induced mouse B cell proliferation in vitro and apoptotic activity of DcR3.Fc on the B cells. This phenomenon collagen-specific IgG2a production in a collagen-induced arthritis is well demonstrated in human B cells (Fig. 4A). In the case model. Altogether, their and our findings support the idea that of mouse B cells, we consistently observed a marked decrease DcR3 may regulate B cell proliferation. in the number of apoptotic mouse B cells upon the addition of DcR3 was found to prevent the FasL- or LIGHT-mediated ap- Pam3CSK4 (Fig. 4B). This phenomenon seems to be in contrast optosis of tumor cells (1, 6, 7) and the activation-induced cell with the dogma that activation induces apoptosis in lymphocytes. death of T cells (10). On the contrary, a high concentration of Under our experimental conditions, mouse B cells undergo DcR3.Fc (10 mg/ml) activates DC apoptosis: it induced a 3.2-fold spontaneous apoptosis without stimulation. The addition of the and 2.4-fold increase of apoptotic DCs at 36 and 72 h, respectively TLR2 agonist induces massive cell division, thereby increasing 8 DcR3 ATTENUATES B CELL ACTIVATION VIA SUPPRESSING NF-kB

FIGURE 5. The antiproliferation activity of DcR3 is not related to any known ligands of DcR3. (A) RT-PCR of FasL, LIGHT, and TL1A in human B cells. 2,H2O control; +, positive control; Ramos, cDNA from Ramos B cell line; PBMC-B, cDNA from purified human blood B cells; b2m, b2-microglobulin. One representative of six experiments is shown. (B) Heparin does not block the binding of DcR3.Fc to human B cells. The THP-1 or PBMC cells were incubated with 10 mg/ml biotin-labeled DcR3. Fc in the absence or presence of various concentrations of heparin (1–100 mg/ml). After washing, streptavidin-PE was added before flow cytometry analysis. Additional anti-CD20–APC Ab was used to mark the B cells in PBMCs, and the analysis was gated on the CD20+ cells. The DcR3.Fc-bound cells were gated to determine the percentage of positive-staining cells. A representative experiment of five is shown. (C Downloaded from and D) The inhibitory effect of DcR3.Fc is not mediated by any known ligands. The proliferation of peripheral human B cells was performed as described in Fig. 2, except that 100 mg/ml heparin or 10 mg/ml Fas.Fc, LT-bR.Fc, or DR3.Fc was used in the assay. One representative dataset of three independent experi- http://www.jimmunol.org/ ments is shown.

the total cell number, whereas the nondividing cells keep dying. increased (Fig. 1C). Whether the enhanced binding of DcR3 can be When the speed of cell division exceeds that of cell death, an attributed to LIGHT, FasL, or TL1A requires further investigation. increase in the number of live cells after stimulation should be However, Fas.Fc, DR3.Fc, and LT-bR.Fc were unable to inhibit B detected. In the literature, activation-induced cell death is trig- cell proliferation (Fig. 5D). Collectively, our data indicate the ex- gered by BCR or CD40 signaling alone (32, 33). Although CpG istence of a novel DcR3 ligand on B cells, and we favor the hy- does not regulate mouse splenic B cell death, LPS is able to pothesis that DcR3 binds to a new receptor(s) and suppresses B cell by guest on September 24, 2021 suppress cell apoptosis (33, 34). TLR2 has been reported to me- activation. We do not yet know the identity of this novel receptor. diate apoptosis in the human THP-1 monocytic cell line and in The data presented in this study reveal differences between the mouse microglia cells (35, 36), but its role in B cell apoptosis myeloid cells and B cells in response to DcR3. Most obviously, remains largely unknown. Our observations suggest that the ac- apoptosis and MHC class II expression (Y.-C. Chang, S.-L. Hsieh, tivation of the TLR2 signaling pathway may not induce B cell and C.-M. Leu, unpublished observations) in B cells were not apoptosis. Whether Pam3CSK4 inhibits apoptosis requires further regulated by exogenous DcR3. This may be due to the differential investigation. In the presence of Pam3CSK4, the DcR3.Fc treat- ligand expression in these two lineages. Monocytes and DCs ment did not affect mouse B cell apoptosis at 24 and 48 h. We also express HSPGs (including CD44v3 and Syndecan-2) and TL1A did not observe an induction of B cell apoptosis by the addition (40–42), and HSPGs are important in the induction of apoptosis of of DcR3.Fc alone. Compared to its significant effect on DC ap- DCs (30) and reduction of MHC class II expression in monocytes. optosis, we conclude that DcR3.Fc may not regulate spontaneous TL1A can be secreted or expressed on the surface of monocytes cell death of primary B cells, although we cannot rule out the and DCs. Because the membrane-bound form of TL1A contains possibilities that DcR3 may block Fas- or activation-induced a cytoplasmic region, DcR3 binding to the surface TL1A may B cell apoptosis. transduce a signal. The influence of DcR3 on monocytes and DCs We have shown four lines of evidence indicating the existence may be caused by the combination of all of the signals evoked of a novel DcR3 receptor(s) on B cells. First, we were unable to by HSPGs and TL1A. However, there is no TL1A, CD44v3, or detect the expression of any known ligands of DcR3 in the resting Sydecan-2 expression in B cells, and our results demonstrated that B cells. Secondly, heparin, heparan sulfate, and all of the carbo- the antiproliferative effect of DcR3 is independent of HSPGs. This hydrates tested failed to block the binding of DcR3.Fc to the B cells. suggests that there are distinct regulatory roles for DcR3 on the Third, heparin failed to reverse the inhibitory effect of DcR3 on the different APCs. The different subsets of DcR3 ligands expressed human B cells. Fourth, Fas.Fc, LT-bR.Fc, and DR3.Fc did not show on the APCs might help fine-tune the functions of the APCs. a similar suppression effect as DcR3 did. The last result was es- In this study, we demonstrated that DcR3 suppressed NF-kB pecially critical because the upexpression of FasL and LIGHT in activation in B cells. B cells under various conditions has been reported before (37–39). In addition, our unpublished observations (M.-H. Chen, Z.-M. We have detected low levels of LIGHT mRNA in the CD38+ Huang, C.-M. Leu, and H.-Y. Lin) indicate that high level of DcR3 pregerminal and germinal center cells from two tonsil samples in the synovial fluid from rheumatoid arthritis (RA) patients is (C.-Y. Chen and C.-M. Leu, unpublished observations), which associated with low RA disease activity (Disease Activity Score raises the possibility that the activated B cells may express known 28). Together with the previous findings that DcR3 promotes Th2- ligands of DcR3. Consistent with this observation, we found that but suppresses Th1- and Th17-type responses (10, 11), these data the DcR3.Fc binding to the pregerminal center cells subsequently lead us to propose that the transient upregulation of DcR3 may The Journal of Immunology 9 Downloaded from

FIGURE 6. DcR3.Fc reduces NF-kB activity in the mouse B220+ B cells. (A) The NF-kB and PI3K inhibitors block the TLR2- or TLR4-induced B cell http://www.jimmunol.org/ + proliferation. The purified splenic B220 cells were incubated with Pam3CSK4 or LPS in the absence or presence of various concentrations of the inhibitor of PI3-K (LY294002) and NF-kB (BAY11-7082) for 72 h in the [3H]thymidine uptake assay. One representative of three experiments is shown. (B) DcR3. + Fc does not affect the activation of ERK or Akt. The B220 cells were stimulated with 0.25–1 mg/ml Pam3CSK4 in the presence or absence of DcR3.Fc, and the cell lysate was harvested after 30 min to detect the active forms of ERK or Akt (pERK or pAkt). The levels of pERK and pAkt were measured by specific ELISA assays and calculated according to standard controls. One representative of two experiments is shown. (C) DcR3 reduces the DNA-binding + activity of the NF-kB p65 subunit. The purified mouse B220 B cells were either left untreated (0), or treated with 0.3 mg/ml Pam3CSK4 in the presence of 30 mg/ml hIgG1, 10 mg/ml DcR3.Fc, or 30 mg/ml DcR3.Fc for 60 min. The nuclear extract was prepared and used to measure the DNA-binding activity of p65 by an ELISA assay. The result is shown as the OD readout of the samples with the blank subtracted. One representative result from three independent experiments is shown. (D) DcR3.Fc reduces the TLR2-induced NF-kB reporter activity. The purified mouse B220+ B cells were stimulated with 10 mg/ml by guest on September 24, 2021 LPS for 24 h and transduced with lentiviruses containing the NF-kB reporter. After resting in fresh medium for 2 h, the cells were plated and treated with the indicated reagents for 6 h. The cell lysates were harvested and used to measure luciferase activity. One representative of five independent experiments is + 6 shown. (E) DcR3.Fc suppresses the Pam3CSK4-induced cytokine expression. B220 cells (10 ) were seeded and treated as follows: lane 1, medium only; lane 2, 0.3 mg/ml Pam3CSK4; lane 3,Pam3CSK4 plus 10 mg/ml hIgG1; and lane 4, Pam3CSK4 plus 10 mg/ml DcR3.Fc. Eight hours later, the RNA from the treated cells was extracted for the measurement of cytokine mRNA levels by real-time RT-PCR. The data were normalized with the amount of 18S rRNA in each sample. One representative of three experiments is shown. *p , 0.05, **p , 0.01, ***p , 0.005. help attenuate inflammation. The DcR3 level in healthy donors is In this study, we used peripheral blood B cells and found an extremely low. The elevated expression of DcR3 was reported in inhibitory effect of DcR3 on cell activation. Peripheral blood the tissues from patients with Crohn’s diseases (43) and in the B cells consist of naive B and memory B cells. We are not sure if serum and/or synovial fluid of patients with systemic lupus both populations of B cells respond to DcR3.Fc because we did not erythematosus (SLE) and RA (44–46 and M.-H. Chen, Z.-M. separate these two populations to perform the experiments. Be- Huang, C.-M. Leu, and H.-Y. Lin, unpublished observations). The cause most of the mouse splenic B cells are at the naive stage, we induction of DcR3 in autoimmune patients seems to be stimulated assume that at least naive B cells are influenced by DcR3. In by TNF-a (or by other inflammatory cytokines) via the NF-kB addition to naive B cells, DcR3.Fc binds to all other stages of pathway (47). Therefore, DcR3 might be a negative-feedback B cells, including germinal center cells, memory cells, and plasma regulator of inflammation. Although the level of DcR3 is in- cells. It is possible that DcR3 might modulate B cell differentia- creased in the serum of SLE patients, its local concentration may tion or Ab secretion. We have tested the effect of DcR3.Fc on the not be high enough to attenuate inflammation. This might be the LPS-stimulated Ab secretion by mouse B cells in vitro and found reason why DcR3 fails to suppress active inflammation despite its that LPS-induced IgM production per cell was blocked (J.-K. increased circulation levels. In contrast, sustained elevated levels Kang and C.-M. Leu, unpublished observations). We found that of DcR3 may protect activated lymphocytes from Fas-mediated the B cell activation in vitro was suppressed and speculated similar apoptosis via binding FasL, leading to the accumulation of self- effects in vivo. As expected, we found a general reduction of reactive T and B cells and to the pathogenesis of autoimmune specific IgG secretion in the DcR3 transgenic mice immunized diseases. Our findings in this study support the notion that DcR3 with T-dependent or -independent Ags (W.-J. Hsu and C.-M. Leu, may impair B cell proliferation and inflammation; however, the unpublished observations). In accordance with our observations, long-term effect of continuous increase of DcR3 in the patho- the injection of DcR3 plasmid into collagen-induced arthritis genesis of autoimmune diseases remains to be investigated. Ad- mice decreases the serum level of collagen-specific IgG2a (31). ministration of DcR3.Fc fusion protein into autoimmune animal Therefore, DcR3 is able to suppress B cells both in vitro and models with arthritis or SLE-like syndromes may help clarify the in vivo. Our data support the role of DcR3 as a suppressor of role of DcR3 in the regulation of inflammation. the TLR- or BCR-mediated B cell activation/proliferation. 10 DcR3 ATTENUATES B CELL ACTIVATION VIA SUPPRESSING NF-kB

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