A Novel in Vivo Role for Osteoprotegerin Ligand in Activation of Monocyte Effector Function and Inflammatory Response*

Osteoprotegerin Ligand (OPGL) is a member of the tumor necrosis factor ligand superfamily and has been shown to be involved in interactions between T cells and dendritic cells. Its role in monocyte effector function, however, has not been defined. In the present study a role for OPGL in activating monocytes/macrophages has been characterized. OPGL was found to up-regulate receptor activator of NF- (cid:1) B (RANK) receptor expression on monocytes, regulate their effector function by inducing cytokine and chemokine secretion, activate antigen presentation through up-regulation of co-stimulatory molecule expression, and promote survival. This activation is mediated through the MAPK pathway as evidenced by activation of p38 and p42/44 MAPK and up-regulation of BCL-XL protein levels. A physiological role for OPGL in monocyte activation and effector function was tested in a model of lipopolysaccharide-induced endotoxic shock. Administration of receptor activator of NF- (cid:1) B (RANK)-Fc to block OPGL activity in vivo was able to protect mice from death induced by sepsis, indicating a hitherto undescribed role for OPGL in monocyte function and in mediating inflammatory response. This was further tested in an animal model of inflamma-tion-mediated arthritis. Treatment with RANK-Fc significantly

Osteoprotegerin ligand (OPGL) 1 (also called TNF-related activation-induced cytokine, receptor activator of NF-B (RANK) ligand, and osteoclast differentiation factor) was discovered almost simultaneously by two groups during attempts to clone novel genes involved in the regulation of apoptosis and function of dendritic cells (1,2). OPGL is a member of the TNF ligand superfamily. Most TNF/TNFR superfamily proteins, including CD40L/CD40, TNF/TNFR, or lymphotoxin-␤/lymphotoxin-␤ receptor, are expressed in the immune system and are known to regulate immune response by co-coordinating homeostasis, T cell activation, dendritic cell function, or the formation of germinal centers and lymphoid organs such as Peyer's patches and lymph nodes (3)(4)(5). Sequence analysis has shown that the extracellular domain of OPGL shares 18 -28% amino acid identity with other members of the TNF superfamily and the greatest identity with CD40L (2). High levels of OPGL mRNA are detectable in T cells in the lymph nodes and bone osteoblastic cells. OPGL binds to its specific receptor, RANK, a transmembrane member of the TNFR superfamily (2). Although RANK mRNA can be detected in skeletal muscle, thymus, liver, colon, small intestine, and adrenal gland, at the protein level RANK expression to date has been detectable only on the surfaces of mature dendritic cells and osteoclasts. OPGL has also been shown to bind an alternate receptor, osteoprotegerin (OPG) (6,7). OPG acts as a soluble decoy receptor for OPGL and has been shown to neutralize the activity of OPGL (8).
Initial functional studies on OPGL revealed its ability to activate dendritic cells and inhibit apoptosis, resulting in an increase in dendritic cell-mediated T cell proliferation in a mixed leukocyte reaction (9). This increase was likely mediated by increased expression of proinflammatory cytokines, such as IL-6 and IL-1, and T cell growth factors, such as IL-12 and IL-15, by dendritic cells (10). In vitro studies have also revealed that OPGL, in combination with colony-stimulating factor 1, could activate mature osteoclasts and mediate osteoclastogenesis (6,7). Evidence for an essential role for OPGL in the immune system and bone development was provided by using OPGL Ϫ/Ϫ mice (11,12). Although dendritic cells appeared normal, OPGL-deficient mice exhibited defects in early differentiation of T and B cells and lacked all lymph nodes. Null mice also exhibited severe osteopetrosis and a complete lack of osteoclasts. RANK Ϫ/Ϫ mice expressed similar phenotypes suggesting that OPGL-RANK interactions provided critical signals necessary for lymph node organogenesis and osteoclast differentiation (13).
An important connection between bone and the immune system was reported by Kong et al. (14,15) when they observed that activated T cells could directly activate osteoclastogenesis through OPGL. This connection was further strengthened following a report that this T cell-mediated regulation could be suppressed by interferon-␥ produced by T cells themselves (16). These findings suggested that bone metabolism is regulated by the immune system through complex and dynamic interactions. In addition, activation of T cells in vivo could lead to an OPGL-mediated increase in osteoclastogenesis and bone loss, suggesting that blocking OPGL activity may serve as an effi-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Immunology, Genentech Inc., 1 DNA Way, MS  cient therapeutic approach to attenuate bone loss observed in various malignant bone disorders (8).
Thus, while a major role for OPGL in regulation of osteoclastogenesis and chondrocyte differentiation has been established, the exact nature of how OPGL regulates inflammation and the immune system has not been determined. Osteoclasts and monocytes are derived from a common myeloid progenitor and are known to utilize similar signaling pathways involving TNF receptor-associated factors, MAPK, and NF-B, indicating that they may share a common mechanism of OPGL regulation of their function. This coupled with the fact that OPGL is most closely related in sequence to CD40L, a molecule crucial to activation of antigen-presenting cells, suggests a role for OPGL in immune response.
In the present study, we examined a novel role for OPGL in activating monocytes. RANK protein expression was detected on freshly isolated monocytes, and treatment with OPGL was shown to activate monocytes, resulting in MAPK activation, cytokine secretion, and up-regulation of co-stimulatory molecule expression. OPGL was also able to protect monocytes from apoptosis and induced up-regulation of BCL-2 pro-survival family members such as BCL-XL and BCL-2. These in vitro findings were confirmed in an in vivo model of LPS-induced septic shock through the use of a receptor fusion protein approach to specifically block OPGL activity. Administration of RANK-Fc was able to protect mice from death induced by sepsis, indicating a novel role for OPGL in monocyte function in vivo. In addition, treatment with RANK-Fc significantly ameliorated disease development in a model of inflammation-mediated arthritis, suggesting therapeutic potential in inflammatory disease. Thus, the identification of a novel immune cell population regulated by OPGL opens up the possibility that OPGL may play a key role in inflammatory immune response.

EXPERIMENTAL PROCEDURES
Cell Culture-Monocytes were isolated from human peripheral blood using the Monocyte Isolation kit (Milteny Biotec) according to the manufacturer's recommendations. Briefly lymphocytes were isolated from human peripheral blood using Lymphocyte Separation medium (ICN Pharmaceuticals). A magnetic labeling system (MACS Mi-croBeads) was used for the isolation of untouched monocytes from peripheral blood by depleting non-monocytes. Cells were maintained in complete medium (RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 g/ml streptomycin) and cultured at 37°C with 5% CO 2 .
FACS Analyses-Monocytes were adjusted to 5 ϫ 10 5 cells/ml and incubated in the presence or absence of OPGL (5 g/ml) for 24 h. Cells were then harvested, washed with phosphate-buffered saline containing 2% heat-inactivated fetal bovine serum, and incubated with one of the following antibodies for 15 min at 4°C: PE-conjugated ␣-hCD80, FITC-conjugated ␣-hCD86, or PE-conjugated ␣-hClass II or ␣-hRANK. Cells stained with ␣-hRANK were washed and incubated with FITCconjugated ␣-mouse IgG1 antibody for 15 min at 4°C. Following another wash step, cells were analyzed by FACS using CELLQUEST software (BD Biosciences).
Taqman Analyses-Monocytes were adjusted to 1 ϫ 10 6 cells/ml and cultured for 24 h with (or without) various concentrations of OPGL. Total RNA was then isolated from OPGL-treated and control cells using TRIzol reagent (Invitrogen). Quantitative reverse transcription-PCR analyses were performed with 50 ng of total RNA sample and 40 l of a reaction mixture. The reaction mixture in the Taqman Core kit contained 10ϫ buffer A; 10 units of RNase inhibitor; 200 M dATP, dCTP, dGTP, and dTTP; 4 mM MgCl 2 , 1.25 units of Taq Gold polymerase; and 25 units of murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences). Each well contained a 10-l primer/probe mixture of 200 nM gene-specific hybridization probe and 300 nM gene-specific amplification primers. Thermal cycling conditions were as follows: 30 min at 48°C, then 2 min at 50°C, and 10 min at 95°C. The reactions then cycled 40 times for 15 s at 95°C and 1 min at 60°C. Reactions and sequence detection was conducted with the ABI Prism 7700 sequence detector. The sequences of the RANK/GAPDH Taqman primer/probe set used are as follows: RANK forward primer, 5Ј-AGTGGTGCGATT-ATAGCCCG-3Ј; RANK reverse primer, 5Ј-GAAGGTTGAGGTGGGAG-GATC-3Ј; RANK probe, 5Ј-AGCCTCTAACTCCTGGGCTCAAGCAATC-3Ј; GAPDH forward primer, 5Ј-TGGGCTACACTGAGCACCAG-3Ј; GAPDH reverse primer, 5Ј-CAGCGTCAAAGGTGGAGGAG-3Ј; GAPDH probe, 5Ј-TGGTCTCCTCTGACTTCAACAGCGACAC-3Ј.
Detection of Cytokines-Monocytes were induced with various concentrations of OPGL for 24 h, and supernatants were harvested. Levels of secreted cytokines in the cell culture supernatants were determined using enzyme-linked immunosorbent assays (BD Pharmingen kits for IL-12 and IL-6, and R&D Systems kits for TNF-␣, MIP-1␣, and IL-1␤).
Survival Assays-For in vitro assays, monocytes were adjusted to 5 ϫ 10 5 c/ml and incubated with 0.5 mg/ml LPS, 1 g/ml CD40 ligand, or 1 g/ml OPGL. At the indicated time points, cells in the respective cultures were stained with Annexin V-FITC (Clontech) and analyzed by FACS. For in vivo assays, 8-week-old C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were administered RANK-Fc or control IgG (100 g intraperitoneally) daily starting on day 0, injected intraperitoneally with 30 mg kg Ϫ1 of body weight LPS from Escherichia coli serotype O55:B5 (Calbiochem) on day 1, and monitored for survival.
Induction of Antibody-mediated Arthritis-Arthritis was induced in two groups of 8-week-old female BALB/c mice (The Jackson Laboratory) by intravenous injection (2 mg/mouse, subarthritogenic dose) of a combination of four different monoclonal antibodies generated by the Arthrogen-CIA® mouse B hybridoma cell lines (Chemicon, Temecula, CA). Disease development was aided by an intraperitoneal injection of 50 g/mouse LPS the following day. Groups 1 and 2 were administered RANK-Fc and control IgG starting the same day as the injection of the monoclonal antibodies (day 0). Mice were administered 100 g of RANK-Fc/control IgG intraperitoneally daily. Mice were sacrificed on day 14.
X-ray and Microcomputed Tomography (CT) Acquisition-Planar x-ray images of the front and hind paws were acquired after sacrifice. The paws were severed with an axial cut of the distal radius/ulna (front paw) and tibia/fibula (hind paw). The samples were then imaged with a digital planar x-ray system (MX-20, Faxitron X-ray, Inc., Wheeling, IL). X-ray images were acquired with an x-ray tube current of 300 A and a voltage of 26 kV. The extracted mouse samples (front and hind paws) were also imaged with a CT40 (SCANCO Medical, Basserdorf, Switzerland) x-ray CT system. A sagittal scout image, comparable to a conventional planar x-ray, was obtained to define the start and end point for the axial acquisition of a series of computed tomography images. The location and number of axial images were chosen to provide complete coverage of the joints of interest for each paw. The target joints of the front paw were the metacarpophalangeal and interphalangeal joints (PIP and DIP) for digits 2-5. The metatarsophalangeal joints for digits 2-5, PIP joints for digits 2-5, and DIP joints of the hind paw were also evaluated. The CT images were generated by operating the x-ray tube at an energy level of 50 kV, a current of 160 A, and an integration time of 300 ms . Axial images were obtained at an isotropic resolution of 16 m.
Radiographic Analysis-X-ray images and CT were evaluated for bone abnormalities by application of a semiquantitative scoring system that was based on the scoring system for radiographs described by Genant et al. (17). A three-dimensional surface rendering was created from the CT data with Analyze (AnalyzeDirect Inc., Lenexa, KS), an image analysis software package. The x-ray image and corresponding CT three-dimensional rendering were simultaneously view and evaluated by a single reader (W. P. Lee) who was blinded to treatment. The degree of erosions and periarticular osteoporosis were graded with a six-point score ranging from 0 (normal) to 5 (severe). A cumulative score was determined for each paw by summing the scores of the individual joints: front paw (metacarpophalangeal, PIP, and DIP; digits 2-5) and hind paw (metatarsophalangeal, PIP, and DIP; digits 2-5).
Histological Analysis-Joints from all four feet from each animal were scored in three categories (synovial, bone, and cartilage changes) and added to achieve a final total score. Scores were derived as follows: 1 ϭ severity is minimal and distribution is multifocal to diffuse, OR severity is mild but distribution is focal; 2 ϭ severity is mild and distribution is diffuse, OR severity is moderate but distribution is focal; 3 ϭ severity is moderate and distribution is multifocal; 4 ϭ severity is moderate and distribution is diffuse, OR severity is severe but distribution is focal; 5 ϭ severity is severe and distribution is multifocal to diffuse.
The criteria for lesion severity scores were as follows. Synovium, bone, and cartilage lesions were scored separately. Synovial lesion scores were based on the amount of synovial proliferation (pannus) and inflammation. Bone lesion scores were based on the amount of bone destruction/loss (occasional evidence of osteoclastic activity in areas of pannus or inflammation as minimal; segmental complete loss of bone and replacement by pannus or new/noncortical bone as severe). Cartilage lesion scores were based on the amount of cartilage destruction/ loss (loss of nuclei and preservation of smooth cartilage surface as minimal; complete fragmentation and/or loss as severe).

RANK Is Expressed on Monocytes and Is Up-regulated upon
Stimulation with OPGL-To investigate potential roles for OPGL in monocyte and macrophage effector function, we first wanted to determine whether RANK is expressed on monocytes. Toward this end, monocytes were freshly isolated from peripheral blood, stained with a monoclonal antibody to RANK, and analyzed by FACS. As shown in Fig. 1A, RANK expression on the cell surface of resting monocytes was clearly detected over control isotype staining. To determine whether OPGL can regulate expression of RANK, monocytes were treated with various concentrations of OPGL (0, 0.3, 0.6, 1.25, and 2.5 g/ ml) for 24 h. Total RNA was isolated, and quantitative reverse transcription-PCR analyses were performed (Fig. 1B). Upon stimulation with OPGL, a dose-dependent increase in RANK mRNA expression was observed. The highest concentration of OPGL used, 2.5 g/ml, stimulated a 4-fold increase in RANK expression over unstimulated monocytes after 24 h. This upregulation in RANK expression was specific to OPGL and was not observed when monocytes were stimulated with LPS or CD40L (data not shown). Consistent with an increase in mRNA levels, up-regulation of RANK protein cell surface expression was also observed in FACS analyses upon OPGL treatment after 24 h (Fig. 1C).
OPGL Induces Effector Function of Monocytes by Up-regulating Secretion of Cytokines and Chemokines-Activation of monocytes by physiological stimuli such as LPS and TNF family members like CD40L has been shown to induce their effector function resulting in secretion of proinflammatory cytokines such as IL-1␤ and TNF-␣. To test whether OPGL could functionally activate monocytes, we stimulated monocytes for 24 h with incremental doses of OPGL and looked for production of proinflammatory cytokines such as TNF-␣ and IL-1␤, T cell activation cytokines including IL-12 and IL-6, and chemokines such as MIP-1␣. As shown in Fig. 2, A-E, OPGL was able to induce secretion of these cytokines from freshly isolated monocytes in a dose-dependent manner. At the highest concentration of OPGL used (5 g/ml), levels of cytokine concentration in the supernatant were 213 pg/ml for IL-12, 7704 pg/ml for IL-6, 13.4 pg/ml for TNF-␣, 803 pg/ml for IL-1␤, and 8740 pg/ml for MIP-1␣. These levels were comparable to those induced by LPS at 5 g/ml (373 pg/ml for IL-12, 6193 pg/ml for IL-6, 21.3 pg/ml for TNF-␣, 658 pg/ml for IL-1␤, and 10,395 pg/ml for MIP-1␣).
OPGL Activates Antigen Presentation Function of Monocytes by Inducing Expression of Co-stimulatory Molecules-In addition to playing a dominant role in innate immunity by phagocytosis of microorganisms, monocytes and macrophages play a very important role in activating the adaptive immune response by activating T lymphocytes. This is accomplished by the secretion of activation cytokines such as IL-12 and IL-6, by presenting antigens, and by providing co-stimulatory signals to T cells. Antigen presentation is accomplished by molecules such as major histocompatibility complex Class II, while members of the B7 family such as B7.1/CD80 and B7.2/CD86 are involved in T cell co-stimulation. To study whether OPGL is involved in activation of the above functions, freshly isolated monocytes were incubated with OPGL (5 g/ml) and analyzed by FACS. As shown in Fig. 3, OPGL was able to efficiently up-regulate expression of CD80 (Fig. 3A), CD86 (Fig. 3B), and major histocompatibility complex Class II (Fig. 3C) molecules on monocytes after a 24-h stimulation. OPGL was also able to

FIG. 1. RANK is expressed on monocytes and is up-regulated upon OPGL treatment.
A, freshly isolated monocytes were stained for RANK expression (bold line). Isotype control staining is indicated in gray. B, monocytes were treated with (or without) the indicated concentrations of OPGL for 24 h, and total RNA was isolated. Quantitative reverse transcription-PCR analyses were performed, and -fold increase in RANK mRNA levels of OPGL-treated over control untreated cells is shown. GAPDH levels were used to normalize loading. C, monocytes were analyzed for RANK protein expression at 0 and 24 h after incubation in the presence or absence of OPGL (5 g/ml). RANK cell surface expression at 0 and 24 h is shown in gray and bold lines, respectively. moderately up-regulate CD40 expression on monocytes (data not shown). The above results thus indicate a novel role for OPGL in activating various in vivo functions of monocytes and macrophages such as proinflammatory cytokine secretion and T cell activation.
OPGL Activates the MAPK Pathway in Monocytes-To determine whether monocyte activation by OPGL involves the MAPK pathway, we induced monocytes with OPGL (1 g/ml) for 1, 3, 10, 30, and 60 min and looked for activation of MAPK pathways (Fig. 4). Phosphorylation of p38 MAPK (Fig. 4A) and p42/44 ERK (Fig. 4B) was observed at 1 min and peaked at 10 min for p38, while it remained at elevated levels for p42/44 MAPK even at 60 min. Interestingly, phosphorylation of p52/54 JNK/SAPK could not be detected in monocytes upon OPGL treatment (data not shown) suggesting a differential utilization of MAPK pathways.
OPGL Enhances Survival of Monocytes-Based on previous studies suggesting a role for OPGL as a survival factor for dendritic cells, we induced apoptosis in monocytes by serum withdrawal and looked for protection from apoptosis induced by OPGL. Annexin V staining analyses revealed that OPGL protected monocytes from apoptosis at levels comparable to LPS (0.5 mg/ml) and CD40L (1 g/ml) (Fig. 5A). Similar results were also obtained for monocytes treated with ␣-Fas antibody and dexamethasone (data not shown). To determine possible mechanisms of OPGL-induced survival in monocytes, we looked for induction of antiapoptotic proteins belonging to the BCL-2 family. Robust activation of BCL-XL protein expression was observed starting at 4 h post-OPGL stimulation (1 g/ml) in monocytes, and a moderate but reproducible up-regulation of BCL-2 protein expression was also observed (Fig. 5B).
Blocking OPGL Activity Results in Decreased Susceptibility to Endotoxic Shock-The above results suggest an important role for OPGL in the activation of effector function of monocytes during inflammation, and this was tested in an in vivo model of LPS-induced endotoxic shock (18). A soluble receptor form, RANK-Fc, comprising the extracellular domain of murine RANK was used to block OPGL function in vivo. RANK-Fc was chosen based on its ability to bind OPGL exclusively as opposed to the soluble decoy receptor OPG, which can also bind an alternate TNF ligand, TNF-related apoptosis-inducing ligand (TRAIL) (19). 8-week-old C57/Bl6 mice were injected intraperitoneally with 30 mg/kg LPS and 100 g of RANK-Fc or control IgG and monitored for survival. As shown in Fig. 6, administration of RANK-Fc was able to significantly block death induced by septic shock. After 72 h, while only 20% of mice in the control group survived, 80% of RANK-Fc-treated mice were still alive. These results indicate a dominant intrinsic role for OPGL in mediating inflammatory response and potential clinical applications in combating septic shock.
Therapeutic Potential for Blocking OPGL Activity in Inflammatory Arthritis-Based on the above observed ability of RANK-Fc to block OPGL function in activating monocytes/ macrophages, we tested roles for RANK-Fc in blocking development of inflammation-mediated arthritis in vivo (20,21). This model is distinct from conventional collagen-induced arthritis models in that inflammation induced by LPS is one of the major determinants of disease development. In this model, 8-week-old BALB/c mice were injected with a mixture of four different monoclonal antibodies to collagen on day 0 and 50 g of LPS on day 1 to activate inflammation. Mice were treated with 100 g of RANK-Fc or control IgG daily and monitored for inflammation in the joints and arthritis. As shown in Fig. 7A, swelling in the joints of mice administered RANK-Fc was significantly lower than that in control IgG-treated mice; this was also reflected in the histology scores (Fig. 7B). Mice were sacrificed on day 14 representing the end of the study, and radiographic examinations were performed. Digital planar x-rays of fore and hind paws revealed that the affected joints in control IgG-treated mice had severe osteolysis and osteophyte production accompanied by disfigurement, and this was absent in the RANK-Fc-treated mice (Fig. 7, C and D). Disease was scored using conventional radiographic methods (scale of 0 -4, with 4 representing the most severe disease) with x-rays and is shown in Table I. Individual paw scores for RANK-Fc-treated mice range from 0 to 3, while those for Ig-treated mice are primarily 4 or 3. In addition, joints were also scanned by CT to generate three-dimensional rendered images. X-rays and CT renderings were simultaneously viewed and visually evaluated for bone destruction by the application of a modified radiographic scoring system (17). Development of arthritis was significantly inhibited in RANK-Fc-treated mice as evidenced by suppressed bone erosion and loss of bone density (Fig. 8A). The degree of erosions and periarticular osteoporosis were graded with a six-point score ranging from 0 (normal) to 5 (severe). A cumulative score was determined for each paw by summing the scores of the individual joints, front paw (metacarpophalangeal, PIP, and DIP; digits 2-5) and hind paw  6. Blocking OPGL activity with RANK-Fc protects mice from LPS-induced endotoxic shock. 8-week-old C57/Bl6 mice were injected intraperitoneally with 30 mg/kg LPS serotype O55:B1 and 100 g/day RANK-Fc (or control IgG) and monitored for survival. Group 1 administered RANK-Fc is represented by black squares, and group 2 (control IgG) is represented by white squares.
(metatarsophalangeal, PIP, and DIP; digits 2-5), and is graphed for each mouse (five per group) in control IgG-and RANK-Fc-treated groups (Fig. 8B). Mice treated with RANK-Fc had significantly reduced bone destruction (mean score, 6 Ϯ 2.5) compared with the control group (mean score, 33.4 Ϯ 11.3). These results thus demonstrate good efficacy for RANK-Fc in treatment of inflammation-mediated arthritis. DISCUSSION Interactions between the TNF superfamily ligands and their cognate receptors are essential in regulating immune response both by promoting cell survival and proliferation and through activation-induced cell death (3). OPGL and its receptor RANK, members of the TNF superfamily, are known to be key regulators of bone metabolism and essential for the development and function of osteoclasts (6,7). Osteoclasts and monocytes are derived from a common myeloid progenitor indicating that there may be common mechanisms of regulation of their function (22). In addition, OPGL is very closely related to CD40L (1, 2), which is known to be crucial for activation of monocytes/ macrophages (23). This suggests a potential role for OPGL in regulation of effector function by these cells. In the present study, we have defined a novel role for OPGL in activating monocytes/macrophages. OPGL was found to regulate their effector function by inducing cytokine and chemokine secretion, activate antigen presentation through up-regulation of co-stimulatory molecule expression, and inhibit serum withdrawalinduced apoptosis. These in vitro findings were confirmed in an in vivo model of LPS-induced septic shock through the use of a receptor fusion protein approach to block OPGL activity. Administration of RANK-Fc was able to protect mice from death induced by sepsis, indicating a novel, important role for OPGL in monocyte function and in mediating inflammatory response. This was further tested in an animal model of antibody-induced arthritis. Treatment with RANK-Fc significantly ameliorated disease development and attenuated bone destruction, suggesting therapeutic potential in inflammatory disease. OPGL and its receptor RANK were initially discovered based on their expression in T cells and dendritic cells, respectively (2,9). Interactions between ligand and receptor were shown to be important for activation of dendritic cell function and in regulating cross-talk between T cells and dendritic cells. Dendritic cells serve primarily to present antigens to T cells and form an important part of the antigen-presenting cell population. Monocytes/macrophages represent another important component of the antigen-presenting cell population and in addition also perform key effector functions such as inducing inflammation and directly killing microorganisms. They also serve as an important bridge between innate and adaptive immune responses by priming T cells through secretion of activation cytokines and presentation of antigens. To date, OPGL has not been implicated in the regulation of this key immune population, and roles for OPGL in activation of monocyte/macrophage function have not been defined. Our results describe a novel function for OPGL in monocyte activation and define important roles in regulating their diverse functions including secretion of proinflammatory and T cell activation cytokines, antigen presentation, and co-stimulation.
Results from our studies indicate that the biochemical pathways involved in OPGL signaling in monocytes appear to be mediated through p38 MAPK and p42/44 ERK. In mammalian cells, at least three MAPK pathways have been identified: ERK1/2, JNK1/2/SAPK, and p38 MAPK pathways. Various cell growth and differentiation stimuli have been shown to activate the ERK1/2 pathway, leading to proliferation and differentiation responses (24). The JNK1/2 and p38 MAPK pathways are primarily activated by inflammatory cytokines and environmental stress and lead to inflammatory, apoptotic, or developmental responses (24). Previous studies in monocytes have linked T cell-dependent secretion of the proinflammatory cytokines IL-1␤ and TNF-␣ to activation of the ERK1/2 pathway (25). Our studies suggest that this T cell-dependent signal may be mediated through OPGL, and the subsequent ERK1/2 activation in monocytes may play a central role in the secretion of proinflammatory cytokines.
Circulating monocytes have a limited life span and when recruited to a site of inflammation will undergo apoptosis in the absence of further survival stimuli. CD40L, present on CD4 ϩ T cells, has been shown to inhibit apoptosis of circulating monocytes and promote their survival (26). Our results indicate that OPGL may also be important in vivo for monocyte survival. A key parameter that determines whether a cell will respond to an apoptotic signal is the ratio of death antagonists (BCL-2, BCL-XL, BCL-W, MCL-1, and BFL-1) to agonists (BAX, BAK, BCL-XS, BAD, BID, and BIM) belonging to the BCL-2 family (27). In this context, up-regulation of BCL-XL and BCL-2 induced by OPGL may well account for the decrease in the number of monocytes undergoing apoptosis at the site of inflammation in vivo. Enhanced survival of inflammatory cells, including monocytes, may be an important factor in the establishment of chronic inflammation that characterizes both atopic and autoimmune diseases (28,29). Thus OPGL may play a role in the persistence of inflammatory responses associated with these disorders by prolonging monocyte/macrophage survival.
Classical activation signals for monocytes/macrophages include LPS and another TNF family member, CD40L. LPS is a component of cell walls of Gram-negative bacteria and is a potent stimulator of inflammatory response. CD40, the receptor for CD40L, is expressed on primarily on B cells, antigen-presenting cells, and endothelial cells (30 -32). In monocytes/macrophages, activation of CD40 by its ligand, CD154, has been shown to induce secretion of proinflammatory cytokines and chemokines such as IL-1␤, IL-6, IL-8, IL-10, IL-12, TNF-␣, and MIP-1␣ (33,34). Activation of CD40 signaling in monocytes/macrophages also results in up-regulation of co-stimulatory molecules such as B7.1/ B7.2 for T cell activation (34), nitric oxide generation (35), and induction of metalloproteinase production (36) for killing of microorganisms. Thus, there are many similarities in the various physiological roles for CD40L and OPGL, including expression patterns of ligand/receptors and effects on monocytes/macrophages. Results from our and previous studies strongly indicate that CD40L and OPGL may play important roles in mediating the primary immune response through direct killing and the induction of inflammation. However, OPGL has been implicated as an immediate early gene in T cells (1) and may be acting prior to CD40L during innate and adaptive immune response. Rapid up-regulation of OPGL upon activation of the T cell receptor on T cells could specifically activate antigen-presenting cells and promote their survival. Both antigen-specific T cells and the antigenpresenting cells would therefore depend on each other for activation and survival. Mature antigen-presenting cells that fail to present antigen to T cells would not receive T cell help and would therefore die of neglect. This feedback loop may play an important role in the initiation and maintenance of immune response.

FIG. 8. RANK-Fc blocks arthritis by inhibiting bone erosion and loss of bone density in an inflammation-mediated model of arthritis.
Mice in control IgG-and RANK-Fc-treated groups were sacrificed 14 days after ␣-collagen antibody injection, and joints were scanned by CT. A, bone erosion and loss of bone density in joints of mice representative of RANK-Fc and control IgG groups are shown. The images are a three-dimensional surface rendering created from the CT data using Analyze image analysis software. B, x-ray images and CT were evaluated for bone abnormalities by application of a semiquantitative scoring system that was based on the scoring system for radiographs described by Genant et al. (17). The x-ray image and corresponding CT three-dimensional rendering were simultaneously viewed and evaluated by a single reader who was blinded to treatment. The degree of erosions and periarticular osteoporosis were graded with a six-point score ranging from 0 (normal) to 5 (severe). A cumulative score was determined for each paw by summing the scores of the individual joints: front paw (metacarpophalangeal, PIP, and DIP; digits 2-5) and hind paw (metatarsophalangeal, PIP, and DIP; digits 2-5).
Previous studies have used the decoy receptor OPG as a therapeutic reagent for various malignant bone disorders (37)(38)(39). OPG, however, is also known to bind another TNF ligand, TNF-related apoptosis-inducing ligand (TRAIL), thus complicating interpretation of results. In our study, we used RANK-Fc to block OPGL activity since RANK binds to OPGL exclusively. Results from both in vivo models of septic shock and inflammation-mediated arthritis described in our study demonstrate the therapeutic efficacy of RANK-Fc in these applications, establishing a dominant role for OPGL in mediating sepsis and inflammatory arthritis. Furthermore the ability of RANK-Fc to block monocyte/macrophage function strongly indicates that it may ameliorate disease in additional clinical indications where these cell populations are known to play major roles.