Osteoprotegerin, a Crucial Regulator of Metabolism, Also Regulates Development and Function

This information is current as Theodore J. Yun, Michelle D. Tallquist, Alexandra Aicher, of September 23, 2021. Katherine L. Rafferty, Aaron J. Marshall, James J. Moon, Maria K. Ewings, Mariette Mohaupt, Susan W. Herring and Edward A. Clark J Immunol 2001; 166:1482-1491; ;

doi: 10.4049/jimmunol.166.3.1482 Downloaded from http://www.jimmunol.org/content/166/3/1482

References This article cites 46 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/166/3/1482.full#ref-list-1 http://www.jimmunol.org/

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Osteoprotegerin, a Crucial Regulator of Bone Metabolism, Also Regulates B Cell Development and Function1

Theodore J. Yun,*§ Michelle D. Tallquist,¶ Alexandra Aicher,†§ Katherine L. Rafferty,‡ Aaron J. Marshall,*§ James J. Moon,* Maria K. Ewings,†§ Mariette Mohaupt,ʈ Susan W. Herring,‡ and Edward A. Clark2*†§

Osteoprotegerin (OPG) is a CD40-regulated gene in B cells and dendritic cells (DCs). We investigated the role of OPG in the immune system by generating opg؊/؊ mice. Like its role as a regulator of bone metabolism, OPG also influences processes in the immune system, notably in B cell development. Ex vivo, opg؊/؊ pro-B cells have enhanced proliferation to IL-7, and in opg؊/؊ spleen, there is an accumulation of type 1 transitional B cells. Furthermore, opg؊/؊ bone marrow-derived DCs are more effective in stimulating allogeneic T cells than control DCs. When challenged with a T-dependent Ag, opg؊/؊ mice had a compromised

ability to sustain an IgG3 Ag-specific response. Thus, in the immune system, OPG regulates B cell maturation and development Downloaded from of efficient Ab responses. The Journal of Immunology, 2001, 166: 1482–1491.

reviously, we characterized a molecule from a human fol- and bone marrow cells (8, 9, 13, 14), implying that it functions within licular (DC) line called follicular DC-de- the immune system in addition to bone metabolism. TNF-related ap- rived receptor-1 (1), which is identical with osteoprote- optosis-inducing ligand is known to cause of a variety of P 3 gerin (OPG) (2). OPG is a member of an emerging subgroup of cell lines by ligating one of its several death receptors (15), yet its in http://www.jimmunol.org/ the TNFR family that can function as soluble decoy receptors vivo role still remains to be elucidated (15–17). (DcR). Examples of other members of this subgroup are DcR-1, -2, The RANKL-RANK system influences processes in the immune and -3 (3). A major function of these soluble inhibitors is to com- system. Both RANKL- and RANK-deficient mice have defects in pete with membrane receptors, thereby inhibiting apoptosis induc- T and B cell development and lymphorganogenesis (11, 18), im- tion by their ligands. For example, the recently described CD95 plicating these molecules in lymphocyte and lymph node (LN) DcR3 may inhibit Fas ligand- and LIGHT-induced apoptosis of development. Also, in vitro studies suggest that RANKL and certain tumors (4, 5), and it is possible that amplification of DcR3 RANK may play a role in DC survival and function (13, 14, 19). by tumor cells may select for apoptosis-resistant cells. Indeed, both RANK and RANKL were originally described in

Bone metabolism is one of the in vivo processes regulated by studies of T cell and DC activation (13, 14, 20). The expression by guest on September 23, 2021 OPG. OPG has a dramatic effect on both differentiation pattern of these molecules also suggests they are involved in the and activation (2, 6, 7). Addition of rOPG to osteoclast cultures immune system. While RANK and OPG have been detected on a inhibits the differentiation of precursors to mature, multinucleated variety of cells, they are also expressed on B lymphocytes and DCs (2). OPG can also directly inhibit the function of ma- (1, 13, 21). Furthermore, the expression of OPG is up-regulated in ture osteoclasts in bone slice cultures (7). Furthermore, opg trans- DC and primary B cells by activation through CD40 (1), a receptor genic mice develop osteopetrosis (2). Clearly, this molecule reg- required for germinal center (GC) formation (22, 23). Thus, OPG ulates osteoclastogenesis in the bone marrow. may be involved in regulating B cell or DC functions. OPG has two known TNF family ligands: receptor activator of To address the in vivo role of OPG in the immune system, we NF-␬B ligand (RANKL) and TNF-related apoptosis-inducing ligand generated OPG-deficient mice using targeted genetic recombina- (8–10). RANKL, like OPG, regulates final stages of osteoclast dif- tion in embryonic stem (ES) cells. Mice homozygous for the dis- ferentiation (8, 9, 11, 12). RANKL is primarily expressed by T cells rupted opg allele are viable and progressively developed severe as they age. Further analysis revealed perturbations in

Departments of *Immunology, †Microbiology, and ‡Orthodontics, and §Regional Pri- central and peripheral B compartments, including an accumulation mate Research Center, University of Washington, Seattle, WA 98195; ¶Division of of type 1 transitional (T1) B cells (24). Ex vivo, pro-B cells and Basic Sciences and Program in Developmental Biology, Fred Hutchinson Cancer ʈ DCs were hyperresponsive in functional assays. These immune Research Center, Seattle, WA 98109; and Max Delbrueck Center for Molecular Ϫ/Ϫ Medicine, Berlin, Germany phenotypes are converse to that found in RANKL mice, thus Received for publication July 19, 2000. Accepted for publication October 31, 2000. providing a genetic link between these molecules in the immune system. When challenged with a T-dependent (TD) Ag, the The costs of publication of this article were defrayed in part by the payment of page Ϫ/Ϫ charges. This article must therefore be hereby marked advertisement in accordance opg mice were less effective in their ability to isotype switch. with 18 U.S.C. Section 1734 solely to indicate this fact. We propose that these differences are attributable to dysregulation 1 This work was supported by National Institutes of Health Grants AI44257, of RANK stimulation of both DC and B cells and discuss possible DE13061, HD24875, and HD25326. mechanisms that can account for these observations. 2 Address correspondence and reprint requests to Dr. Edward A. Clark, Department of Microbiology, Regional Primate Research Center, University of Washington, Seattle, WA 98195. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: OPG, osteoprotegerin; DC, dendritic cell; GC, Disruption of the opg locus germinal center; DcR, decoy receptor; ES, embryonic stem; T1, type 1 transitional; T2, type 2 transitional; TD, T-dependent; BAC, bacterial artificial ; Mouse opg cDNA was isolated using RT-PCR using the following primers: KLH, keyhole limpet hemocyanin; WT, wild type; LN, lymph node; MZ, marginal 5Ј-GAGGTTTCTCGAGGACCACAATGAACAA-3Ј (upstream) and 5Ј- zone; RANKL, receptor activator of NF-␬B ligand. GGCCCATCTAGAAGAAACAGCCCAGTG-3Ј (downstream). Using the

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 1483

mouse opg cDNA fragment as a probe, we screened filters representing San Diego, CA), anti-CD43 (PE-conjugated S7; PharMingen), anti-IgM clones from a mouse genomic bacterial artificial chromosome (BAC) li- (FITC-, biotin-conjugated Bet-2, PE-conjugated R6-60.2; PharMingen), anti- brary (strain 129S6/SvEvTac; RPCI-22; Research Genetics, Huntsville, CD3 (biotin-conjugated 145-2C11; PharMingen), anti-CD8 (PE-conjugated AL) following the manufacturer’s protocol. We identified a BAC clone that 53-6.7; PharMingen), anti-CD4 (FITC-conjugated H129.19; PharMingen), anti- was positive for the mouse opg genomic locus. From this clone, we sub- CD19 (FITC-conjugated ID3; PharMingen), anti-CD21 (FITC-conjugated cloned two XbaI-SmaI fragments that were ϳ2.5 kb and 9 kb into pBlue- 796; PharMingen), anti-IgD (biotin-conjugated JA12.5), anti-CD11c (PE-con- script II (SKϩ; Stratagene, La Jolla, CA) and obtained partial sequences of jugated HL3; PharMingen), anti-CD86 (biotin-conjugated GL1; PharMin- both. Next, we subcloned a 2-kb SnaBI-EcoRI fragment into the targeting gen), anti-IAb (biotin-conjugated Y3P, a generous gift from A. Rudensky; vector pKMCS, upstream of the PGK-neor gene. For the long arm, we biotin-conjugated AF6-120.1; PharMingen), and anti-CD40 (biotin-conju- subcloned a 6-kb SmaI-StuI fragment downstream of PGK-neor and up- gated 1C10). Staining by biotinylated mAbs was visualized by streptavidin stream of PGK-dta. The construct was linearized by digestion with XhoI. conjugated to PerCP (Becton Dickinson, Mountain View, CA). Appropri- As previously described (25), AK7 ES cells were transfected with 20 ␮gof ate FITC, PE, and biotinylated isotype controls were run in each experi- the linearized targeting construct. Transfected ES cell clones that had in- ment to determine gates. tegrated the targeting construct were selected by their resistance to neo- mycin. Cells that had randomly integrated the construct were negatively Pro-B cell isolation and culture selected by their expression of the PGK-dta gene. Over 300 clones were Pro-B cell proliferation assays were performed as previously described screened and one ES cell clone had a successfully targeted disruption of an (28). Using FACS, pro-B cells (B220ϩCD43ϩ) cells were collected from opg allele, which was confirmed by southern blotting of ES cell DNA. opgϩ/ϩ, opgϩ/Ϫ, and opgϪ/Ϫ littermates. For the experiments reported, Southern blotting 7500 cells were aliquoted into each well and incubated with 10 ng/ml of rIL-7 (R&D Systems, Minneapolis, MN) for 3 or 4 days. On the specified Briefly, DNA was extracted from spleen cells. Purified genomic DNA (10 days, the cells were pulsed with 1 ␮Ci/well of [3H]thymidine. Cells were ␮g) was completely digested with XbaI. Fragments were resolved by elec- harvested and [3H]thymidine incorporation was measured. trophoresis through a 0.7% agarose gel in TAE buffer, then transferred to Downloaded from GeneScreenPlus membrane (NEN Life Sciences Products, Boston, MA) as Derivation of bone marrow-derived DCs 32 previously described (25). The DNA was hybridized to a P-labeled probe DCs were prepared from mouse bone marrow as previously described (29), generated from random hexamer primer extension of a genomic opg frag- with some modifications. Briefly, bone marrow from opgϩ/ϩ, opgϩ/Ϫ, and ment. The probe was prepared using a random primers DNA labeling sys- opgϪ/Ϫ littermates was flushed from femurs and tibia, adherent cells were tem (Life Technologies, Gaithersburg, MD) following the manufacturer’s removed, and nonadherent cells were cultured in medium in the presence protocol. of GM-CSF (ϳ40 ng/ml). Mouse GM-CSF was produced by infection of http://www.jimmunol.org/ Polymerase chain reaction NIH-3T3 cells with Psi2-pM5DGM#6 (M. Mohaupt, manuscript in prep- aration) The concentration of GM-CSF in the supernatant was determined PCR was used to genotype mice harboring the disrupted allele. An opg by ELISA (Dianova, Hamburg, Germany) to be 80 ng/ml. locus-specific primer (5Ј-GGTCCTCCTTGATTTTTCTATGCC-3Ј) was On day 1 and 3 of culture, the nonadherent cells were discarded and the used in combination with upstream primers specific for neor (5Ј-TGAC remaining adherent cells were washed with warm RPMI 1640, than fed CGCTTCCTCGTGCTTTAC-3Ј)oropg (5Ј-TGCCCTGACCACTCT with fresh medium and GM-CSF containing supernatant every second day. TATACGGAC-3Ј). Approximately 2 ␮g of genomic DNA was used as a The DCs were used at days 8–9 for additional experiments. For induction template in a 50-␮l reaction composed of 2.5 U Taq polymerase (Promega, of maturation, DC were incubated for 24 h (days 8–9) with 10 ␮g/ml LPS ϫ 2ϩ Madison, WI), 10 Mg -free PCR buffer (Promega), and 1.5 mM MgCl2. (Sigma-Aldrich, Deisenhofen, Germany). Northern blotting T cell hybridoma assay by guest on September 23, 2021 Livers from mice were harvested and quick-frozen in liquid nitrogen. The T cell hybridoma assays were performed as previously described (30, 31). frozen livers were ground using a mortar and pestle, then lysed in TRIzol Triplicate cultures of 1 ϫ 103 cells/well of LPS-activated opgϪ/Ϫ or (Life Technologies). Total liver RNA was extracted following the manu- opgϩ/Ϫ bone marrow-derived DCs were incubated with variable concen- facturer’s protocol. RNA was resolved on a 1.5% agarose gel in MOPS trations of peptide (E␣(52–68) or hClip), and 1 ϫ 105 cells/well of T cell buffer, then transferred to GeneScreenPlus, following the manufacturer’s hybrids specific for E␣(52–68):I-Ab or hClip:I-Ab. After 24 h of culture, protocol. The RNA was hybridized by standard methods using 32P-labeled the supernatants were assayed for production of IL-2 by HT-2 proliferation. probe generated from random hexamer primer extension of the specified The degree of proliferation was assessed by the Alamar blue colorimetric opg cDNA fragments. assay. The results are expressed in arbitrary OD units (A550-A600 nm). Fluorescent label of newly deposited bone Mixed lymphocyte reaction A total of 13 mice were used to examine the effects of OPG on the mor- For the MLR, the day 9 bone marrow-derived DCs were further purified phology and patterns of growth/remodeling of the skeleton. The mice using FACS. DCs were washed, then incubated with dialyzed anti- ranged in age from 1 to 9 mo and consisted of littermate groups of opgϩ/ϩ, CD11cϩ. Cells were initially seeded at 10,000 cells/well, then serially di- opgϩ/Ϫ, and opgϪ/Ϫ animals. Ten days before sacrifice, the mice were luted. The opgϪ/Ϫ and control mice were H-2b. T cells were isolated from injected i.p. with a solution of 30 mg/kg calcein (Sigma, St. Louis, MO) LN and spleens of BALB/c mice (H-2d). For the spleen, RBCs were lysed that was neutralized with a 1-N solution of NaOH and forced through a using Gey’s hemolysis solution. CD4ϩ T cells were enriched by comple- 0.22-␮m syringe filter. A second solution of alizarin complexone (Sigma) ment lysis. Cells were incubated with 10 ␮g/ml of Bet-2 (anti-IgM) and was administered in a similar manner 7 days later. The animals were eu- 3.155 (anti-CD8) (at 1:200 dilution) for 30 min at 4°C. Cells were then thanized 3 days following the last injection. Both calcein and alizarin com- incubated with 20 ␮g/ml of MAR185 (Rat anti-mouse Ig), for 30 min at plexone bind with calcium ions on the surfaces of newly forming apatite 4°C. Cells were then treated with guinea pig Low-Tox complement (Ce- crystals, thus labeling the bone undergoing mineralization during the pe- darlane Laboratories, Westbury, NY) for 45 min at 37°C. The cells were riod of exposure (26). Following euthanasia the skeletons were stripped of washed, then overlayed onto a Ficoll-Hypaque cushion (Pharmacia, Pisca- soft tissue, and the individual elements were radiographed and photo- taway, NJ). The cells at the interface were collected and washed. The graphed before being embedded in methyl methacrylate. The plastic blocks enriched CD4ϩ T cells were incubated with 0.5 ␮g/ml 2C11 (anti-CD3) for were sectioned to a thickness of 30–40 ␮m with a Leica (Deerfield, IL) 18 h. The cells were then washed, then ϳ10,000 cells were seeded into SP1600 saw microtome and the mounted sections were viewed using the wells containing DCs. The reaction was pulsed with 0.5 ␮Ci/well of fluorescent mode of a Nikon (Melville, NY) Eclipse E400 microscope. [3H]thymidine after 3 days. After 18 h, cells were harvested and [3H]thy- midine incorporation was measured. Flow cytometry Immunization and serum Ab assays For flow cytometry analyses, cell suspensions were prepared from the spec- ified organs of 1.5- to 4-mo-old mice from opgϩ/ϩ, opgϩ/Ϫ, and opgϪ/Ϫ Immunization with 100 ␮g of DNP-keyhole limpet hemocyanin (KLH; littermates. Inguinal and/or cervical LNs were collected. RBCs from spleen Calbiochem, La Jolla, CA), and measurement of serum DNP-specific Ab ␮ and bone marrow cells were lysed using ACK (0.155 M NH4Cl, 0.1 mM isotypes were performed as previously described (27). Briefly, 100 g Ϫ/Ϫ EDTA, 0.01 M KHCO3). Flow cytometric analysis was performed as pre- alum-precipitated DNP-KLH was injected i.p. opg and control litter- viously described (27). The mAbs used for the experiments reported are as mate mice of 4–6 wk of age were chosen, so that the last time point would follows: anti-B220 (PE-, FITC-, biotin-conjugated RA3-6B2; PharMingen, be before the onset of severe osteoporosis. Serum was collected and was 1484 THE IMMUNE PHENOTYPE OF OPG-DEFICIENT MICE

assayed for DNP-specific Ab by ELISA. Nunc-Immuno Plate MaxiSorp band, as predicted (Fig. 1B). A PCR-based screen was developed 96-well plates (Van Waters and Rogers, Bisbane, CA) were coated with 4 to facilitate genotyping (Fig. 1B). ␮ g of DNP-BSA. Plates were washed, then blocked with 1% BSA and To demonstrate that the intact OPG product was not expressed, 0.05% Tween 20. Serial dilutions of sera were prepared and added in trip- licate to plates. The specified isotypes were detected with 450 ng/ml sheep we performed Northern blot analysis of total RNA from the liver antisera specific for the indicated mouse isotype (The Binding Site, Bir- isolated from opgϪ/Ϫ or opgϩ/Ϫ mice. Using a probe specific for mingham, U.K.) followed by 375 ng/ml HRP-conjugated donkey anti- the region deleted (Fig. 1C), we could not detect a transcript from sheep serum. Titers of DNP-specific isotypes were revealed by peroxidase Ϫ/Ϫ reaction using o-phenylenediamine as a substrate. Variation among plates the opg mice (Fig. 1D). When the entire cDNA was used as a was controlled by pooling anti-DNP antiserum from immunized mice, pre- probe, we detected a smaller transcribed product (Fig. 1D). This paring bulk serial dilutions as in the test samples, and using it as a relative indicates that the disrupted allele is transcribed, and suggests that standard on each plate. Relative isotype-specific Ab response to DNP of the PGK-neor gene is eliminated by splicing. Based on the each test sample was calculated by normalizing the value at half-maximal OD of the test sample against the value at half-maximal OD of the standard genomic sequence of opg (32), the spliced product would no dilutions. longer be in frame, and thus would produce a nonfunctional protein. Results Because OPG plays a critical role in regulating osteoclast for- Targeted disruption of opg results in osteoporosis mation, opgϪ/Ϫ mice were expected to develop severe osteoporo- Using mouse opg cDNA as a probe, we isolated a mouse genomic sis (12, 33). To test whether the disrupted allele was indeed a ϩ/Ϫ Ϫ/Ϫ BAC clone including the mouse opg locus. The targeting construct functional null, we radiographed opg and opg littermates (Fig. 2A). The bone density of the 3-mo-old opgϪ/Ϫ male mouse is

was designed so that a targeted recombination event would elim- Downloaded from Ϫ/Ϫ inate the coding sequence for the first two Cys-rich repeats of the dramatically decreased compared with the control. Older opg mature protein. In addition, 8 of 21 residues in the signal pep- mice often exhibit fractures in long bone diaphyses, particularly tide and the first half of the third Cys-rich region would be the humerus, femur, and fibula (K.L.R., S.W.H., and T.J.Y., un- deleted (Fig. 1A). published observations). Animals as young as 2 mo showed short- The recombination of the targeting construct with the opg locus ened femoral necks, suggesting common traumatic fracture at this introduces a XbaI site that could distinguish the disrupted allele location (Fig. 2B). Surprisingly, the outer diameters of the long Ϫ Ϫ http://www.jimmunol.org/ from the wild-type (WT) allele, using a probe specific for a 3-kb bone diaphyses and the mandibular corpus are greater in opg / region 3Ј of the 6-kb SmaI StuI long arm. In XbaI-digested mice compared with their opgϩ/Ϫ littermates (Fig. 2, B and C). To genomic DNA, the mutant band is ϳ4 kb smaller than the WT follow up on the radiographic results, mice were injected with by guest on September 23, 2021

FIGURE 1. Generation of opgϪ/Ϫ mice. A, The opg locus (top) was targeted by constructing a vector with PGK-neor gene flanked by the indicated fragments from the opg genomic locus (middle). The region from the genomic opg locus that was used as a flanking probe in Southern blots is indicated by the white bar (top). The recombined locus introduces an XbaI site (bottom). B, Southern blot and PCR strategy to determine recombination of targeting construct into the genomic opg locus. Splenic genomic DNA was isolated from opgϩ/ϩ, opgϩ/Ϫ, and opgϪ/Ϫ littermates, digested with XbaI, then blotted with the flanking probe as indicated in A (top). A PCR-based screening method was developed to facilitate genotyping of mice. DNA from opgϩ/ϩ, opgϩ/Ϫ, opgϪ/Ϫ or no template (nt) was tested. The primers amplify a 200-bp fragment from the WT opg locus and a 500-bp fragment from the disrupted allele. C, The indicated regions from the opg cDNA were used as probes for Northern blotting. A probe generated from the full length cDNA (I) and a probe generated from the deleted region (II) were used. D, Northern blots of opgϪ/Ϫ mice. Liver total RNA was isolated from opgϩ/Ϫ and opgϪ/Ϫ littermates and analyzed with probes specific for the full length or the deleted region. The Journal of Immunology 1485

FIGURE 2. opgϪ/Ϫ mice develop profound osteoporosis, and have gross deformations in bone structure. A, Ra- diographs of 3-mo-old opgϩ/Ϫ and opgϪ/Ϫ littermates reveal a decrease in bone density of opg-deficient mice, particularly in the long . For den- sity comparisons, the mice were radio- graphed together. B, Isolated femurs from 9-mo-old opgϩ/Ϫ and opgϪ/Ϫ lit- termates were radiographed. Gener- ally, long bones from opgϪ/Ϫ mice have altered physical dimensions, in- cluding increased diaphyseal diame- Downloaded from ters (upper). Also apparent is the lack of a femoral neck (lower). C, Superior view of mandibles from opgϩ/Ϫ and opgϪ/Ϫ 9-mo-old littermates. There is bony hypertrophy in the mandibular corpus of opgϪ/Ϫ mice (upper, white http://www.jimmunol.org/ arrows). Cross sections of the mandi- ble from the region indicated by the arrows (upper) shown in the bottom panel. Calcein (green fluorescence) and alizarin complexone (red fluores- cence) were injected into 9-mo-old opgϩ/Ϫ and opgϪ/Ϫ littermates to ob- serve newly deposited bone (lower). Note the greater porosity and in- Ϫ/Ϫ creased deposition in the opg by guest on September 23, 2021 mouse, compared with its littermate control.

fluorochromes, which label newly mineralized bone (26). Histo- in the presence of IL-7 for 3 or 4 days, then measured differences logically, the cortical bone of opgϪ/Ϫ mice is porous compared in proliferation (Fig. 3). opgϪ/Ϫ pro-B cells gave a greater prolif- with that of opgϩ/Ϫ littermates and shows a greater quantity of erative response to IL-7 than either opgϩ/ϩ or opgϩ/Ϫ controls. fluorescing label (Fig. 2C). This finding suggests that increased Interestingly, a gene dosage effect was evident because cells with osteogenesis accompanies the increased osteoclast activity in one copy of the intact locus had an intermediate response to IL-7. opgϪ/Ϫ mice. Generally, opgϪ/Ϫ pro-B cells had a 1.7- to 2-fold increase in IL-7 responsiveness compared with opgϩ/ϩ pro-B cells. OPG regulates early B cell development Because pro-B cells from the opgϪ/Ϫ bone marrow had an en- In long bones, the marrow cavity is the site of hematopoiesis and hanced proliferative response to IL-7 in vitro, we next questioned B lymphopoiesis. In opgϪ/Ϫ mice, the dysregulation of osteoclast whether this population would be increased in vivo. Using flow production leads to drastic changes in the bone architecture. Fur- cytometry we observed no significant differences in the numbers of thermore RANKL-deficient mice have a defect in B cell develop- nonlymphoid lineage (B220Ϫ) and lymphoid lineage (B220ϩ) ment from the pro-B to pre-B cell transition (11), suggesting that cells in opgϪ/Ϫ and control mice (Table I). However, RANKL/RANK/OPG may play a role in regulating B cell B220lowCD43ϩIgMϪ pro-B cells (36), were increased in opgϪ/Ϫ development. mice compared with littermate controls. Similar results were ob- To test whether OPG can regulate the pro-B cell population, we tained using CD25 to distinguish pro- and pre-B populations (data compared the ability of ex vivo opgϪ/Ϫ, opgϩ/Ϫ, and opgϩ/ϩ not shown). Because the mean percentages of other B220ϩ pop- pro-B cells to respond to rIL-7. Pro-B cells are dependent on the ulations were similar, these data show that there is an increase in presence of IL-7 for survival and proliferation (28, 34, 35). We pro-B cells in the opgϪ/Ϫ bone marrow. The observed increase isolated and cultured pro-B cells from opgϪ/Ϫ or opgϩ/Ϫ controls in the pro-B cell compartment is unlikely to be due to the 1486 THE IMMUNE PHENOTYPE OF OPG-DEFICIENT MICE

but no significant difference in the mature splenic B cell population. The IgMhighIgDlow population includes the T1 and MZ B cells (24). These two subpopulations can be distinguished by CD21 ex- pression; T1 B cells are CD21low and MZ B cells are CD21high (24, 37). Based on the expression of IgM and IgD on mature B cells in LN and immature B cells in bone marrow, we determined the gates for the IgMhighIgDlow, IgMhighIgDhigh,orIgMlowIgDhigh popula- tions in the spleen (Fig. 4A). In Fig. 4B, the expression level of CD21 was measured in the IgMhighIgDlow population. The per- centage of IgMhighIgDlowCD21low population was consistently in- creased in the opgϪ/Ϫ mice, compared with opgϩ/ϩ mice. This increase in the percentage of T1 B cells was reflected by a signif- icant increase in the absolute numbers (Fig. 4C). We also observed a modest increase in numbers of T2 and mature B cells in opgϪ/Ϫ mice and also to some extent in opgϩ/Ϫ mice, compared with ϩ/ϩ Ϫ/Ϫ opg mice, again suggesting a gene dosage effect. In contrast, FIGURE 3. Ex vivo opg pro-B cells are more responsive to IL-7. there was no discernable difference in the numbers of MZ B cells CD43ϩ pro-B cells were purified from bone marrow of opgϩ/ϩ, opgϩ/Ϫ,or Ϫ Ϫ or T cells (Fig. 4C). Downloaded from opg / littermates by FACS. Seven-thousand five-hundred cells per well were seeded in triplicate in the presence of 10 ng/ml of IL-7. Proliferation Absence of OPG results in enhanced stimulatory capacity of DC was then measured on the indicated day after plating. The mean of each Ϫ/Ϫ triplicate culture and the SEM are represented. Similar results were ob- Given these lymphoid developmental perturbations in opg tained in three independent experiments. mice, we next investigated the effect of OPG deficiency on im- mune responses. Both RANK and OPG are up-regulated by

CD40 ligation in DCs (1, 13), which suggests that during T cell http://www.jimmunol.org/ Ϫ Ϫ expansion of the marrow cavity in opg / osteoporotic mice activation by DCs, OPG may be expressed as a negative regu- because the absolute number of lymphoid lineage cells is not lator to modulate the T cell responses (38). According to this increased. Together with the enhanced proliferative response of model, we hypothesized that in the absence of OPG, dysregu- Ϫ Ϫ opg / pro-B cells in vitro, this result suggests that OPG nega- lated RANK signaling could alter stimulatory capabilities of tively regulates expansion of the pro-B cell pool. opgϪ/Ϫ DCs. Bone marrow cells from opgϪ/Ϫ or opgϩ/Ϫ littermates were Increase in frequency of peripheral B cell subpopulations cultured in the presence of GM-CSF. After 7 days, an enriched Because both RANK and OPG are expressed in B lineage cells population of functional DCs differentiated from monocytic pre- (1, 13) and both are up-regulated by CD40 cross-linking, we cursors. Flow cytometric analysis of these cells revealed coexpres- by guest on September 23, 2021 next analyzed peripheral B cell populations. We consistently sion of CD11c, MHC class II, and CD86 (data not shown). Mat- observed a greater percentage of peripheral B cells uration of these cultured DCs was induced by incubation with LPS, (B220ϩCD19ϩ)inopgϪ/Ϫ mice vs controls. This difference as measured by up-regulation of costimulatory molecules (39). The was evident in the absolute numbers of splenic and LN B cells levels of expression of MHC class II or CD86 were not different on in opgϪ/Ϫ mice vs controls (Table II). In contrast, there were no opgϪ/Ϫ and opgϩ/Ϫ CD11cϩ cells, both on stimulated and un- significant differences in mean numbers of LN or splenic CD8ϩ stimulated cells (Fig. 5A). Also, when tested for their ability to and CD4ϩ T cells. present exogenous peptide to T cell hybridomas expressing a pep- Based on levels of membrane IgM and IgD expression, the ma- tide-Iab complex-specific TCR, we observed no difference in this jority of splenic B cells can be subdivided into IgMhighIgDlow, early presentation event (Fig. 5B). However, in 3-day MLRs, we IgMhighIgDhigh,orIgMlowIgDhigh (24). These three populations found that DCs from opgϪ/Ϫ mice consistently had a 2- to 5-fold have been classified as T1 plus marginal zone (MZ), type 2 tran- enhanced ability to stimulate allogeneic T cell proliferation (Fig. sitional (T2), and mature B cells, respectively (24). Using IgM and 5C) compared with control opgϩ/Ϫ DCs. IgD expression levels, we found that the mean percentage and absolute numbers of the IgMhighIgDlow B cells were significantly Absence of OPG results in isotype class switch defects during increased in opgϪ/Ϫ vs controls (Table II). On average, there were the primary immune response 53% more of this population in opgϪ/Ϫ mice than controls. There Next, we measured Ab response to TD Ag mounted by opgϪ/Ϫ was also a modest increase in the IgMhighIgDhigh B cell population, mice. We immunized mice with the TD Ag, DNP-KLH, and

Table I. Bone marrow populations in opgϪ/Ϫ and opgϩ/Ϫ mice

Population opgϩ/Ϫ opgϪ/Ϫ No. p Value

Absolute Number per Femur (ϫ10Ϫ6) B220Ϫ cells (ϫ10Ϫ6) 14.2 Ϯ 1.4 15.2 Ϯ 1.6 31 Ͻ0.2 B220ϩ cells (ϫ10Ϫ6) 8.2 Ϯ 0.8 8.6 Ϯ 0.7 31 Ͻ0.3

Percent of B220ϩ Cells Pro-B (B220ϩCD43ϩIgMϪ) (%) 15.4 Ϯ 0.8 16.8 Ϯ 0.9 31 Ͻ0.04 Pre-B (B220ϩCD43ϪIgMϪ) (%) 46.3 Ϯ 1.8 46.8 Ϯ 1.8 31 Ͻ0.33 The Journal of Immunology 1487

Table II. Peripheral lymphoid populations in opgϪ/Ϫ and opgϩ/Ϫ mice

Population opgϩ/Ϫ opgϪ/Ϫ No. p Value

Lymph Node CD8 (ϫ10Ϫ6) 1.35 Ϯ 0.22 1.69 Ϯ 0.4 9 Ͻ0.13 CD4 (ϫ10Ϫ6) 1.93 Ϯ 0.29 2.37 Ϯ 0.44 9 Ͻ0.1 B cells (ϫ10Ϫ6) 0.84 Ϯ 0.11 1.13 Ϯ 0.13 15 Ͻ0.005

Spleen CD8 (ϫ10Ϫ6) 10.94 Ϯ 1.64 11.71 Ϯ 2.1 10 Ͻ0.35 CD4 (ϫ10Ϫ6) 18.01 Ϯ 1.9 18.63 Ϯ 3.17 10 Ͻ0.43 B cells (ϫ10Ϫ6) 38.6 Ϯ 6.0 50.9 Ϯ 8.4 20 Ͻ0.008 IgMhighIgDlow (ϫ10Ϫ6) 7.4 Ϯ 1.3 11.3 Ϯ 2.1 20 Ͻ0.0006 IgMhighIgDhigh (ϫ10Ϫ6) 3.8 Ϯ 0.8 4.8 Ϯ 0.9 20 Ͻ0.035 IgMlowIgDhigh (ϫ10Ϫ6) 15.4 Ϯ 2.7 18.1 Ϯ 3.5 20 Ͻ0.12 measured Ag-specific serum Ig levels of several isotypes from opgϩ/ϩ control mice. When averaged together, the ability of the opgϪ/Ϫ and control mice (opgϩ/ϩ and opg Ϯ ) on days 0, 7, 14, opgϪ/Ϫ mice to mount an IgM, IgG1, IgG2a, and IgG2b re- and 21. On day 21, the mice were boosted with a second chal- sponse to DNP was comparable to the response by opgϩ/ϩ con- Downloaded from lenge Ag and the secondary Ab response was measured on days trol mice. However, three of the eight opgϪ/Ϫ mice assayed had 28 and 35. deficient primary IgG2a, IgG2b, and/or IgG3 responses. One of The Ab responses of each isotype in the opgϩ/ϩ control mice the four mice failed to isotype switch to IgG2a, and another were very similar; however, the immune responses in opgϪ/Ϫ mouse failed to isotype switch to IgG3. This failure to isotype mice were highly variable (Fig. 6). We observed a significant switch was not observed in any of the 11 opgϩ/ϩ or opgϩ/Ϫ difference in the amount of anti-DNP IgG3 produced by the control mice. Thus OPG may influence the ability of mice to http://www.jimmunol.org/ opgϪ/Ϫ mice on days 14, 21, and 35, as compared with the sustain an IgG3 response to a TD Ag. by guest on September 23, 2021

FIGURE 4. The T1 B cell population is in- creased in opgϪ/Ϫ mice. A, Cells from the specified organs of 5- or 6-wk-old littermates were isolated. Using IgM and IgD expression of mature and im- mature B cells in the LN or bone marrow, respec- tively, we determined the gates of these popula- tions in the spleen. B, Gating on the splenic IgMhighIgDlow B cells, the expression of CD21 re- solves this population into CD21low T1 B cells and CD21high MZ B cells. The percentage (of total events) of T1 cells IgMhighIgDlowCD21low is con- sistently higher in opgϪ/Ϫ mice, compared with opgϩ/ϩ littermates. This figure is representative of four experiments. C, The absolute number of the indicated populations was calculated. The mean number from four experiments and the SEM are represented. 1488 THE IMMUNE PHENOTYPE OF OPG-DEFICIENT MICE

Discussion OPG regulates both osteoclastogenesis and B lymphopoiesis Previous studies of opg-transgenic and opgϪ/Ϫ mice have revealed that OPG plays a vital role in regulating osteoclast production in the bone marrow (2, 12, 33). In this study, we have shown that OPG also regulates B lymphopoiesis, most notably at the pro-B cell and transitional B cell stages. Clues as to how OPG may regulate B lymphopoiesis may come from examining OPG’s role in regulating bone metabolism. In the absence of OPG’s normal “braking” of osteoclast maturation, the opgϪ/Ϫ mice exhibited drastic increases in cortical bone porosity that was accompanied by a striking acceleration of new apposition on periosteal and intracortical surfaces. Preliminary data indicate most pronounced increases in mineralization in those parts of the skeleton under greatest mechanical load, the long bones and the mandibular body, leading to bone hypertrophy on a gross morpho- logical level. We hypothesize that because the quality and, there-

fore, strength of bone is compromised by increased bone resorp- Downloaded from tion in the opgϪ/Ϫ mice, the compensatory response of increased apposition is most pronounced in biomechanically critical regions. This “coupling” of resorption and formation is a well known com- ponent in the pathophysiology of osteoporosis, although the un- derlying biochemical mechanisms are not fully understood (40). Physiologically, the defects in bone metabolism are the most strik- http://www.jimmunol.org/ ing phenotype of OPG deficiency; however, multiple processes in the immune system are dysregulated as well. Could the same OPG dependent regulatory mechanism affect the production of pro-B or transitional B cells in opgϪ/Ϫ mice? De- spite drastic alterations in medullary bone architecture in these mice (12, 33), the overall composition and proportions of lym- phoid and nonlymphoid cells is not changed in the absence of OPG. However, the pro-B cells and transitional B cells recently

arising from the bone marrow are disrupted (Tables I and II, and by guest on September 23, 2021 Fig. 4). A likely explanation of our results is that the B lymphoid com- partment itself is directly regulated by OPG. Not only are pro-B cells and transitional T1 B cells expanded in opgϪ/Ϫ mice, but opgϪ/Ϫ pro-B cells have a greater in vitro proliferative response to IL-7 than heterozygous or WT pro-B cells (Fig. 3). Our results suggest that pro-B cells isolated from opgϪ/Ϫ mice are intrinsically different in their proliferative response to IL-7 because pro-B cells were cultured in the absence of stromal cells or other growth fac- tors, and because addition of rOPG had no effect on pro-B cell proliferation (data not shown). Also, although the proliferative re- sponse of opgϪ/Ϫ pro-B cells may be affected, the ability of these cells to differentiate into pre-B cells does not appear to be affected (A.J.M. and T.J.Y., unpublished observation). The expansion of peripheral B cells and pro-B cells in our opgϪ/Ϫ mice is the op- posite to that observed in the RANKLϪ/Ϫ mice (11) or RANKϪ/Ϫ mice (18). Both of these mutant mice have fewer mature B cells Ϫ/Ϫ FIGURE 5. Bone marrow-derived DCs from opgϩ/Ϫ and opgϪ/Ϫ and RANKL mice have an arrest in the pro-B to pre-B tran- mice express identical levels of CD86 and MHC class II; however, they sition. Collectively, these results suggest that RANKL/RANK is differ in their ability to stimulate allogeneic T cells. A, Using flow involved in the proliferation or proliferative expansion of pro-B cytometry, levels of costimulatory molecules on DCs derived from cells, and that OPG regulates this process. ϩ Ϫ Ϫ Ϫ opg / (thin line) or opg / (thick line) mice were analyzed. Levels of There is a striking negative regulation of B lymphopoiesis by CD86 in immature (upper left panel) or LPS-stimulated DCs (lower left sex steroids, particularly by (41). This observation was panel). Levels of MHC class II in immature (upper right panel)or noted by Kincade et al. (41) while studying B lymphopoiesis LPS-stimulated DCs (lower right panel). B, No difference in the abil- ϩ Ϫ Ϫ Ϫ during pregnancy. Smithson et al. (42) demonstrated that estro- ities of opg / and opg / DCs to present exogenous peptide to pep- tide-Iab complex-specific T cell hybidomas. C, MLR using splenic and gen stimulates bone marrow stroma to secrete factors that neg- LN T cells from BALB/c mice and LPS-stimulated DCs derived from atively regulate B lymphopoiesis. Interestingly, opg expression opgϪ/Ϫ or opgϩ/Ϫ littermate mice. The mean of each triplicate culture is positively regulated by estrogen (43). Also, we have observed and the SEM are represented. The data is representative of six inde- by RT-PCR analysis that human stromal cells (kindly provided pendent experiments. by T. LeBien, University of Minnesota, Minneapolis, MN) and The Journal of Immunology 1489 Downloaded from

FIGURE 6. Ab response to DNP-KLH, a http://www.jimmunol.org/ TD Ag. opgϪ/Ϫ mice (m, n ϭ 8) and litter- mate opgϩ/ϩ (F, n ϭ 4) or opgϩ/Ϫ (U, n ϭ 7) controls were immunized with 100 ␮gof alum-precipitated DNP-KLH in PBS on day 0 and day 21. Each point represents the re- sponse from an individual mouse. The mean and SEM are shown. Asterisks indicate a statistically significant difference between the mean response of opgϪ/Ϫ and opgϩ/ϩ mice (nonpaired t test with equal variance; by guest on September 23, 2021 .(p ϭ 0.03 ,ءءء ;p ϭ 0.004 ,ءء ;p ϭ 0.04 ,ء

mouse stromal cell lines (S10 and S12), which are capable of likely at the pro-B cell stage or at a discrete stage before pro-B supporting B lymphopoiesis, can express opg (data not shown). cells. It is tempting to speculate that estrogen stimulates stromal Finally, Medina et al. (44) demonstrated that estrogen affects elements to produce OPG that negatively regulates B proliferation of developing B cells at an early stage in vivo, lymphopoiesis. 1490 THE IMMUNE PHENOTYPE OF OPG-DEFICIENT MICE

In the spleen, the numbers of T1 B cells, which represent the lack of OPG affects the ability of B cells to sustain an IgG3 re- newly produced B cells from the bone marrow (45), are selectively sponse. Interestingly, we observed a lack of IgG2a response in one increased in opgϪ/Ϫ mice. The T1 B cells are thought to be de- mouse, and a lack of IgG3 in another, suggesting that the absence pendent on B cell receptor-induced signals for entry into primary of OPG may affect IgG isotype switch. Perhaps OPG may be mod- follicles where they receive additional maturation signals. T1 B ulating B cell response by altering cytokine patterns or expression cells differentiate into T2 B cells and up-regulate the expression of of membrane costimulatory molecules. Another possibility is that IgD and other surface receptors (24). Additional signals are then the accumulation of T1 B cells in opgϪ/Ϫ mice indirectly influence required for the transition of T2 B cells into mature B cells. Be- GC formation. The possible effects of OPG on GC formation will cause the T2 and mature B cell populations appeared marginally be the subject of future investigation. affected by the lack of OPG, OPG likely regulates the T1 B The evidence in this report genetically establishes the link be- cell pool. tween RANKL, RANK, and OPG in the immune system. opgϪ/Ϫ The increase in LN B cells and splenic T1 B cells may represent mice exhibit a contrasting phenotype to RANKLϪ/Ϫ and an accumulation of cells, as opposed to proliferation, because we RANKϪ/Ϫ mice in terms of osteoclast activity and numbers of could not detect differences in activation markers expressed by peripheral B cells. Also, RANKLϪ/Ϫ B cell development and splenic or LN B cells from opgϪ/Ϫ and control mice. We favor the RANKLϪ/Ϫ T cell–DC interactions, were disrupted. In cells iso- possibility that due to the increase in the proliferative response of lated from opgϪ/Ϫ mice, we see the converse effect of these cell pro-B cells, there is an increase in the production of immature B types in similar functional assays. Physiologically, OPG deficiency cells in vivo. Another explanation is that there is more efficient during a TD-Ag immune response was demonstrated to result in an homing of B cells to these peripheral sites, analogous to the mech- inefficient ability to switch to particular isotypes, implicating Downloaded from anism that OPG regulates mature osteoclast migration into bone OPG’s role in stochastic processes that influence Ab isotype matrix (9, 11, 18). Finally, because T1 B cells that do not receive switch. We conclude that OPG is a normal regulator during B cell maturation signals likely undergo apoptosis (46), another possibil- development and during DC and B cell function in an immune ity is that there is less apoptosis of B cells in opgϪ/Ϫ mice. RANK response. mediated Bcl-XL induction (14) may also occur in B cells so that absence of OPG indirectly augments Bcl-XL expression and pro- Acknowledgments http://www.jimmunol.org/ longs T1 B cell survival. We thank D. Magaletti for excellent technical assistance; members of the Clark laboratory, especially K. L. Otipoby, for intellectual discussion; OPG functions in the immune system as a “molecular brake” Dr. P. Soriano for valuable intellectual input, technical guidance and crit- on the RANKL/RANK pathway ical review of the manuscript; P. Wong, Dr. T. Nakagawa, and Dr. A. The fact that opgϪ/Ϫ mice develop osteoporosis (Fig. 2 and Refs. Rudensky for intellectual discussion, reagents, and technical assistance; 12 and 33) and that both RANKLϪ/Ϫ and RANKϪ/Ϫ mice develop and Dr. W. C. Dougall and Dr. J. J. Peschon for intellectual discussion and osteopetrosis has led to the proposal that OPG functions as a mo- sharing unpublished results. lecular brake during osteoclastogenesis (11, 12, 18, 33). OPG ap- parently has a similar function during some cellular interactions in References by guest on September 23, 2021 the immune system. On a per cell basis, we consistently observed 1. Yun, T. J., P. M. Chaudhary, G. L. Shu, J. K. Frazer, M. K. Ewings, Ϫ/Ϫ S. M. Schwartz, V. Pascual, L. E. Hood, and E. A. Clark. 1998. OPG/FDCR-1, that opg DCs were 2- to 5-fold more effective at stimulating T a TNF receptor family member, is expressed in lymphoid cells and is up-regu- cells (Fig. 5C) than opgϩ/Ϫ DCs. This result is the converse to that lated by ligating CD40. J. Immunol. 161:6113. Ϫ/Ϫ Ϫ/Ϫ 2. Simonet, W. S., D. L. Lacey, C. R. Dunstan, M. Kelley, M. S. Chang, R. Luthy, observed in RANKL mice in that RANKL T cells are im- H. Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Osteoprotegerin: a paired such that they require more allogeneic DCs to stimulate novel secreted protein involved in the regulation of bone density. Cell 89:309. IL-2 production than RANKLϩ/Ϫ T cells. Because RANK stimu- 3. Ashkenazi, A., and V. M. Dixit. 1999. Apoptosis control by death and decoy receptors. Curr. Opin. Cell. Biol. 11:255. lation of DCs by RANKL increases their ability to stimulate T 4. Pitti, R. M., S. A. Marsters, D. A. Lawrence, M. Roy, F. C. Kischkel, P. Dowd, cells (13, 14), collectively, these results suggest that OPG normally A. Huang, C. J. Donahue, S. W. Sherwood, D. T. Baldwin, et al. 1998. Genomic functions as a molecular brake on the RANKL/RANK pathway amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396:699. during T cell-DC interactions. 5. Yu, K. Y., B. Kwon, J. Ni, Y. Zhai, R. Ebner, and B. S. Kwon. 1999. A newly OPG most likely modulates the ability of DCs to stimulate T identified member of tumor necrosis factor receptor superfamily (TR6) sup- cells after Ag processing and later during presentation, because presses LIGHT-mediated apoptosis. J. Biol. Chem. 274:13733. Ϫ/Ϫ ϩ/Ϫ 6. Tsuda, E., M. Goto, S. Mochizuki, K. Yano, F. Kobayashi, T. Morinaga, and there is no difference in the ability of opg and opg DC to K. Higashio. 1997. Isolation of a novel cytokine from human fibroblasts that present exogenous peptides to Ag-specific T hybridomas. Also, specifically inhibits osteoclastogenesis. Biochem. Biophys. Res. Commun. 234: 137. early costimulatory events are probably not affected by the absence 7. Fuller, K., B. Wong, S. Fox, Y. Choi, and T. J. Chambers. 1998. TRANCE is of OPG because the levels of class II and CD86 were identical necessary and sufficient for -mediated activation of bone resorption in between mature opgϪ/Ϫ and opgϩ/Ϫ DCs. Using RNase protection osteoclasts. J. Exp. Med. 188:997. assays, we observed no difference in a selected set of early T cell 8. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, ϩ Ϫ Ϫ Ϫ A. Tomoyasu, K. Yano, M. Goto, A. Murakami, et al. 1998. Osteoclast differ- cytokine gene induction between opg / and opg / DCs, includ- entiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory fac- ing IL-2, IL-4, IL-13, and IFN-␥ (data not shown). What is more tor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95:3597. 9. Lacey, D. L., E. Timms, H. L. Tan, M. J. Kelley, C. R. Dunstan, T. Burgess, likely is that in the absence of OPG, the quality and duration of R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Osteoprotegerin ligand RANKL/RANK interactions are altered. Because RANK stimula- is a cytokine that regulates osteoclast differentiation and activation. 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