The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yun, T. J.
Right arrow Articles by Clark3, E. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yun, T. J.
Right arrow Articles by Clark3, E. A.
The Journal of Immunology, 1998, 161: 6113-6121.
Copyright © 1998 by The American Association of Immunologists

OPG/FDCR-1, a TNF Receptor Family Member, Is Expressed in Lymphoid Cells and Is Up-Regulated by Ligating CD404

Theodore J. Yun*, Preet M. Chaudhary{dagger}, Geraldine L. Shu{ddagger}, J. Kimble Frazer1,¶, Maria K. Ewings{ddagger}, Stephen M. Schwartz§, Virginia Pascual, Leroy E. Hood{dagger} and Edward A. Clark3*,{ddagger}

Departments of * Immunology, {dagger} Molecular Biotechnology, {ddagger} Microbiology, and § Pathology, University of Washington, Seattle, WA 98195; and Molecular Immunology Center, University of Texas Southwestern Medical Center, Dallas, TX 75235


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have cloned a TNFR family member from a follicular dendritic cell (FDC)-like cell line, FDC-1. This molecule, FDC-derived receptor-1 (FDCR-1), is identical to osteoprotegerin (OPG), a soluble cytokine that regulates osteoclast differentiation. Recently, OPG/FDCR-1 has been characterized as a second receptor for receptor activator of NF-{kappa}B ligand (RANKL)/TNF-related activation-induced cytokine (TRANCE), a primarily T-cell restricted TNF family member that augments dendritic cell (DC) function. In this report, we demonstrate that OPG/FDCR-1 is membrane bound on the surface of transfected baby hamster kidney (BHK) and untransfected FDC-1 cells. We also found a restricted OPG/FDCR-1 expression pattern in lymphoid cells, specifically in B cells, DCs and FDC-enriched fractions, which in B cells and DCs is up-regulated by CD40 stimulation. Because OPG/FDCR-1 shares some properties with RANK, the first RANKL/TRANCE receptor, we discuss how the balance between RANK and OPG/FDCR-1 expression could influence immune responses and, ultimately, germinal center formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Germinal centers (GCs)3 are specialized microenvironments within secondary lymphoid organs where the immune system coordinates a specific response against thymus-dependent Ags. Interactions between an intricate network of cells coordinate GC formation and memory B lymphocyte creation. Dendritic cells (DCs) initiate the immune response by transporting and presenting processed Ags to T cells. Newly produced B cells emigrated from the bone marrow to peripheral lymphoid T cell zones. Most B cells will die unless they are rescued by external signals, such as cytokines or membrane-bound molecules (1, 2, 3, 4, 5, 6, 7, 8). While most of these signals are not fully understood, one is almost certainly a positive signal by Ag (9). Rare B cells with receptors for the foreign Ag bind, process, and present soluble Ag. Activated T cells, and probably also DCs, then interact through cell-adhesion receptors or cytokines to induce a few Ag-specific B cells to migrate to primary follicles. There, B cells associate with a dense network of follicular dendritic cells (FDCs) which provide them with trapped Ag and costimulatory signals leading to B cell maturation and differentiation. Notably, B cells begin to proliferate and undergo affinity maturation (2). Those B cells that develop highly specific Ig receptors to Ag are selected. Through additional costimulation from GC CD4+ T cells and FDCs, these mutated, highly specific B cells differentiate by isotype switching and mature into memory or plasma cells.

The molecular signals that direct B cells to live, to proliferate, or to die are not well understood. The TNFR family members (reviewed in 10 , which include CD95 (Fas) and CD40, have been recently implicated as critical receptors regulating B cell fate. For example, CD40, CD154 (CD40 ligand (CD40L)), TNFRI, or lymphotoxin-{alpha}-deficient mice have defects at some stage of GC formation (11, 12, 13, 14). Despite the overt phenotype of these mice, almost nothing is known about the regulation of TNF/TNFR family members on FDCs or stromal cells and how they function in GC creation. Also, members of the TNF/TNFR families often can affect the expression of other family members. For example, ligating CD40 on B cells or DCs can up-regulate the expression of RANK (15), CD95 (Fas) (16, 17) and lymphotoxin-{alpha} (18).

One major obstacle in trying to characterize molecules involved in GC formation has been the difficulty in isolating FDC. Most of our knowledge about FDCs comes from observations made in situ (19, 20). However, several groups have had some success in purifying FDCs or isolating FDC lines (21, 22, 23, 24, 25, 26, 27, 28). FDCs can associate with B cells via Ag-Ig receptors, and through adhesion molecules such as CD54/CD11a and VCAM/VLA-4. FDC-mediated costimulation can maintain memory B cells and influence the fate of GC B cells. For example, isolated centroblasts or centrocytes undergo apoptosis in culture unless rescued by coculture with FDCs (27, 28, 29, 30).

Previously, our laboratory isolated and characterized a cell line, FDC-1, that has morphologic and functional similarities to FDCs in vivo (22, 23). FDC-1 cells are able to bind B cells, but not T cells, in a CD54-dependent fashion. FDC-1 cells, through soluble and membrane-bound signals, are able to promote B cells to proliferate and secrete IgM and IgG. To define novel molecules that play a role in GC formation, we prepared a cDNA library from FDC-1 cells and sequenced FDC-associated cDNAs. One of these clones encodes a TNFR family member which we termed FDCR-1, for follicular dendritic cell-derived receptor-1. This cDNA is identical to osteoprotegerin (OPG) and osteoclastogenesis inhibitory factor (OCIF), recently reported by Simonet et al. (31) and Yasuda et al. (32), respectively. Currently, this molecule is thought to influence bone metabolism and possibly vascular development (33). OPG/FDCR-1 is a second receptor for RANKL/TNF-related activation-induced cytokine (TRANCE) (34, 35), a T cell restricted molecule that augments DC survival and function (15, 36, 37). Here, we demonstrate that OPG/FDCR-1 is expressed as both a membrane-bound form and secreted form. OPG/FDCR-1 has several properties in common with RANK, the first characterized receptor for RANKL/TRANCE. OPG/FDCR-1 has a restricted expression pattern in cells of the immune system, including DCs, EBV-transformed B cell lines, and tonsillar B cells. Furthermore, it is regulated by CD40 stimulation, suggesting that it could play a role in DC and B cell function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of cDNA library and sequencing of clones

FDC-1 cells were used to prepare an FDC cDNA library using the UNIZAP XR vector (Stratagene, La Jolla, CA). Preparation of the cDNA library was performed according to the manufacturer’s recommendation. The cDNA library was screened by the partial sequencing of a set of 500 cDNA clones through the use of automated fluorescent sequencing, following the recommendation of the manufacturer (Applied Biosystems, Foster City, CA). The sequenced clones were analyzed for relatedness to previously identified genes using public domain programs available through the National Human Genome Project, National Institutes of Health, which can be accessed on the World Wide Web. These programs included Blast-n, Blast-x and Fasta that used BLAST and FASTA sequence comparison algorithms.

Surface biotinylation

Tissue culture dishes (100 mm2) of near confluent cells (OPG/FDCR-1 transfected OPG/FDCR-1-baby hamster kidney (BHK) cells or untransfected FDC-1 cells) were washed twice with bicarbonate/saline buffer (20 mM bicarbonate (pH 8.0)/150 mM NaCl). The dishes were incubated with 4% of biotinylation reagent (Amersham, Arlington Heights, IL) in bicarbonate/saline buffer at 4°C for 45 min with constant rocking. The cells were washed twice with PBS, then lysed with 1 ml of 1% Nonidet P-40 lysis buffer (250 mM NaCl, 25 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 1% Nonidet P-40, 2 µg/ml aprotinin, and 0.02% NaN3) at 4°C for 15 min. Lysates were centrifuged at 14,000 rpm at 4°C for 10 min. The supernatants were precleared with a pre-immune rabbit serum at a dilution of 1 µl/ml of lysate and 50 µl of packed protein A-Sepharose at 4°C for 4 h with constant rocking. The supernatants were then incubated with anti-FDCR-1 serum at a dilution of 1:1000, or the preimmune serum at a dilution of 1:1000, at 4°C for 30 min with constant rocking. A total of 50 µl of packed protein A-Sepharose was added, and then precipitated at 4°C overnight with constant rocking. The beads were washed three times with lysis buffer and then boiled 5 min. The proteins were separated on a 10% polyacrylamide gel, under reducing or nonreducing conditions, and then transferred to nitrocellulose. The blots were blocked with 1x TBST-B (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Tween-20, and 5% BSA), at 4°C overnight with constant rocking, and then washed five times with TBST (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween-20). Blots were incubated with 0.5 µg/ml of streptavidin/horseradish peroxidase for 1 h at 4°C and washed as before. Biotinylated proteins were visualized by enhanced chemiluminescence (ECL) (Amersham).

Construction of expression vectors and FACS analysis of transfected BHK cells

The full-length OPG/FDCR-1 was cloned into the mammalian expression vector PCI-neo (Promega, Madison, WI). A portion of FDCR-1 was amplified by PCR using 5'-CCACCCAGGATCCGTTTCCTCCAAAGTA-3' as the upstream primer and a downstream primer specific for the T3 promoter. The resulting product was digested with BamHI and NotI, and then ligated into a modified pSecTag A vector (Invitrogen, San Diego, CA) where a region encoding a 5' c-myc epitope had been inserted downstream of the murine Ig-{kappa} chain signal peptide. The resulting construct encoded the Ig-{kappa} signal peptide, the c-myc epitope and human FDCR-1, residues 24–401. To construct soluble extracellular domain of FDCR-1, amino acids 24–202 were amplified with a 5' primer (5'-CCACCCAGGATCCGTTTCCTCCAAAGTA-3') and a 3' primer (5'-AAGAATGCCTCGAGACACAGGGTAACATCTATTCCAC-3') so that the resulting product had a BamHI and XhoI site at its 5' and 3' ends, respectively. This product was digested with BamHI and XhoI and cloned in the BamHI-XhoI sites of the modified pSecTag A vector. This produced an open reading frame encoding the murine Ig-{kappa} chain signal peptide, amino acids 24–202 of FDCR-1, c-myc epitope and 6x His epitope. The myc-DR3 expression construct was described previously (38). Briefly, residues 27–418 were cloned in a similar fashion as the c-myc-OPG/FDCR-1 construct such that the protein had the Ig-{kappa} chain signal peptide, c-myc epitope and residues 27–418 of DR3.

Cells were grown in 100 mm2 culture dishes to semiconfluency. The cells were washed with PBS, incubated with 0.5 mM EDTA in PBS, and then removed by cell scraping. The harvested cells were centrifuged, the pellet was resuspended in FACS media (2% FCS in RPMI 1640 and 0.1% sodium azide), then divided into 10–20 wells of a 96-well plate for flow cytometry experiments. Cells were incubated with anti-myc (9E20), anti-FDCR-1 sera, at a dilution of 1:1000, or preimmune anti-sera, at a dilution of 1:1000, at 4°C for 40 min. The cells were washed, then incubated with FITC conjugated, F(ab')2 fragments of goat anti-rabbit Ab (BioSource International, Camarillo, CA) at a 1:80 dilution at 4°C for 40 min. The live, unfixed cells were analyzed by flow cytometry. The mean peak fluorescence (MPF) was calculated.

Pulse chase of transfected OPG/FDCR-1-BHK cells and untransfected FDC-1 cells

Adherent cell lines were grown to near confluency. The cells were washed with PBS and then incubated with medium lacking cysteine and methionine for 2 h. The cells were pulsed with media containing 0.2 mCi/ml of [35S]Met,Cys for 1 h and then chased with 5 ml of complete medium. At the indicated times, the medium was removed and the cells lysed with 0.5% Nonidet P-40 lysis buffer. Lysates or cell supernatants were precleared with preimmune sera at a concentration of 1:1000 and 50 µl of packed protein A Sepharose for 4 h at 4°C. Samples were precipitated with anti OPG/FDCR-1 sera (1:1000 dilution) and 50 µl of packed protein A Sepharose overnight at 4°C. Samples were washed with Nonidet P-40 lysis buffer and then separated on a 10% acrylamide gel under nonreducing conditions. Gels were analyzed by autoradiography.

Isolation of primary cells

As described by Grouard et al. (27), to isolate enriched FDC populations, we minced human tonsils in FDC medium (RPMI 1640 with 10% FCS, 25 mM EDTA (pH 7.4), 0.1 mg/ml DNase I, and 2 mg/ml collagenase IV). Tissue fragments were incubated for 12 min at 37°C. The cell suspension was collected after two successive digestions and then separated over Ficoll-Hypaque. The interface cells were removed and washed then rosetted to remove T cells. The Er- cells were washed in FDC medium and then placed on a 15/35% Percoll gradient and spun 800 x g for 20 min. Cells from the interface were recovered and washed in PBS. Cells were resuspended in RPMI 1640 with 10% FCS, 1 mM EDTA, and 1% human serum, and then incubated with phycoerythrin-conjugated anti-CD21 (BioSource International) and FITC-conjugated anti-CD14 (Becton Dickinson, San Jose, CA). Samples were washed and then sorted using flow cytometry. CD21brightCD14bright cells were collected.

As described in Clark et al. (22), tonsillar B cells were isolated by disaggregation of human tonsils with 10% FCS in RPMI and then centrifuged through Ficoll-Hypaque. The mononuclear cells were harvested and then rosetted. The Er- cells were fractionated by a discontinuous percoll gradient. B cells from the following interfaces were collected: <45%, 45/50%, 50/55%, 55/60%, 60/65%, and 65/70%.

DCs were differentiated in vitro from CD14+ peripheral blood monocytes using established methods. Briefly, human blood was separated over Ficoll-Hypaque and the mononuclear cells were collected. PBMCs were washed twice in PBS, 0.5% BSA, and 2 mM EDTA. Cells (20 µl/10 x 106) were sorted by MACS with CD14 conjugated microbeads (Miltenyi Biotec, Sunnyvale, CA). CD14+ PBMCs were washed and resuspended at 2 x 106 cells/ml in RPMI 1640, 10% FCS, 100 ng/ml granulocyte-macrophage CSF, and 15 ng/ml IL-4. Fresh medium with cytokines was added after 3 days. After 7 days of culture, DCs were collected.

Cell stimulations

Tonsillar B cells were suspended 10 x 107 cells/ml or 1 x 107 cells/ml (for cells collected from 60/65% and 65/70% Percoll interfaces) with 1 µg/ml each of goat anti-human IgM F(ab')2 (Jackson ImmunoResearch, West Grove, PA) and anti-CD40 (G28-5) for 18 h at 37°C. DCs were cultured with either anti-CD40 (G28-5) or IgG1 isotype control (MOPC-21) at 10 µg/ml for 72 h at 37°C.

RNA isolation

RNA samples from cell lines, isolated PBMCs, tonsillar B cell Percoll fractions, or DCs were prepared using TRIzol reagent (Life Technologies, Grand Island, NY) following the manufacturer’s instructions.

Northern blot analysis of human cell lines, isolated cells, and human tissue

Total cellular RNA (5 µg) was resolved by electrophoresis in an 0.8% formaldehyde agarose gel in 1x MOPS buffer and then transferred to a nylon membrane (Nytran, Schleicher & Schuell, Keene, NH) by capillary blotting. RNA was cross-linked by UV irradiation. 32P-labeled probe was generated with random hexamer primers. Hybridization was performed at 42°C in hybridization buffer (50% formamide, 1x Denhardt’s solution, 5x SSC, and 250 µg/ml denatured salmon sperm DNA). Blots were washed four times with 2x SSC and 0.2% SDS for 5 min at room temperature and then washed at high stringency, twice in 0.1x SSC and 0.2% (w/v) SDS for 30 min at 50°C. Membranes were exposed to autoradiographic film at -70°C.

For the multiple tissue Northern blot of lymphoid tissues (see Fig. 5GoD) (Clontech, Palo Alto, CA), 32P-labeled probe was generated using the PCR radioactive labeling system (Life Technologies) and following the manufacturer’s instructions. Hybridization and washing were performed following the manufacturer’s instruction.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 5. Northern blot analysis of OPG/FDCR-1 transcription in cell lines and normal tissues. A and B, OPG/FDCR-1 is not detectable by Northern blot analysis in B lineage (BJAB, Daudi, Raji, Ramos, CESS), T lineage (Jurkat, Hut78, thymocytes), myelomonocytic (HL60, K562, U937), or epithelial cells (COS, HeLa). FDC-1 is used as the positive control for OPG/FDCR-1 expression and ß-actin is the loading control. C, OPG/FDCR-1 is expressed in human heart, lung, liver, and kidney, but not brain, skeletal muscle, or pancreas. D, OPG/FDCR-1 is detected in lymphoid tissues, such as spleen, lymph node, thymus, bone marrow, and fetal liver, but not in peripheral blood lymphocytes (PBLs). E, OPG/FDCR-1 is expressed in smooth muscle cells (SMC) from a range of sources.

 
RT-PCR

RNA from isolated CD21brightCD14bright FDC was reverse transcribed and amplified using the Smart cDNA synthesis kit (Clontech) following the manufacturer’s instructions. The amplified cDNA was used in the following RT-PCR experiments.

RT-PCR analysis was performed using the titan one-tube RT-PCR system (Boehringer Mannheim, Indianapolis, IN) following the manufacturer’s instructions. Briefly, 0.75–1 µg of total RNA from cells was subjected to reverse transcription with AMV-RT at 55°C for 30 min. The PCR was performed using 400 nM each of the following primers: 5'-GTTCTGCTTGAAACATAGGAGCTGCCCTCCTGG-3' or 5'-GCGGCCCCGGGGAGGTTTGCTGTTCCTACAAAGTTT-3' ("upstream primers"), and 5'-GCGGTCGACTAAGCAGCTTATTTTTACTGA-3' ("downstream primer"). For RNA loading controls, oligonucleotides specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript were used: 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3'. For tonsillar B cells analysis, the PCR products were transferred to Genescreen membrane (NEN Life Sciences, Boston, MA) then probed with an oligonucleotide with the following sequence: 5'-GGTTTGCTGTTCCTACAAAGTT-3'. A total of 100 ng of the oligonucleotide was end labeled with 0.02 mCi of [32P-{gamma}]dATP using DNA polynucleotide kinase. The probe was added to 50 ml of 5x SSPE plus 1% SDS. After overnight hybridization at 57°C, the membranes were washed with 2x SSPE for 30 min and then 2x SSPE plus 1% SDS for 1 min. Specific hybridization of the oligonucleotide probe to DNA fragments was visualized by autoradiography.

Representational difference analysis (RDA) was performed as described (39, 40). The cell substrates used in RDA were kindly provided by Dr. Francine Briere (Schering-Plough, Dardilly, France). Bm2 cells were purified from human tonsils based on the expression of follicular mantle markers, such as surface IgD and CD23, and the absence of GC markers, such as CD38, as described (8, 41). Purified cells were resuspended at 5 x 105 cells/ml in RPMI 1640 medium with 10% FCS. A total of 100–200 ml of the suspension was dispensed in microwells. SAC particles (0.001–0.005%, v/v) or sCD40L (16) were added to the cultures and the cells were harvested after 48 h. Amplicons were constructed by digesting double-stranded cDNA from either SAC or sCD40L-activated Bm2 cells with DpnII for 4 h at 37°C. After digestion R12 (5'-GATCTGCGGTGA-3') and R24 (5'-AGCACTCTCCAGCCTCTCACCGCA-3') linkers were ligated to the double-stranded cDNA with T4 DNA ligase. Amplicons were obtained after 20 cycles of PCR amplification using as template 1/10 of the purified double-stranded cDNA plus linkers ligation reactions. The products of these amplifications were then pooled and purified by phenol extraction and ethanol precipitation, and 3–10 µg of each amplicon product were electrophoresed through a 1% agarose gel, the immobilized on Zeta probe membranes (Bio-Rad, Hercules, CA). After capillary transfer and UV cross-linking, the blots were hybridized with a 32P-labeled double-stranded cDNA probe generated by the PCR radioactive labeling system (Life Technologies), followed by stringent washing, and developed by using a PhosphoImager and the Imagequant software package (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FDC-1 cells express a molecule that is identical to OPG

To characterize novel FDC-associated molecules, we constructed a size selected library from FDC-1 cells and randomly sequenced the cDNA inserts. One of these clones encoded a TNFR family member, which we called FDCR-1. Concurrent with our finding, others reported an identical amino acid sequence for OPG/OCIF, a molecule found to influence osteoclastogenesis (31, 42). When we compared the cysteine-rich amino terminus, FDCR-1 had 46% amino acid similarity to CD40, a key molecule regulating B cell maturation and GC formation (43, 44). FDCR-1 also had high similarity to TNFRII. An alignment with RANK, CD40, and TNFRII is shown in Fig. 1GoA. When we compared the carboxy terminus, we did not find high homology with any known protein; however, FDCR-1 had some residues at corresponding positions that are commonly found in death domains of other TNFR family members (Fig. 1GoB). Interestingly, there is no long region in this sequence that corresponds to a classical transmembrane region. However, Yamaguchi et al. (34) have found that the putative cytoplasmic death domain homology region of OPG/OCIF can transduce an apoptotic signal, strongly suggesting that OPG/OCIF may be a transmembrane protein. There are several possibilities which might explain these observations (see Discussion).



View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 1. Amino acid comparison of OPG/FDCR-1 with other TNFR family members. A, Deduced amino acid alignment of the cysteine-rich regions of OPG/FDCR-1, RANK, TNFRII, and CD40. B, Alignment of carboxy terminus of FDCR-1 with death domains of other TNFR family members. Amino acid residues that are identical or similar are shown in black or grey, respectively. The region with homology to other death domains is indicated below the alignment.

 
Membrane and soluble forms of OPG/FDCR-1

To test the possibility that OPG/FDCR-1 was expressed on cell membranes, we generated rabbit heteroantisera to recombinant FDCR-1 and expressed recombinant, myc epitope-tagged FDCR-1 in BHK cells. Using these reagents, we found that FDCR-1 is expressed on cell membranes using flow cytometry (Fig. 2Go), surface biotinylation (Fig. 3Go), and surface iodination (data not shown). We transfected BHK cells with a myc epitope-tagged FDCR-1 or a similar construct of a related molecule myc epitope-tagged DR3, a known membrane bound member of the TNFR family (45). These cells were indirectly stained with either an anti-myc Ab or the rabbit anti-FDCR-1 serum, followed by goat anti-rabbit F(ab')2-conjugated to FITC. Samples were analyzed by flow cytometry (Fig. 2Go). The anti-myc epitope Ab clearly detected the full-length myc/FDCR-1 construct, the myc/DR3 construct, but not a truncated version of myc/FDCR-1 containing only the amino terminus. The MPF fold increase for the epitope-specific Ab over the negative control was 2.8 times and 3.2 times for the myc/FDCR-1 and myc/DR3 constructs, respectively. The anti-FDCR-1 serum detected the FDCR-1 molecule on the surface of the transfected cells but not the truncated version or myc/DR3. The MPF fold increase for the specific antisera over the negative control was 2.1. Neither Ab reacted against cells transfected with vector alone. Thus, the full-length FDCR-1, including the carboxy region, is required for expression on the cell membrane. Also, the antiserum is able to detect FDCR-1 on the surface of FDC-1 cells, the cell line from which we isolated the cDNA (Fig. 2GoB). The MPF fold increases for the epitope specific Ab over the normal rabbit serum (NRS) or secondary Ab alone negative controls were 2.1 times and 2.3 times, respectively. Thus, OPG/FDCR-1 is expressed on the surface of some untransfected cells.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. Flow cytometric analysis of cell surface OPG/FDCR-1 using specific anti-FDCR-1 sera. A, FDCR-1 on the cell surface of transfected BHK-OPG/FDCR-1 cells. Using anti-myc (left panels) or anti-FDCR-1 (right panels), full-length myc-FDCR-1 is detected (top), but not truncated FDCR-1 (sol-myc-FDCR-1, middle), myc-DR3 (middle), or vector only (bottom). B, FDCR-1 is on the cell surface of FDC-1 cells. Anti-FDCR-1 stains positive for cell surface OPG/FDCR-1 on FDC-1 cells compared with negative staining by normal rabbit serum or FITC-conjugated F(ab')2 goat anti-rabbit alone.

 


View larger version (82K):
[in this window]
[in a new window]
  		FIGURE 3. Immunoprecipitation of cell-surface biotinylated OPG/FDCR-1 from BHK-OPG/FDCR-1 transfectants or FDC-1 cells. Using anti-FDCR-1 sera to immunoprecipitate biotinylated surface molecules, then blotting with streptavidin/horseradish peroxidase, a 120-kDa protein is revealed under nonreducing conditions (left) and a 60-kDa protein is revealed under reducing conditions (right).

 
Next we biotinylated extracellular regions of FDCR-1 on either BHK cells transfected with FDCR-1 cDNA (FDCR-1-BHK) or on FDC-1 cells. Proteins were immunoprecipitated with anti-FDCR-1 serum or preimmune serum as the negative control. Anti-FDCR-1 serum precipitated a protein that was not present in control lanes precipitates (Fig. 3Go). Under reducing conditions, this protein migrated at ~60 kDa, slightly higher than a nonspecific protein. Under nonreducing conditions, the anti-FDCR-1 serum precipitated a protein of ~120 kDa, suggesting that OPG/FDCR-1 is a dimer on cell membranes.

Simonet et al. (31) and Tsuda et al. (42) found that OPG/FDCR-1 was present in supernatants of transfected cells or untransfected human embryonic lung fibroblasts. We tested whether FDCR-1 was expressed as a soluble form in our hands. Using the FDCR-1-BHK cells or nontransfected FDC-1 cells, we performed a pulse-chase analysis. In Fig. 4Go, FDC-1 cells were metabolically labeled with [35S]Met,Cys. Using the anti-FDCR-1 serum, we immunoprecipitated proteins from either the cell supernatants or whole cellular lysates at several time points after addition of cold medium. FDCR-1 was detected in the supernatant within 30 min after addition of cold medium, and persisted in the supernatant for 10 h (Fig. 4Go). Similar results were obtained with FDCR-1-BHK cells. The m.w. of the soluble form was 60–65 kDa, confirming the results of Simonet et al. (31). Under nonreducing conditions, the m.w. of soluble OPG/FDCR-1 was ~120 kDa, consistent with it being a homodimer.



View larger version (100K):
[in this window]
[in a new window]
  		FIGURE 4. Pulse-chase analysis of OPG/FDCR-1 production by metabolically labeled FDC-1 cells. After 1 h pulse with [35S]Met,Cys, OPG/FDCR-1 was immunoprecipitated at the indicated times from the lysates or supernatants as a 120-kDa protein from FDC-1 cells, under nonreducing conditions.

 
Expression of OPG/FDCR-1

Next we probed isolated RNA from either human tissues or cell lines for their ability to express OPG/FDCR-1 (Fig. 5Go). In lymphoid tissues, we detected strong OPG/FDCR-1 expression lymph node, fetal liver, bone marrow, and, to a lesser extent, in spleen and thymus (Fig. 5GoD). It is also expressed in thyroid and spinal cord (data not shown). We detected OPG/FDCR-1 transcripts in human heart, lung, liver, kidney, and pancreas. Simonet et al. (31) did not detect OPG expression in human liver, which is the only difference from this report. The reason for this difference is not clear. Although the poly(A)+ RNA was obtained from the same source, we are not certain whether the tissues were isolated from the same individual or whether technical reasons can account for this observation. OPG/FDCR-1 does not appear to be expressed in brain, skeletal muscle, thymocytes, or peripheral blood lymphocytes (Fig. 5Go, B–D). Expression was not detected after activation of peripheral blood lymphocytes with PMA or after separation into unstimulated T cell or B cell populations (data not shown).

We also evaluated whether OPG/FDCR-1 was expressed by various cell lines. We were unable to detect OPG/FDCR-1 transcripts in a number of hematopoietic lineages or epithelial cell lines (Fig. 5Go, A and B). In independent experiments with a different isolate of HeLa RNA, we detected expression of OPG/FDCR-1, suggesting subline differences in HeLa cells. OPG/FDCR-1 was expressed in all stages of smooth muscle cells evaluated (Fig. 5GoE). The strong OPG/FDCR-1 expression by heart and smooth muscle cells supports the possibility that OPG/FDCR-1 has a role in vascular development. In the developing mouse embryo (E15) Simonet et al. (31) demonstrated that OPG/FDCR-1 was highly expressed in several regions of the developing vasculature, and Bucay et al. (33) demonstrated that opg-/- mice have arterial calcification.

We then extended our expression analysis by using RT-PCR, a method more efficient and sensitive than Northern blot analysis. Because we isolated OPG/FDCR-1 from an FDC-like cell line, we wanted to test whether it is expressed in normal human FDCs. Using Percoll gradient fractionation and cell sorting for CD21highCD14+ cells, we isolated highly enriched FDCs (27). OPG/FDCR-1 transcription was detected in enriched FDCs (Fig. 6GoA). Theoretically, some B cells may still be associated with the FDC clusters (27), so we cannot be entirely sure whether the FDCs, B cells associated with FDCs, or both cell types express OPG/FDCR-1. Nevertheless, we can conclude OPG/FDCR-1 is expressed in FDC clusters.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 6. A, OPG/FDCR-1 is expressed in highly enriched FDC populations. From two independent sorting runs, FDCs were isolated via the method of Liu et al. (10). Low density tonsillar cells from 15/35% Percoll gradients were sorted based on CD21highCD14high. The slower migrating fragment is derived from genomic DNA contamination because this fragment is not observed after RNase-free DNase I treatment of the RNA template (data not shown). B, OPG/FDCR-1 is expressed in EBV-transformed lymphoblastoid cells, MP-1, CESS, and T5–1, but not in Burkitt lymphoma derived B cells such as BJAB.

 
To test whether B cells are able to express OPG/FDCR-1, we next assayed for OPG/FDCR-1 expression in various B cell lines using RT-PCR. Our RT-PCR results confirmed our Northern analysis results where FDC-1 express OPG/FDCR-1, but Jurkat cell lines (Fig. 7GoA), and EBV B lymphomas, such as BJAB (Fig. 6GoB), Ramos and B104 (data not shown), do not express OPG/FDCR-1. Interestingly, we were able to detect OPG/FDCR-1 expression in EBV-transformed lymphoblastoid cell lines, CESS, T5-1, and MP-1 (Fig. 6GoB). Detection of OPG/FDCR-1 expression in CESS using RT-PCR but not by Northern blot analysis probably reflects the difference in sensitivity between these two assays. Thus, B lineage cell lines do express OPG/FDCR-1, and its expression could be differentially regulated during the B cell activation and maturation process.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 7. CD40 up-regulation of FDCR-1 in isolated normal B lymphoid cells. A, Expression of OPG/FDCR-1 in human tonsillar Percoll fractions as detected by RT-PCR, followed by Southern blot analysis of products. Each cell fraction was stimulated or not for 18 h with CD40 mAb plus anti-µ sera. B, RDA assay of isolated tonsillar naive Bm2 cells that have been stimulated with SAC or sCD40L suggests that OPG/FDCR-1 can be up-regulated in B cells by CD40 activation. C, In vitro differentiated DC were stimulated or not by CD40 ligation. The slower migrating fragment is derived from genomic DNA contamination because this fragment is not observed after RNase-free DNase I treatment of the RNA template (data not shown).

 
CD40 regulates OPG/FDCR-1 expression

To test whether activated normal B cells express OPG/FDCR-1, we isolated tonsillar B cells and fractionated them on a Percoll gradient. We stimulated each fraction with anti-IgM and anti-CD40 and then compared changes in OPG/FDCR-1 transcripts by RT-PCR. Amplification products were probed with an OPG/FDCR-1-specific oligonucleotide to assure specificity. After activation, OPG/FDCR-1 transcription was up-regulated in all B cell fractions (Fig. 7GoA). The less dense, in vivo-activated B cells had higher levels of constitutive OPG/FDCR-1 transcription than the dense fractions. In the dense fractions, ligation with anti-IgM and anti-CD40 clearly increased expression.

Because EBV-transformed cell lines generally resemble cells that are activated through the CD40 pathway, this raised the possibility that OPG/FDCR-1 expression was regulated by CD40 ligation of B cells. To test this, we performed representational difference analysis on primary tonsillar B cells. Using defined markers, we isolated naive follicular mantle B cells (Bm2) (39). These cells were stimulated with either SAC or sCD40L and then assayed for expression of OPG/FDCR-1. As shown in Fig. 7GoB, we were able to detect a high level of OPG/FDCR-1 expression in CD40-stimulated naive B cells compared to SAC-activated cells.

DCs are also known to become activated and enhance their T cell stimulatory capabilities upon CD40 ligation (46, 47). We tested whether DCs could also up-regulate FDCR-1 expression after CD40 ligation. DCs were isolated from human peripheral blood mononuclear cells and differentiated in culture using granylocyte-macrophage CSF and IL-4 (46, 48). We stimulated DCs for 72 h with anti-CD40. By RT-PCR, we observed a clear up-regulation of FDCR-1 transcription (Fig. 7GoC).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated expression of the RANKL/TRANCE receptor, OPG/FDCR-1, in cells of the immune system, including B cells, DCs, and FDC clusters. In B cells and DCs, expression of OPG/FDCR-1 can be up-regulated by CD40, a key molecule involved in B cell activation. Along with CD95 (Fas) and RANK, OPG/FDCR-1 represents another TNFR family member regulated by CD40. Because of its expression and regulation patterns, OPG/FDCR-1 is an excellent candidate to be a molecular signal involved in the process of B cell differentiation and maturation. We have observed two forms of OPG/FDCR-1, membrane bound and soluble, and whether these two forms mediate different functions is currently unknown.

Currently, OPG/FDCR-1 has been reported to function in bone metabolism. OPG/FDCR-1 has been demonstrated by two groups to inhibit osteoclastogenesis. Yasuda et al. (32, 49) and Lacey et al. (35) proposed that OPG/FDCR-1 acts as a soluble inhibitor that prevents RANKL/TRANCE interaction with its receptor. In this respect, OPG/FDCR-1 would behave in an analogous way that TRAIL-R3 (TRID) (50, 51) or soluble CD95 (Fas) do in blocking nonlocal interactions between their soluble ligands, i.e., TRAIL or Fas ligand (FasL), respectively, and their membrane bound receptors, TRAIL-R1, -R2, -R4 (52) and membrane CD95 (53), respectively. Another possibility is that RANK and OPG/FDCR-1 may have different abilities to signal through surface bound RANKL/TRANCE. There is evidence that transmembrane forms of TNF family molecules are capable of transducing an activation signal, such as FasL and CD154 (CD40L) (54, 55).

Another model that has not been fully explored is the possibility that OPG/FDCR-1 exists as a transmembrane protein. We have demonstrated that OPG/FDCR-1 is expressed on the surface of normal or transfected cells. The inhibitory activity of the molecule on osteoclastogenesis resides in its amino half, from residues 1 to 198 (31, 34). The function of the remaining carboxy half and the reason for its high evolutionary conservation need to be elucidated. OPG/FDCR-1 may exist on the membrane either bound by the extracellular matrix or as a transmembrane protein. By comparing elution rates from a heparin column of mutant vs intact forms of the molecule, Yamaguchi et al. (34) suggest that OPG/FDCR-1 can bind to the extracellular matrix protein, heparin. The carboxy-terminal region, which we found necessary for membrane expression on transfected BHK and nontransfected FDC-1 cells, is necessary for OPG/FDCR-1 binding to the heparin column. Thus, it is possible that membrane OPG/FDCR-1 functions analogous to extracellular matrix trapped cytokines (56).

However, Yamaguchi et al. (34) also found that the carboxy region of OPG/FDCR-1, when anchored to a classic transmembrane domain, can transduce an apoptotic signal, thus suggesting that OPG/FDCR-1 is a transmembrane protein. We have transfected OPG/FDCR-1 into several cell types but have detected neither increases in cell death nor NF-{kappa}B activation (data not shown). Our biochemical results are consistent with either a matrix-bound and/or a transmembrane form of the protein. Interestingly, while OPG/FDCR-1 does not have a classical hydrophobic region separating its NH2 and COOH regions, when we plotted one region on a helical wheel, we noted that the charged residues aligned on one face of an {alpha}-helix. Thus, transmembrane OPG/FDCR-1 molecules might form homodimers that could possibly associate via these amphipathic helices and then insert into the membrane. Further biochemical studies will be necessary to test this possibility. Another explanation is that an alternatively spliced transcript may encode an OPG/FDCR-1 transmembrane form, analogous to the related family member, CD95 (Fas), which is produced in two forms by alternate spliced transcripts (57). In any case, the function of membrane associated OPG/FDCR-1 is not yet clear. Possibly, a transmembrane form of OPG/FDCR-1 could transduce a signal that imparts a differentiation program to the cell which expresses it. The secreted form of OPG/FDCR-1 prevents systemic effects of the ligand, effectively making the local interactions between ligand and the membrane form the relevant ones.

Our observation that OPG/FDCR-1 is regulated by CD40 in B cells and DCs further suggests a role for OPG/FDCR-1 in immune functions. The importance of CD40 in various stages of GC formation, B cell differentiation, and memory B cell development is well documented (3, 8, 44, 58, 59). RANK and OPG/FDCR-1 share the features of both being receptors for RANKL/TRANCE and both being up-regulated by CD40 ligation. Also, the expression pattern of OPG/FDCR-1 in hematopoietic tissues somewhat parallels that of RANK; i.e., it can be observed in EBV lymphoblastoid cells, DCs, and certain stromal cells. In DCs, OPG/FDCR-1 expression, like RANK expression, is also up-regulated by CD40 stimulation. In terms of the immune system, the significance of RANK and OPG/FDCR-1 coexpression and coregulation will likely require understanding how the interplay between the two receptors and their ligand, RANKL is regulated.

How OPG/FDCR-1 functions in the regulation of the immune response is unknown. However, OPG/FDCR-1 may function in an analogous manner as it does in bone metabolism. During osteoclastogenesis, RANKL/TRANCE binding to osteoclast progenitors (possibly through RANK) may be an essential signal for their maturation. OPG/FDCR-1 may also block development of progenitor osteoclasts (31, 32). Simonet et al. (31) demonstrated that overexpression of the soluble form of OPG can inhibit osteoclastogenesis, and Yasuda et al. (49) found that soluble OPG can inhibit binding of RANKL/TRANCE to progenitor osteoclasts. According to this model, osteoclast production is balanced by relative amounts of RANK and OPG/FDCR-1. Bone-resorbing signals stimulate osteoblasts or stromal cells to express more RANKL/TRANCE, which results in increased osteoclast differentiation.

From this model, we can propose a similar and testable model for the function of OPG/FDCR-1 in the immune response. In the immune system, RANKL/TRANCE expression is relative to T cell restricted (15, 35, 36, 37), and RANKL/TRANCE can enhance DC aggregation and allostimulation in vitro (15, 37). CD40L+ RANKL+ T cells may interact with cells that express OPG/FDCR-1; i.e., DCs, B cells, and FDCs. DCs and B lineage cells can express both RANK and OPG/FDCR-1, and both molecules are up-regulated after CD40 stimulation. On T cells, RANKL/TRANCE can activate JNK (36) and NF-{kappa}B, which may play a role in T cell viability (15). Analogous to the bone metabolism model OPG/FDCR-1 could be a negative regulator of RANKL/TRANCE by interfering with RANKL/TRANCE signaling to DCs or T cells. In this model, OPG/FDCR-1 would attenuate the RANKL/TRANCE signal given by T cells. Another possibility is that OPG/FDCR-1 exists as a transmembrane receptor and directly transduces a negative signal via its death domain (34).

Recently OPG/FDCR-1 was reported to be a fifth receptor for TRAIL (60). TRAIL-R1 and -R2 have active death domain regions (38), and TRAIL-R3, -R4, and soluble OPG/FDCR-1 may serve as decoy receptors for TRAIL (52, 60). However, how TRAIL and its five receptors function in the in vivo immune response is by no means clear. Whether there is a complex cross regulation between ligands RANKL and TRAIL with OPG/FDCR-1 and the other TRAIL receptors will be subject of further investigation.

In summary, the expression of OPG/FDCR-1, the second receptor for RANKL/TRANCE, in lymphoid tissues and by cells of the immune system implicates its involvement in immune responses, which adds to its characterized role in bone metabolism and vascular development. The fact that it is has a very similar expression pattern to the first RANKL/TRANCE receptor, RANK, opens the possibility that OPG/FDCR-1 plays a role in activation of DCs and B cells during the GC response.


   Acknowledgments
 
We thank Kevin Otipoby, Dr. Alexandra Aicher, Dr. Geraldine Grouard, Dr. Raymond Goodwin, Dr. Andrew Craxton, Dr. Aaron Marshall, Dr. Che-Leung Law, Dr. Svetlana Sidorenko, Kevin Draves, Sasha Solow, and Dr. Michelle Tallquist for helpful discussions.


   Footnotes
 
1 Current address: Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104. Back

2 Address correspondence and reprint requests to Dr. Edward A. Clark, Department of Microbiology, University of Washington Medical School, 1959 NE Pacific, Box 357242, Seattle, WA 98195. E-mail address: Back

3 Abbreviations used in this paper: GC, germinal center; DC, dendritic cell; FDC, follicular dendritic cell; OPG, osteoprotegerin; OCIF, osteoclastogenesis inhibitory factor; RDA, representational difference analysis; SAC, staphylococcus aureus Cowan I; TRANCE, TNF-related activation-induced cytokine; MPF, mean peak fluorescence; RANK, receptor activator of NF-{kappa}B; RANKL, RANK ligand; MACS, magnetic cell sorting; AMV-RT, avian myeloblastosis virus-reverse transcriptase. Back

4 This work was supported by grants DE08229, RR00166, GM37905. P.M.C. is supported by a fellowship from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation. Back

Received for publication May 21, 1998. Accepted for publication August 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Parker, D. C.. 1993. T cell-dependent B cell activation. Annu. Rev. Immunol. 11:331.[Medline]
  2. MacLennan, I. C. M.. 1994. Germinal centers. Annu. Rev. Immunol. 12:117.[Medline]
  3. Clark, E. A., J. A. Ledbetter. 1994. How B and T cells talk to each other. Nature 367:425.[Medline]
  4. Kelsoe, G.. 1996. Life and death in germinal centers (redux). Immunity 4:107.[Medline]
  5. Ahmed, R., D. Gray. 1996. Immunologic memory and protective immunity: understanding their relation. Science 272:54.[Abstract]
  6. Lindhout, E., G. Koopman, S. T. Pals, C. de Groot. 1997. Triple check for antigen specificity of B cells during germinal center reactions. Immunol. Today 18:573.[Medline]
  7. MacLennan, I. C. M., A. Gulbranson-Judge, K. M. Toellner, M. Casamayor-Palleja, E. Chan, D. M. Sze, S. A. Luther, H. A. Orbea. 1997. The changing preference of T and B cells for partners as T-dependent antibody responses develop. Immunol. Rev. 156:53.[Medline]
  8. Liu, Y.-J., C. Arpin. 1997. Germinal center development. Immunol. Rev. 156:111.[Medline]
  9. Gu, H., D. Tarlinton, W. Muller, K. Rajewsky, I. Forster. 1991. Most peripheral B cells in mice are ligand selected. J. Exp. Med. 173:1357.[Abstract/Free Full Text]
  10. Gruss, H.-J., S. K. Dower. 1995. Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas. Blood 85:3378.[Abstract/Free Full Text]
  11. Ramesh, N., T. Morio, R. Fuleihan, M. Worm, A. Horner, E. Tsitsikov, E. Castigli, R. S. Geha. 1995. CD40-CD40 ligand (CD40L) interactions and X-linked hyper-IgM syndrome (HIGMX-1). Clin. Immunol. Immunopathol. 76:S208.[Medline]
  12. Matsumoto, M., Y. X. Fu, H. Molina, D. D. Chaplin. 1997. Lymphotoxin-{alpha}-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers. Immunol. Rev. 156:137.[Medline]
  13. Liu, Y.-J., J. Banchereau. 1996. Mutant mice without B lymphocyte follicles. J. Exp. Med. 184:1207.[Free Full Text]
  14. Kosco-Vilbois, M. H., J. Y. Bonnefoy, Y. Chvatchko. 1997. The physiology of murine germinal center reactions. Immunol. Rev. 156:127.[Medline]
  15. Anderson, D. M., E. Maraskovsky, W. L. Billingsley, W. C. Dougall, M. E. Tometsko, E. R. Roux, M. C. Teepe, R. F. DuBose, D. Cosman, L. Galibert. 1997. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175.[Medline]
  16. Garrone, P., E. M. Neidhardt, E. Garcia, L. Galibert, C. van Kooten, J. Banchereau. 1995. Fas ligation induces apoptosis of CD40-activated human B lymphocytes. J. Exp. Med. 82:1265.
  17. Schattner, E. J., K. B. Elkon, D. H. Yoo, J. Tumang, P. H. Krammer, M. K. Crow, S. M. Friedman. 1995. CD40 ligation induces Apo-1/Fas expression on human B lymphocytes and facilitates apoptosis through the Apo-1/Fas pathway. J. Exp. Med. 182:1557.[Abstract/Free Full Text]
  18. Worm, M., R. S. Geha. 1995. CD40-mediated lymphotoxin-{alpha} expression on human B cells is tyrosine kinase dependent. Eur. J. Immunol. 25:2438.[Medline]
  19. Tew, J. G., J. Wu, D. Qin, S. Helm, G. F. Burton, A. K. Szakal. 1997. Follicular dendritic cells and presentation of antigen and costimulatory signal to B cells. Immunol. Rev. 156:39.[Medline]
  20. Liu, Y.-J., G. Grouard, O. de Bouteiller, J. Banchereau. 1996. Follicular dendritic cells and germinal centers. Int. Rev. Cytology 166:139.
  21. Tsunoda, R., N. Nakayama, K. Onozaki, E. Heinan, N. Cormann, C. Kinet-Denoël, M. Kojima. 1990. Isolation and long-term cultivation of human tonsil follicular dendritic cells. Virchows Arch. B Cell Pathol. 59:95.[Medline]
  22. Clark, E. A., K. Grabstein, G. L. Shu. 1992. Cultured human follicular dendritic-like cells: growth, characteristics and interactions with B lymphocytes. J. Immunol. 148:3327.[Abstract]
  23. Clark, E. A., K. H. Grabstein, A. M. Gown, M. Skelly, T. Kaisho, T. Hirano, G. L. Shu. 1995. Activation of B lymphocyte maturation by a human follicular dendritic cell line, FDC-1. J. Immunol. 155:545.[Abstract]
  24. Kim, H.-S., X. Zhang, Y. S. Choi. 1994. Activation and proliferation of follicular dendritic cell-like cells by activated T lymphocytes. J. Immunol. 153:2951.[Abstract]
  25. Lindhout, E., A. Lakeman, M. L. C. M. Mevissen, C. de Groot. 1994. Functionally active Epstein-Barr virus-transformed follicular dendritic cell-like cell lines. J. Exp. Med. 179:1173.[Abstract/Free Full Text]
  26. Orscheschek, K., H. Merz, B. Schlegelbereger, A. C. Feller. 1994. An immortalized cell line with features of human follicular dendritic cells: antigen and cytokine expression analysis. Eur. J. Immunol. 24:2682.[Medline]
  27. Grouard, G., O. de Bouteiller, J. Banchereau, Y.-J. Liu. 1995. Human follicular dendritic cells enhance cytokine dependent growth and differentiation of CD40-activated B cells. J. Immunol. 155:3345.[Abstract]
  28. Schriever, F., A. S. Freedman, G. Freeman, E. Messner, G. Lee, J. Daley, L. M. Nadler. 1989. Isolated follicular dendritic cells display a unique antigenic phenotype. J. Exp. Med. 169:2043.[Abstract/Free Full Text]
  29. Lebecque, S., O. de Bouteiller, C. Arpin, J. Banchereau, Y.-J. Liu. 1997. Germinal center founder cells display propensity for apoptosis before the onset of somatic mutation. J. Exp. Med. 185:563.[Abstract/Free Full Text]
  30. Lindhout, E., A. Lakeman, C. de Groot. 1995. Follicular dendritic cells inhibit apoptosis in human B lymphocytes by a rapid and irreversible blockade of preexisitng endonuclease. J. Exp. Med. 181:1985.[Abstract/Free Full Text]
  31. Simonet, W. S., D. L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Lüthy, H. Q. Nguyen, S. Wooden, L. Bennet, T. Boone, et al 1997. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309.[Medline]
  32. Yasuda, H., N. Shima, N. Nakagawa, S. Mochizuki, K. Yano, N. Fujise, Y. Sato, M. Goto, K. Yamaguchi, M. Kuriyama, et al 1998. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 139:1329.[Abstract/Free Full Text]
  33. Bucay, N., I. Sarosi, C. R. Dunstan, S. Morony, J. Tarpley, C. Capparelli, S. Scully, H. L. Tan, W. Xu, D. L. Lacey, W. J. Boyle, W. S. Simonet. 1998. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 12:1260.[Abstract/Free Full Text]
  34. Yamaguchi, K., M. Kinosaki, M. Goto, F. Kobayashi, E. Tsuda, T. Morinaga, K. Higashio. 1998. Characterization of structural domains of human osteoclastogenesis inhibitory factor. J. Biol. Chem. 273:5117.[Abstract/Free Full Text]
  35. Lacey, D. L., E. Timms, H.-L. Tan, M. J. Kelley, C. R. Dunstan, T. Burgess, R. Elliot, A. Colombero, G. Elliott, S. Scully, et al 1998. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165.[Medline]
  36. Wong, B. R., J. Rho, J. Arron, E. Robinson, J. Orlinick, M. Chao, S. Kalachikov, E. Cayani, F. S. Bartlett, W. N. Frankel, S. Y. Lee, Y. Choi. 1997. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J. Biol. Chem. 272:25190.[Abstract/Free Full Text]
  37. Wong, B. R., R. Josien, S. Y. Lee, B. Sauter, H.-L. Li, Steinman R. M., Y. Choi. 1997. TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J. Exp. Med. 186:2075.[Abstract/Free Full Text]
  38. Chaudhary, P. M., M. Eby, A. Jasmin, A. Bookwalter, J. Murray, L. Hood. 1997. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-{kappa}B pathway. Immunity 7:821.[Medline]
  39. Hubank, M., D. G. Schatz. 1994. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 22:5640.[Abstract/Free Full Text]
  40. Frazer, K. J., V. Pascual, J. D. Capra. 1997. RDA of lymphocyte subsets. J. Immunol. Methods 207:1.[Medline]
  41. Liu, Y.-J., O. de Bouteiller, C. Arpin, I. Durand, J. Banchereau. 1994. Five human mature B cell subsets. Adv. Exp. Med. Biol. 355:289.[Medline]
  42. Tsuda, E., M. Goto, S.-I. Mochizuki, K. Yano, F. Kobayashi, T. Morinaga, K. Higashio. 1997. Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem. Biophys. Res. Commun. 234:137.[Medline]
  43. Stamenkovic, I., E. A. Clark, B. Seed. 1989. A B-lymphocyte activation molecule related to the nerve growth factor receptor and induced by cytokines in carcinoma. EMBO J. 8:1403.[Medline]
  44. Banchereau, J., F. Bazan, D. Blanchard, F. Briere, J. P. Galizzi, C. van Kooten, Y.-J. Liu, F. Rousset, S. Saeland. 1994. The CD40 antigen and its ligand. Annu. Rev. Immunol. 12:881.[Medline]
  45. Chinnaiyan, A. M., K. O’Rourke, G. L. Yu, R. H. Lyons, M. Garg, D. R. Duan, L. Xing, R. Gentz, J. Ni, V. M. Dixit. 1996. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science 274:990.[Abstract/Free Full Text]
  46. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
  47. Pinchuk, L. M., P. S. Polacino, M. B. Agy, S. J. Klaus, E. A. Clark. 1994. Role of CD40 and CD80 accessory cell molecules in dendritic cell-dependent HIV-1 infection. Immunity 1:317.[Medline]
  48. Aicher, A., J. Westermann, S. Cayeux, G. Willimsky, K. Daemen, T. Blankenstein, W. Uckert, B. Dorken, A. Pezzutto. 1997. Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells. Exp. Hematol. 25:39.[Medline]
  49. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S.-I. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, et al 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95:3597.[Abstract/Free Full Text]
  50. Pan, G., J. Ni, Y.-F. Wei, G.-L. Yu, R. Gentz, V. M. Dixit. 1997. An antagonistic decoy receptor and a death domain-containing receptor for TRAIL. Science 277:815.[Abstract/Free Full Text]
  51. Sheridan, J. P., S. A. Marsters, R. M. Pitti, A. Gurney, M. Skubatch, D. Baldwin, L. Ramakrishnan, C. L. Gray, K. Baker, W.I. Wood, et al 1997. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277:818.[Abstract/Free Full Text]
  52. Degli-Esposti, M. A., W. C. Dougall, P. J. Smolak, J. Y. Waugh, C. A. Smith, R. G. Goodwin. 1997. The novel receptor TRAIL-R4 induces NF-kB and protects against TRAIL-mediated apoptosis yet retains an incomplete death domain. Immunity 7:813.[Medline]
  53. Cheng, J., T. Zhou, C. Liu, J. P. Shapiro, M. J. Brauer, M. C. Kiefer, P. J. Barr, J. D. Mountz. 1994. Protection from Fas-mediated apoptosis by a soluble form of the fas molecule. Science 263:1759.[Abstract/Free Full Text]
  54. Suzuki, I., P. J. Fink. 1998. Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand. J. Exp. Med. 187:123.[Abstract/Free Full Text]
  55. van Essen, D., H. Kikutai, D. Gray. 1995. CD40 ligand-transduced co-stimulation of T cells in the development of helper function. Nature 378:620.[Medline]
  56. Tanaka, Y., D. H. Adams, S. Shaw. 1993. Proteoglycans on endothelial cells present adhesion-inducing cytokines to leukocytes. Immunol. Today 14:111.[Medline]
  57. Cascino, I., G. Fiucci, G. Papoff, G. Ruberti. 1995. Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. J. Immunol. 154:2706.[Abstract]
  58. Foy, T. M., A. Aruffo, J. Bajorath, J. E. Buhlmann, R. J. Noelle. 1996. Immune regulation by CD40 and its ligand gp39. Annu. Rev. Immunol. 14:591.[Medline]
  59. Arpin, C., J. D’echanet, C. Van Kooten, P. Merville, G. Grouard, F. Briere, J. Banchereau, Y.-J. Liu. 1995. Generation of memory B cells and plasma cells in vitro. Science 268:720.[Abstract/Free Full Text]
  60. Emery, J. G., P. McDonnell, M. B. Burke, K. C. Deen, S. Lyn, C. Silverman, E. Dul, E. R. Appelbaum, C. Eichman, R. DiPrinzio, et al 1998. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J. Biol. Chem. 273:5117.



This article has been cited by other articles:


Home page
 Endocr. Rev.Home page
J. Lorenzo, M. Horowitz, and Y. Choi
Osteoimmunology: Interactions of the Bone and Immune System
Endocr. Rev., June 1, 2008; 29(4): 403 - 440.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
H K Datta, W F Ng, J A Walker, S P Tuck, and S S Varanasi
The cell biology of bone metabolism
J. Clin. Pathol., May 1, 2008; 61(5): 577 - 587.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
A. E. Kearns, S. Khosla, and P. J. Kostenuik
Receptor Activator of Nuclear Factor {kappa}B Ligand and Osteoprotegerin Regulation of Bone Remodeling in Health and Disease
Endocr. Rev., April 1, 2008; 29(2): 155 - 192.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Stolina, D. Dwyer, M. S. Ominsky, T. Corbin, G. Van, B. Bolon, I. Sarosi, J. McCabe, D. J. Zack, and P. Kostenuik
Continuous RANKL Inhibition in Osteoprotegerin Transgenic Mice and Rats Suppresses Bone Resorption without Impairing Lymphorganogenesis or Functional Immune Responses
J. Immunol., December 1, 2007; 179(11): 7497 - 7505.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
Y. Li, G. Toraldo, A. Li, X. Yang, H. Zhang, W.-P. Qian, and M. N. Weitzmann
B cells and T cells are critical for the preservation of bone homeostasis and attainment of peak bone mass in vivo
Blood, May 1, 2007; 109(9): 3839 - 3848.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Munoz-Fernandez, F. J. Blanco, C. Frecha, F. Martin, M. Kimatrai, A. C. Abadia-Molina, J. M. Garcia-Pacheco, and E. G. Olivares
Follicular Dendritic Cells Are Related to Bone Marrow Stromal Cell Progenitors and to Myofibroblasts
J. Immunol., July 1, 2006; 177(1): 280 - 289.
[Abstract] [Full Text] [PDF]

Home page
 J. Dent. Res.Home page
P. Valverde, T. Kawai, and M.A. Taubman
Potassium Channel-blockers as Therapeutic Agents to Interfere with Bone Resorption of Periodontal Disease
J. Dent. Res., June 1, 2005; 84(6): 488 - 499.
[Abstract] [Full Text] [PDF]