Impaired skeletal development in interleukin-6transgenic miceA model for the impact of chronic inflammation on the growing skeletal system.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 54, No. 11, November 2006, pp 3551–3563 DOI 10.1002/art.22175 © 2006, American College of Rheumatology Impaired Skeletal Development in Interleukin-6–Transgenic Mice A Model for the Impact of Chronic Inflammation on the Growing Skeletal System Fabrizio De Benedetti,1 Nadia Rucci,2 Andrea Del Fattore,2 Barbara Peruzzi,2 Rita Paro,2 Maurizio Longo,2 Marina Vivarelli,1 Flaminia Muratori,1 Silvia Berni,3 Paola Ballanti,3 Serge Ferrari,4 and Anna Teti2 creased osteoblast and increased osteoclast number and activity. Increased osteoclastogenesis and reduced osteoblast activity, secondary to decreased precursor proliferation and osteoblast function, were present. IL-6– transgenic mice also showed impaired development of growth plates and epiphyseal ossification centers. Intramembranous and endochondral ossification and the mineral apposition rate were markedly affected, showing the presence of defective ossification. Conclusion. Chronic overexpression of IL-6 alone induces a skeletal phenotype closely resembling growth and skeletal abnormalities observed in children with chronic inflammatory diseases, pointing to IL-6 as a pivotal mediator of the impact of chronic inflammation on postnatal skeletal development. We hypothesize that IL-6–modifying drugs may reduce skeletal defects and prevent the growth retardation associated with these diseases. Objective. To identify the mediator responsible for the impact of chronic inflammation on skeletal development in children (bone loss, defective peak bone mass accrual, stunted growth), we evaluated the effects of chronic interleukin-6 (IL-6) overexpression on the skeletons of growing prepubertal mice. Methods. We studied IL-6–transgenic mice that had high circulating IL-6 levels since birth. Trabecular and cortical bone structure were analyzed by microcomputed tomography. Epiphyseal ossification, growth plates, and calvariae were studied by histology/ histomorphometry. Osteoclastogenesis, osteoblast function/differentiation, and the effects of IL-6 on bone cells were studied in vitro. Osteoblast gene expression was evaluated by reverse transcriptase–polymerase chain reaction. The mineral apposition rate was evaluated dynamically in cortical bone by in vivo double fluorescence labeling. Results. In prepubertal IL-6–transgenic mice, we observed osteopenia, with severe alterations in cortical and trabecular bone microarchitecture, as well as uncoupling of bone formation from resorption, with de- Children with juvenile idiopathic arthritis (JIA), particularly those with systemic JIA, may present with a decrease in bone mass and an increased risk of fractures. Later in life, they may reach a suboptimal bone mass peak, which is a well-known risk factor for developing osteoporosis (1,2). These children also present with inhibition of somatic growth, associated with retarded skeletal development and delayed epiphyseal ossification (3,4). Although malnutrition, physical inactivity, and therapies may contribute to these skeletal abnormalities, several lines of evidence indicate that inflammation itself may play a major role. Low bone mass and stunted growth were demonstrated in JIA patients not treated with glucocorticoids and were found to be associated Supported by an IRCCS grant to Ospedale Pediatrico Bambino Gesù. Dr. Teti’s work was supported by the European Commission grant OSTEOGENE (contract LSHM-CT-2003-502941). 1 Fabrizio De Benedetti, MD, Marina Vivarelli, MD, Flaminia Muratori, PhD: Ospedale Pediatrico Bambino Gesù, Rome, Italy; 2 Nadia Rucci, PhD, Andrea Del Fattore, BSc, Barbara Peruzzi, BSc, Rita Paro, PhD, Maurizio Longo, PhD, Anna Teti, PhD: University of L’Aquila, L’Aquila, Italy; 3Silvia Berni, BSc, Paola Ballanti, PhD: University La Sapienza, Rome, Italy; 4Serge Ferrari, MD: Geneva University Hospital, Geneva, Switzerland. Address correspondence and reprint requests to Anna Teti, PhD, Department of Experimental Medicine, University of L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy. E-mail: firstname.lastname@example.org. Submitted for publication October 27, 2005; accepted in revised form July 24, 2006. 3551 3552 DE BENEDETTI ET AL with disease activity (3,5). In a recent prospective study, decreased levels of markers of osteoblast activity and increased levels of markers of osteoclast activity were found to be independent predictors of defective bone mineral content in JIA patients (6). It is therefore conceivable that an imbalance between osteoblast and osteoclast activities may play a pivotal role in bone loss and in suboptimal bone mass accrual in patients with JIA. However, the molecular and cellular mechanisms underlying this uncoupling and its relationship with stunted growth in children are still unknown. We previously demonstrated that interleukin-6 (IL-6)–transgenic mice, with high circulating IL-6 levels since birth, have a marked prepubertal growth defect, indicating that chronic overexpression of IL-6 causes growth retardation, mimicking the stunted growth observed in human chronic inflammatory diseases (7). IL-6 is a pleiotropic cytokine that plays an important role in the inflammatory response (8). Increased IL-6 levels are present in patients with JIA, particularly in those with systemic JIA, and they appear to mediate several systemic effects of inflammation (9). Moreover, IL-6 is known to promote osteoclast maturation and activation (10,11) and to affect osteoblasts (12). Deletion of the IL-6 gene in mice protects against ovariectomy-induced osteoporosis, with a mechanism involving prevention of osteoclast activation (13). These observations raise the hypothesis that IL-6 may be crucial in mediating the impact of chronic inflammation on the developing skeletal system. We report here that prepubertal IL-6– transgenic mice show decreased trabecular and cortical bone, uncoupled osteoclast and osteoblast activities, and delayed ossification and impaired growth of skeletal segments, pointing to IL-6 as a pivotal mediator of the damage induced by chronic inflammation in postnatal bone development. MATERIALS AND METHODS Animals. IL-6–transgenic mice were previously generated using the NSE/hIL-6 construct, which carries the rat neurospecific enolase (NSE) promoter driving the expression of human IL-6 complementary DNA (7). These mice have a significantly reduced growth rate, reaching 50–70% of the size of their age-matched littermates (7). They have normal food intake and hematic glucose as well as a normal life span, with no peculiar susceptibility to infections and no defects in the numbers of spleen T cells and B cells. They do have a modest increase in the number of monocytes in blood and spleen, increased levels of serum amyloid A, and elevated white blood cell and platelet counts. Histologic analysis of several organs, including joints, did not yield evidence of active inflammatory responses. Unless indicated otherwise, all experiments were performed in 10-day-old mice. Procedures involving animals and their care were conducted in conformity with national and international laws and policies (European Economic Community Council Directive 86/609, Italian Legislative Decree 116/ 92, National Institutes of Health Guide for the Care and Use of Laboratory Animals). Mice were killed by cervical dislocation, and long and parietal bones were removed and cleared of soft tissue. Assessment of trabecular and cortical microarchitecture. Microcomputed tomography (UCT40; Scanco Medical, Basserdorf, Switzerland) was used to assess trabecular bone volume fraction (bone volume/total volume [BV/TV]) and microarchitecture in the metaphyseal region of the tibia and was also used to assess cortical geometry at the mid-tibial diaphysis of bones excised from 10-day-old mice. For trabecular bone in the metaphyseal region, the BV/TV (in %), the trabecular thickness (in m), the trabecular number (the number of plates per unit of length [mm]), the connectivity density (the 3-dimensional [3-D] index of the connectivity of the trabecular bone per unit of volume [mm3]), and the structural model index were assessed on 100 contiguous computed tomography (CT) slides, starting 100 slides below the growth plate. For cortical bone, the average TV inside the periosteal envelope (in mm3), the BV within this same envelope (in mm3), and the average cortical thickness (in mm) were assessed at 6 resolution on 54 contiguous CT slides. Bone histology and histomorphometry. Tibiae and parietal bones were fixed in 4% formaldehyde in 0.1M phosphate buffer (pH 7.2), dehydrated in ethanol or acetone, and processed for paraffin embedding with previous decalcification or for glycol–methacrylate embedding without decalcification, respectively. Histomorphometric measurements were carried out on 2–5-m–thick sections with an interactive image analysis system (IAS 2000; Delta Sistemi, Rome, Italy). Nomenclature, symbols, and units of histomorphometric bone variables are those suggested by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (14). Osteoclast precursors. Bone marrow from long bones of wild-type (WT) and IL-6–transgenic mice was flushed and collected in Hanks’ balanced salt solution. Cells were layered over an equal volume of Ficoll-Histopaque and centrifuged at 400g for 30 minutes. The interface layer was removed, washed twice in culture medium (at 250g for 10 minutes), resuspended in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS), and attached to glass slides by a cytospin procedure, and then tartrate-resistant acid phosphatase (TRAP) activity was detected histochemically (kit no. 387A; Sigma-Aldrich, St. Louis, MO). Osteoclast primary cultures. Primary osteoclasts were differentiated from the bone marrow of WT and IL-6– transgenic littermates. For cocultures with stromal cells as a source of osteoclastogenic factors, bone marrow was flushed from bone cavity and minced in DMEM supplemented with 10% FBS. Cells were recovered and plated in 96-well plates with or without bone slices, in DMEM plus 10% fetal calf serum (FCS). After 24 hours, nonadherent cells were removed by extensive washing, while the adherent cells were cultured for up to 7 days in the presence of 10 ⫺8 M 1,25dihydroxyvitamin D3 (1,25[OH]2D3). IL-6 AND SKELETAL GROWTH To obtain purified osteoclast cultures, bone marrow macrophages isolated from bone marrow by the FicollHistopaque method were resuspended in DMEM containing 10% FCS and plated as described above. After 3 hours, cell cultures were rinsed to remove nonadherent cells and maintained in the same medium in the presence of 25 ng/ml recombinant human macrophage colony-stimulating factor (M-CSF) and 30 ng/ml recombinant human RANKL for 15 days. Osteoclasts were then detected by TRAP histochemical staining. Osteoblast primary cultures. Calvariae from WT and IL-6–transgenic mice were digested 3 times with 1 mg/ml Clostridium histolyticum type IV collagenase and 0.25% trypsin for 20 minutes at 37°C with gentle agitation. Cells from the second and third digestions were grown in DMEM plus 10% FBS, trypsinized by standard procedure, and plated according to the experimental protocol. Alkaline phosphatase (AP) activity was evaluated histochemically using kit no. 85. For the mineralization assay, medium was supplemented with 10% FBS, 10 mM ␤-glycerophosphate, and 50 g/ml ascorbic acid, and cells were cultured for 3 weeks prior to detection of mineralization by von Kossa’s stain. Osteoblast proliferation assay. Osteoblasts (10,000/ well) were cultured in 24-well plates in DMEM plus 10% FBS. Twenty-four hours later, medium was replaced with DMEM plus 1% FBS, and, after 4 hours, 2 Ci/ml 3H-thymidine (specific activity 25 Ci/mmole) was added to each well and incubated overnight. At the end of incubation, the incorporated 3H-thymidine was measured in a beta counter. In vivo osteocalcin and deoxypyridinoline measurements. Mouse serum osteocalcin levels and urinary excretion of deoxypyridinoline were measured by enzyme-linked immunosorbent assay kits according to the instructions of the manufacturers (Biomedical Technologies [Stoughton, MA] and Quidel [San Diego, CA], respectively). The detection limits of the assays were 1 ng/ml for osteocalcin and 1.1 nM for deoxypyridinoline. Reverse transcriptase–polymerase chain reaction (RTPCR). Total RNA was extracted using the acid–phenol technique. One microgram of RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase, and the equivalent of 0.1 g was used for the PCR reactions. These were carried out in a final volume of 50 l containing 200 M of dNTPs, 1.5 mM MgCl2, 10 pM of each primer, and 1 unit Taq DNA polymerase. For quantitative analysis, primers for the housekeeping gene GAPDH were used along with the primers for the gene being analyzed. PCR-amplified products were analyzed on 1.5% agarose gel containing ethidium bromide. Statistical analysis. Data are expressed as the mean ⫾ SEM or mean ⫾ SD of at least 3 independent experiments or 3 animals per group, respectively. Statistical analysis was performed by one-way analysis of variance, followed by Student’s unpaired t-test or the Mann-Whitney U test. P values less than 0.05 were considered significant. RESULTS Reduced trabecular and cortical bone in IL-6– transgenic mice. Radiographic analysis at various ages revealed that the entire skeleton of IL-6–transgenic 3553 Figure 1. Impaired skeletal development and decreased trabecular and cortical bone in interleukin-6 (IL-6)–transgenic (TG) mice. A, Radiographic analysis of total body and tibias of 10-day-old wild-type (WT) and IL-6–transgenic mice. B, Longitudinal measurements of femurs and tibias in WT and IL-6–transgenic mice plotted against age. Values are the mean ⫾ SEM of 3 animals per group. C, Top, Hematoxylin and eosin staining of proximal secondary spongiosa of tibias of 10-day-old WT and IL-6–transgenic mice. GP ⫽ growth plate; * ⫽ bone trabeculae; BM ⫽ bone marrow. (Original magnification ⫻ 10.) Bottom, microcomputed tomography analysis performed in the tibia proximal spongiosa of 10-day-old WT and IL-6–transgenic animals. D, Color panels show coronal sections of tibia midshafts from 10-day-old WT and IL-6–transgenic mice stained with methylene blue/azure II. * ⫽ cortical bone. (Original magnification ⫻ 2.5.) Black and white panels show microcomputed tomography analysis of cortical bone in the tibia midshafts of 10-day-old WT and IL-6–transgenic mice. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org 3554 DE BENEDETTI ET AL Table 1. Microarchitectural parameters and histomorphometric analysis in 10-day-old mice* Trabecular bone, by microcomputed tomography† Bone volume/total volume, % Trabecular number‡ Trabecular thickness, m Connectivity density§ Structural model index Cortical bone, by microcomputed tomography Cortical total volume, mm3 Cortical bone volume, mm3 Cortical thickness, mm Trabecular bone histomorphometry¶ Osteoclast surface/bone surface, % Osteoclast number# Osteoblast surface/bone surface, % Cortical bone histomorphometry** Periosteal mineral apposition rate, m/day Endosteal mineral apposition rate, m/day Growth plate histomorphometry†† Total width, m Hypertrophic zone width, m Calvarial bone histomorphometry‡‡ Cortical thickness, mm Osteoclast surface/bone surface, % Osteoclast number# Osteoblast surface/bone surface, % WT mice IL-6–transgenic mice P 7.1 ⫾ 2.7 3.81 ⫾ 0.46 29 ⫾ 2 229 ⫾ 113 2.056 ⫾ 0.40 2.8 ⫾ 1.3 3.88 ⫾ 0.49 22 ⫾ 1 60 ⫾ 38 3.154 ⫾ 0.272 ⬍0.03 NS ⬍0.0005 ⬍0.03 0.004 0.107 ⫾ 0.013 0.043 ⫾ 0.003 0.065 ⫾ 0.009 0.07 ⫾ 0.011 0.025 ⫾ 0.003 0.044 ⫾ 0.001 0.005 0.0002 ⬍0.005 17.68 ⫾ 4.55 8.31 ⫾ 1.67 28.82 ⫾ 6.53 26.28 ⫾ 4.37 13.52 ⫾ 3.78 17.95 ⫾ 5.96 ⬍0.007 ⬍0.01 ⬍0.01 9.16 ⫾ 0.33 3.77 ⫾ 3.33 6.69 ⫾ 1.13 0.00 ⫾ 0.00 ⬍0.02 NA 394.17 ⫾ 8.29 177.07 ⫾ 14.04 356.50 ⫾ 17.88 116.15 ⫾ 23.03 ⬍0.03 ⬍0.02 59.80 ⫾ 11.65 10.90 ⫾ 4.63 10.11 ⫾ 4.27 47.10 ⫾ 9.57 29.00 ⫾ 3.73 26.66 ⫾ 5.30 25.01 ⫾ 2.01 16.52 ⫾ 0.71 ⬍0.01 ⬍0.02 ⬍0.005 ⬍0.005 * Values are the mean ⫾ SD of at least 3 animals per group. WT ⫽ wild-type; IL-6–transgenic ⫽ interleukin-6–transgenic; NS ⫽ not significant; NA ⫽ not applicable. † Proximal tibia metaphysis. ‡ Number of plates per unit of length (mm). § The 3-dimensional index of the connectivity of the trabecular bone per unit of volume (mm3). ¶ Proximal tibia metaphysis, 100 m from distal end of growth plate excluding the endocortical surfaces, longitudinal sections. # Number of osteoclasts per mm2 of bone surface. ** Mid-diaphysis of the tibia, coronal sections. †† Proximal tibia, longitudinal sections. ‡‡ Coronal sections. animals was smaller than that of WT littermate controls. The defect in skeletal development affected virtually all segments (Figure 1A). In IL-6–transgenic animals, femurs and tibias showed decreased longitudinal growth, with a maximal 20% reduction in bone length at 10 days (Figure 1B). In IL-6–transgenic animals, high-power radiographs demonstrated increased radiolucency in tibias (Figure 1A) and in femurs and vertebrae (not shown). In 10-day-old mice, conventional histology (Figure 1C, top panels) and histomorphometry (not shown) demonstrated a clear-cut reduction of trabecular bone in the proximal metaphysis of the tibia in IL-6–transgenic animals compared with WT littermates. Microcomputed tomography analysis confirmed a significant reduction of 3-D trabecular microarchitecture of the tibial metaphysis in IL-6–transgenic animals (Table 1 and Figure 1C, bottom panels). At the mid-diaphysis of the tibia, there was an obvious thinning of the collar in IL-6–transgenic animals compared with WT animals (Figure 1D), with a consequent decrease of the cortical TV and BV as assessed by both conventional histomorphometry (not shown) and microcomputed tomography (Table 1). These observations demonstrate that chronic IL-6 overexpression causes an osteopenic phenotype. Therefore, we investigated the underlying cellular mechanism by examining osteoclasts and osteoblasts both in vivo and in cell culture. Increased osteoclast activity in IL-6–transgenic mice. Histochemistry for the osteoclast-specific marker TRAP showed an increase of TRAP-positive cells lining the bone trabeculae of the proximal spongiosa of the tibia and of the endosteal surface of the metaphyseal cortex in IL-6–transgenic animals (Figures 2A and B). Higher magnification showed larger and more intensely stained multinucleated osteoclasts in IL-6–transgenic mice, along with abundant putative mononuclear precursors (Figure 2A). Histomorphometry demonstrated increased osteoclast surface and osteoclast number (Ta- IL-6 AND SKELETAL GROWTH 3555 Figure 2. Increased osteoclasts (OC) and decreased osteoblasts (OB) in IL-6–transgenic mice. A, Top, Histochemical detection of the osteoclast-specific marker tartrate-resistant acid phosphatase (TRAP; purple staining) in proximal tibias of 10-day-old WT and IL-6–transgenic mice (original magnification ⫻ 2.5). Bottom, Higher magnification view showing details of osteoclast staining (original magnification ⫻ 20). OCp ⫽ osteoclast precursors. B, TRAP histochemical staining of cortical bone (B). Bidirectional arrows indicate cortical bone width. Osteoclasts line the endosteal surface (original magnification ⫻ 10). C, Top, Histochemical detection of the osteoblast-specific marker alkaline phosphatase (red staining) in proximal tibias of 10-day-old WT and IL-6–transgenic mice (original magnification ⫻ 2.5). Bottom, Semithin sections of the proximal secondary spongiosa of WT and IL-6–transgenic mouse proximal tibias stained with methylene blue/azure II (original magnification ⫻ 20). Shown are osteoblasts lining a bone trabecula as well as a nearby osteoclast. D, Methylene blue/azure II–stained semithin sections of cortical bone of the tibia midshafts of 10-day-old WT and IL-6–transgenic animals (original magnification ⫻ 20). P ⫽ periosteal surface; * ⫽ osteoid tissue; E ⫽ endosteal surface (see Figure 1 for other definitions). Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org ble 1). Similarly, TRAP staining in cortical bone of IL-6–transgenic mice revealed an increased number of endosteal multinucleated osteoclasts compared with that in cortical bone of WT mice (Figure 2B). Consistent with the histologic results, urinary excretion of deoxypyridinoline, a marker of osteoclast activity, was signifi- 3556 cantly increased in IL-6–transgenic mice (mean ⫾ SD 175 ⫾ 73 nmoles/liter; n ⫽ 11) compared with that in WT littermates (mean ⫾ SD 67 ⫾ 20 nmoles/liter; n ⫽ 13) (P ⬍ 0.001), showing increased bone resorption in vivo. Effect of IL-6 on osteoclastogenesis. In freshflushed bone marrow cells, the number of putative TRAP-positive mononuclear precursors per field in IL-6–transgenic mice (mean ⫾ SEM 9.67 ⫾ 1.5) was 4-fold higher than that in WT mice (mean ⫾ SEM 2.67 ⫾ 1.5) (P ⬍ 0.0001; n ⫽ 3 per group). Consistently, unfractionated bone marrow cells cultured with 1,25(OH)2D3 generated a 1.6-fold higher number of mature multinucleated osteoclasts in IL-6–transgenic mice (mean ⫾ SEM 51 ⫾ 6.7 per field) than in WT mice (mean ⫾ SEM 30.67 ⫾ 2.9 per field) (P ⬍ 0.04; n ⫽ 3 per group), with a mean ⫾ SEM of 4.14 ⫾ 0.29 nuclei per osteoclast in both groups. Likewise, FicollHistopaque–purified bone marrow macrophages treated with M-CSF and RANKL generated more mature osteoclasts in cultures from IL-6–transgenic mice than in cultures from WT mice (mean ⫾ SEM 31.67 ⫾ 4.83 per field versus 16.14 ⫾ 2.4 per field, a 1.96-fold increase; P ⬍ 0.02; n ⫽ 3 per group), providing further evidence of the presence of more osteoclast precursors in the bone marrow of IL-6–transgenic mice. Moreover, the addition of 5 ng/ml recombinant human IL-6 to M-CSF– and RANKL-treated bone marrow macrophages from WT and IL-6–transgenic mice induced mean ⫾ SEM fold increases of 1.97 ⫾ 0.29 (P ⬍ 0.03; n ⫽ 3) and 2.08 ⫾ 0.23 (P ⬍ 0.02; n ⫽ 3), respectively, of mature osteoclast formation compared with the vehicle-treated counterparts, demonstrating that IL-6 was able to directly stimulate osteoclastogenesis. Taken together, these results indicate that chronic overexposure to IL-6 in prepubertal animals increases bone marrow osteoclast differentiation and mature osteoclast number and activity. Decreased osteoblast activity in IL-6–transgenic mice. Histochemistry for the osteoblast-specific marker AP in trabecular bone of IL-6–transgenic mice showed a reduction of this enzymatic activity (Figure 2C, top panels) and a lower osteoblast surface per bone surface, as evaluated by histomorphometry (Table 1). Examination of semithin sections revealed that in IL-6– transgenic mice, osteoblasts were smaller and reduced in numbers compared with those in WT littermates, and these osteoblasts lined bone trabeculae presenting with scarce osteoid tissue (Figure 2C, bottom panels). In the same trabeculae, resorbing osteoclasts were often noted (Figure 2C, bottom right panel), suggesting uncoupled DE BENEDETTI ET AL activities between the 2 cell types. In cortical bone in IL-6–transgenic mice, the endosteal surface was more irregularly shaped, with less osteoid tissue, compared with that in WT littermates. Osteoid tissue and osteoblasts were less prominent in the periosteal surface in IL-6–transgenic animals (Figure 2D). In vivo, decreased osteoblast activity was confirmed by low serum levels of osteocalcin in IL-6–transgenic mice (mean ⫾ SD 759 ⫾ 363 ng/ml; n ⫽ 11) compared with those in WT mice (mean ⫾ SD 1,323 ⫾ 325 ng/ml; n ⫽ 13) (P ⬍ 0.04). These results show that chronic overexposure to IL-6 in vivo leads to reduced osteoblast number and activity. Effect of IL-6 on osteoblast function in vitro. To investigate the mechanisms of the decreased osteoblast activity present in IL-6–transgenic mice, we evaluated the in vitro effects of IL-6 on osteoblast phenotype. Calvarial osteoblasts from IL-6–transgenic mice showed decreased AP activity (Figure 3A) and formed fewer mineralized nodules than osteoblasts from WT littermates (Figure 3B). Treatment of WT mouse osteoblasts with IL-6 at the highest concentration present in IL-6– transgenic mice (i.e., 5 ng/ml) decreased osteoblast AP (Figure 3A) and nodule mineralization (Figure 3B), with no additional effect at 10 ng/ml (not shown). Treatment of IL-6–transgenic mouse osteoblasts with recombinant human IL-6 failed to further significantly reduce their activity compared with untreated IL-6–transgenic mouse osteoblasts. We also observed a significantly reduced proliferation (measured by 3H-thymidine incorporation assay) in cultures of IL-6–transgenic mouse osteoblasts (mean ⫾ SEM 66,664 ⫾ 4,747 counts per minute; n ⫽ 3) compared with that in cultures of WT mouse osteoblasts (mean ⫾ SEM 103,240 ⫾ 4,950 cpm; n ⫽ 3) (P ⬍ 0.05), and proliferation of WT mouse osteoblasts could be reduced by treatment with recombinant human IL-6 (mean ⫾ SEM 80,073 ⫾ 4,899 cpm; n ⫽ 3) (P ⬍ 0.05 versus untreated cultures of WT mouse osteoblasts). In addition to decreased proliferation, reduced bone formation may depend on impaired osteoblast differentiation and/or function. To distinguish between these 2 possibilities, IL-6–transgenic mouse cells were examined for the expression of relevant osteoblastspecific genes by RT-PCR. Our analysis showed no changes in the expression of the osteoblast lineage markers Runx2 and AP compared with that in the WT mouse cells, and no modulations of these genes occurred in recombinant human IL-6–treated osteoblasts of either IL-6–transgenic or WT mice. In contrast, the messenger RNA (mRNA) of the matrix proteins osteocalcin and type I collagen ␣2 were down-regulated in IL-6– transgenic mouse osteoblasts and in WT mouse osteo- IL-6 AND SKELETAL GROWTH 3557 Figure 3. In vitro effects of IL-6 on osteoblasts. A, Calvarial osteoblasts from WT and IL-6–transgenic mice were cultured for 1 week in the presence of vehicle or 5 ng/ml recombinant human IL-6 (rhIL-6), then fixed and stained for alkaline phosphatase (ALP) histochemical detection (dark spots). Bottom panel shows the densitometric analysis of alkaline phosphatase staining as observed in the top panels. Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.001 versus WT mouse osteoblasts not treated with recombinant human IL-6. B, Calvarial osteoblasts from WT and IL-6–transgenic mice were cultured as described in A for 3 weeks in the presence of ascorbic acid and ␤-glycerophosphate to favor mineralization. Mineralized nodules were then detected by the von Kossa reaction (dark spots). Bottom panel shows quantitative determination of mineralized nodules, as observed in the top panels, performed by densitometric analysis. Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.001 versus WT mouse osteoblasts not treated with recombinant human IL-6. C and D, RNA was extracted from calvarial osteoblasts cultured as described in A, and the expression of the indicated genes was detected by semiquantitative reverse transcriptase–polymerase chain reaction. Similar results were observed in 3 independent experiments. OCN ⫽ osteocalcin; COLL 1A2 ⫽ Col1a2; OPG ⫽ osteoprotegerin; M-CSF ⫽ macrophage colony-stimulating factor; TGF␤ ⫽ transforming growth factor ␤; TNF␣ ⫽ tumor necrosis factor ␣; mIL-6 ⫽ murine IL-6; hIL-6 ⫽ human IL-6; IGF-1 ⫽ insulin-like growth factor 1 (see Figure 1 for other definitions). blasts treated with recombinant human IL-6 compared with untreated WT mouse cells, again with no further effect of recombinant human IL-6 on IL-6–transgenic mouse osteoblasts (Figure 3C). Taken together, these results indicate that IL-6 affects osteoblast function rather than differentiation. Since IL-6–transgenic mice have increased oste- oclastogenesis, and osteoblasts induce osteoclast differentiation by a number of membrane-bound or soluble factors, we evaluated osteoblast expression of cytokines involved in osteoclast regulation. The mRNA of RANKL, osteoprotegerin (OPG), M-CSF, transforming growth factor ␤ (TGF␤), and tumor necrosis factor ␣ (TNF␣) were comparable in IL-6–transgenic and WT 3558 DE BENEDETTI ET AL Figure 4. Histologic and histochemical evaluation of the endochondral ossification of the proximal tibia of 10-day-old mice. Aa and Aaⴕ, Von Kossa staining. Arrow indicates epiphyseal secondary ossification center (original magnification ⫻ 2.5). Ab and Abⴕ, Methylene blue/azure II staining of semithin sections showing details of the epiphyseal ossification centers and surrounding cartilage. Red-boxed area shows details of a bone trabecula (black arrow) and a blood capillary (red arrowhead) (original magnification ⫻ 10). Ac and Acⴕ, Alkaline phosphatase staining of secondary ossification centers. Arrow indicates alkaline phosphatase–positive staining (original magnification ⫻ 4). Ad and Adⴕ, Tartrate-resistant acid phosphatase staining of secondary ossification centers (original magnification ⫻ 20) OCp ⫽ osteoclast precursors; OC ⫽ osteoclasts. B, Left, Hematoxylin and eosin staining of proximal tibias of WT and IL-6–transgenic mice (original magnification ⫻ 2.5). Right, Methylene blue/azure II staining of semithin sections of the proximal growth plates of WT and IL-6–transgenic mouse tibias showing details of the hypertrophic zone (bidirectional arrows) (original magnification ⫻ 10). See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org mouse osteoblasts and were not modified by treatment with recombinant human IL-6 (Figure 3D). Serum levels of RANKL and OPG were consistently comparable in IL-6–transgenic and WT mice (not shown). In contrast, IL-6–transgenic mouse osteoblasts, as well as WT mouse osteoblasts treated with recombinant human IL-6, showed an increased expression of IL-1, a cytokine known for its pro-osteoclastogenic effect (15). Interestingly, IL-6 mRNA was also increased in IL-6–transgenic mouse osteoblasts compared with WT mouse osteoblasts, and treatment with recombinant human IL-6 increased the expression of the endogenous mouse IL-6 IL-6 AND SKELETAL GROWTH in WT mouse osteoblasts but had no effect in the IL-6–transgenic mouse cells (Figure 3D), suggesting that IL-6–transgenic mouse osteoblasts were already maximally stimulated by the endogenous cytokine. Notably, cultured IL-6–transgenic mouse calvarial cells also expressed the human transgene, suggesting that human IL-6 may contribute to the increased expression of murine IL-6 in the same cultures. Taken together, these results suggest that autocrine IL-6 and paracrine IL-6– induced IL-1 may synergistically contribute to the enhancement of osteoclast formation. The level of circulating insulin-like growth factor 1 (IGF-1) is decreased in IL-6–transgenic mice (16). This decrease is due to increased clearance, with liver IGF-1 production being normal, suggesting that IL-6 may not affect IGF-1 expression. We consistently found normal IGF-1 mRNA expression in cultured IL-6– transgenic mouse osteoblasts compared with cultured WT mouse osteoblasts, with no further change upon IL-6 treatment in vitro (Figure 3D). In addition, treatment with exogenous IGF-1 (25 ng/ml) failed to rescue the IL-6–transgenic phenotype in cultured osteoblasts (mean ⫾ SEM 3H-thymidine incorporation 21,568 ⫾ 590 cpm in untreated IL-6–transgenic mouse osteoblasts versus 20,493 ⫾ 303 cpm in IGF-1–treated IL-6– transgenic mouse osteoblasts; P not significant [NS]; n ⫽ 3) (by AP biochemical assay, mean ⫾ SEM optical density 42.60 ⫾ 1.86 in untreated IL-6–transgenic mouse osteoblasts versus 40.20 ⫾ 2.13 in IGF-1–treated IL-6– transgenic mouse osteoblasts; P NS; n ⫽ 3). Impaired ossification in IL-6–transgenic mice. Because the IL-6–transgenic mice show impaired longitudinal bone growth, we evaluated the endochondral ossification. At age 10 days, WT mice showed welldeveloped epiphyseal ossification centers with mineralized tissue detected by von Kossa’s stain (Figure 4Aa) and thin trabeculae intermingled with soft marrow tissue also containing blood vessels (Figure 4Ab). WT ossification centers were AP positive (Figure 4Ac) and TRAP positive (Figure 4Ad). In contrast, in IL-6–transgenic mice, epiphyseal cartilage had not yet mineralized (Figure 4Aa⬘), contained only hypertrophic chondrocytes with no evidence of bone marrow tissue or blood vessels (Figure 4Ab⬘), and was negative for AP (Figure 4Ac⬘) and TRAP (Figure 4Ad⬘). In IL-6–transgenic mice, the growth plates were thinner with an even more markedly reduced hypertrophic zone (Figure 4B and Table 1). Intramembranous ossification is principally involved in skull development. Radiographic analysis of IL-6–transgenic mice had demonstrated small size of the skull (Figure 1A). We therefore evaluated calvarial bone 3559 Figure 5. Intramembranous ossification. A, Coronal semithin sections of calvariae from 10-day-old WT and IL-6–transgenic mice stained with methylene blue/azure II (top and middle panels) or for histochemical detection of tartrate-resistant acid phosphatase (TRAP) (bottom panels). Aa and Aaⴕ, Sagittal sutures. Ab and Abⴕ, Paramedian areas of the parietal bones. Ac and Acⴕ, TRAP staining of similar areas as in Ab and Abⴕ. OC ⫽ osteoclasts. (Original magnification ⫻ 10.) B, Double in vivo alizarin red S (red fluorescence) and calcein (green fluorescence) labeling of tibias from 10-day-old WT and IL-6– transgenic mice. Midshaft longitudinal sections are shown. E ⫽ endosteal surface; P ⫽ periosteal surface (Original magnification ⫻ 2.5.) See Figure 1 for other definitions. formation and found that it was also significantly affected in IL-6–transgenic mice. The sagittal suture was primordial and not well recognizable in calvariae of IL-6–transgenic mice (Figure 5Aa⬘) compared with that of WT littermates (Figure 5Aa). The bone was thinner and irregular in IL-6–transgenic mice (Figure 5Ab⬘) compared with that in WT mice (Figure 5Ab), and TRAP staining showed a marked increase of osteoclasts on the inner calvarial surface in IL-6–transgenic animals (Figures 5Ac and Ac⬘). Histomorphometry (Table 1) consistently showed decreased bone thickness, a decreased number of osteoblasts, and an increased number 3560 DE BENEDETTI ET AL of osteoclasts in calvariae of IL-6–transgenic animals. It is therefore clear that both endochondral and intramembranous ossification are impaired in developing mice overexpressing IL-6. Dynamic assessment of mineral apposition rate in cortical bone. To investigate dynamically whether osteoblast/osteoclast uncoupling had an impact on ossification, we evaluated the mineral apposition rate by in vivo double fluorescence labeling in cortical bone, where the periosteal tissue presents only with osteoblasts and the endosteal surface shows both osteoclasts and osteoblasts. Therefore, examination of cortical bone allows us to evaluate separately the effects of osteoblasts and osteoclasts on ossification. A 30% decrease in periosteal mineral apposition in the mid-diaphysis was found in IL-6–transgenic mice. In the time frame of the experiment, endosteal mineral apposition was completely abrogated (Figure 5B). This result parallels the histologic findings showing an increased number of osteoclasts (Figure 2B) and a decreased number of osteoblasts (Figure 2D) at the endosteal surface of mid-diaphysis cortical bone. It suggests that the defective ossification is secondary to the marked imbalance between endosteal osteoclast and osteoblast activity, and that this may contribute to stunted growth. We consistently found that in IL-6–transgenic mice, body weight was directly correlated with serum osteocalcin levels (r ⫽ 0.636, P ⬍ 0.03, R2 ⫽ 0.40) and inversely correlated with urinary deoxypyridinoline levels (r ⫽ ⫺0.760, P ⬍ 0.01, R2 ⫽ 0.58), suggesting that the more marked the osteoblast/ osteoclast imbalance, the more severe the growth defect. DISCUSSION Our study shows that during prepubertal development, IL-6 overexpression causes osteopenia, with accelerated bone resorption, reduced bone formation, and defective ossification. Enhanced bone resorption is due to increased osteoclastogenesis, and reduced osteoblast activity is induced with a mechanism affecting precursor proliferation and osteoblast function. In our IL-6–transgenic mice, skeletal abnormalities were not present at birth. This is consistent with IL-6 overexpression in these mice starting at birth (7,17), and it implies that the bone phenotype is not due to abnormalities in skeletal development during fetal life. Mean IL-6 levels in the 10-day-old IL-6–transgenic mice are 2 ng/ml (18), and these levels are in the range of those observed in children with active systemic JIA (19). Similar to other IL-6–transgenic mice, our IL-6– transgenic mice showed increased levels of serum amy- loid A and elevated white blood cell and platelet counts, which are typical systemic effects of IL-6 overproduction. In contrast, serum levels of other proinflammatory cytokines, such as TNF and IL-1, were undetectable (De Benedetti F, et al: unpublished observations). This implies that the observed phenotype is secondary to the selective overexpression of IL-6, at levels attainable in children with chronic inflammation, and is not affected by a generalized inflammatory response with multiple mediators. Osteoclasts respond to IL-6 in vitro, as demonstrated by Kudo et al (20) and by us in the present study, suggesting that the increased osteoclast formation and activity could be due to a direct effect of the cytokine that bypasses inflammation. Alternatively, since osteoblasts affect osteoclast function, the in vivo effect of IL-6 could be mediated through the osteoblasts. However, expression of RANKL, OPG, M-CSF, TGF␤, and TNF␣ was unchanged in IL-6–transgenic mouse osteoblasts, as well as in WT mouse osteoblasts treated with recombinant human IL-6, showing that activation of osteoclasts in IL-6–transgenic mice is mediated neither by an increased production of these cytokines by the osteogenic cells, nor by increased circulating levels that were not found to be changed in vivo. We also observed that IL-1 and IL-6 were up-regulated in IL-6–transgenic mouse osteoblasts, and that recombinant human IL-6 treatment induced both cytokines in WT mouse osteoblasts. Since IL-1 can also stimulate osteoclastogenesis (15,20), it is conceivable that in the IL-6–transgenic mice, IL-6 itself and IL-6–induced IL-1 could jointly contribute locally to the observed increase in osteoclast formation and bone resorption. Data on the in vivo and in vitro effects of IL-6 on osteoblasts are still conflicting, and several models have shown contradictory results (12). In our IL-6–transgenic mice, we have observed a marked decrease in osteoblast activity. The cellular mechanisms of osteoblast inhibition by IL-6 include decreased proliferation of putative osteoblast precursors and reduced expression of genes for bone matrix proteins. Osteoblast differentiation appears to be unaffected, as shown by unaltered Runx2 and AP mRNA expression. The apparent discrepancy between unchanged Runx2 expression and low expression of its downstream gene osteocalcin may be explained by possible modulation by IL-6 of Runx2-specific coactivators/repressors (21). In collagen-induced arthritis (CIA), Hoshino et al (22) have shown that prepubertal rats have loss of trabecular and cortical bone with an increased number of osteoclasts and a decreased number of osteoblasts, a IL-6 AND SKELETAL GROWTH bone phenotype that is very similar to the phenotype induced by overexpression of IL-6 alone in our IL-6– transgenic mice. In the same study, adult rats with CIA still showed a decreased number of osteoblasts but, in contrast to prepubertal animals, had a decreased number of osteoclasts and bone resorption (22). Likewise, in another study, adult IL-6–transgenic mice were reported to have decreased bone formation associated with decreased osteoclast number and bone resorption (23). Similar conclusions were also drawn by Sims et al (24), again in adult mice, using models with altered gp130. Consistent with the results of these studies, we also found in our adult mice that both osteoblast and osteoclast activities were decreased compared with those in WT littermates (mean ⫾ SD osteoblast surface/bone surface 28.53 ⫾ 4.37% in WT mice versus 14.72 ⫾ 3.21% in IL-6–transgenic mice; P ⬍ 0.05) (mean ⫾ SD osteoclast surface/bone surface 38.60 ⫾ 11.79% in WT mice versus 20.46 ⫾ 1.85% in IL-6–transgenic mice; P ⬍ 0.02). It is therefore reasonable to conclude that the impact of IL-6, and possibly more generally of inflammation, on bone cells may depend on the stage of development (i.e., growing versus postpubertal animals), despite similar expression of IL-6 receptors and other relevant receptors (e.g., c-Fms) at the two ages, assessed by immunohistochemistry and RT-PCR (not shown). This may very well reflect a different regulation of the osteoblast/osteoclast balance in growing individuals (23). Indeed, in growing individuals, bone formation must exceed bone resorption in order to gain bone mass, allowing skeletal growth (25,26). On the other hand, during growth, balanced bone resorption is essential for mineralized cartilage and woven bone erosion, two processes that contribute physiologically to longitudinal development (27). Furthermore, resorption often occurs at sites distinct from those of formation in order to ensure bone shaping and enlargement of interior cavities. Therefore, the regulation of osteoblast and osteoclast activities must be different in growing subjects compared with adult subjects. The mechanisms behind this different regulation are not clear. Whatever they might be, our results show that chronic IL-6 overexpression in growing prepubertal animals can pathologically dissociate osteoclast function from osteoblast function, thereby contributing to the development of an osteopenic phenotype. We have also found that IL-6–transgenic mice have defective intramembranous and endochondral ossification as well as a decreased mineral apposition rate in diaphyseal bone, suggesting the presence of a marked 3561 ossification defect. Our observations in diaphyseal ossification are consistent with the hypothesis that uncoupling of osteoblast and osteoclast activities contributes to the defective ossification of IL-6–transgenic mice. This uncoupling, and the subsequent ossification defect, could also contribute to stunted growth. In support of this hypothesis, we found that the body weight of IL-6–transgenic mice was directly correlated with the serum osteocalcin levels and inversely correlated with the urinary deoxypyridinoline levels, showing that the more marked the imbalance between osteoblasts and osteoclasts, the more severe the growth defect. Since the growth plate has a major role in longitudinal bone growth, it is clear that the abnormalities observed in IL-6–transgenic mice at this level could also contribute to stunted growth. Whether these abnormalities are due to a direct effect of IL-6 on the growth plate or whether they are mediated by other factors is still unclear. IL-6 has been shown to affect matrix protein production by articular chondrocytes (28); however, to the best of our knowledge, no reports of studies on growth plate chondrocytes are available. We have previously found markedly decreased IGF-1 levels in IL-6–transgenic mice (16). Since IGF-1 is an important regulator of cartilage growth (29,30), it is possible that the growth plate abnormalities are secondary to the IL-6–induced decrease in circulating IGF-1. In addition, while a role in bone resorption is unlikely (31), reduced IGF-1 could contribute to osteoblast failure and impaired bone formation (30). However, this hypothesis is not supported by the unchanged transcriptional expression of IGF-1 in IL-6–transgenic mouse osteoblasts compared with WT mouse osteoblasts, or by the failure of exogenous IGF-1 to rescue the IL-6– transgenic mouse osteoblast phenotype. Moreover, in vivo administration of IGF-1 or of the IGF-1–IGF binding protein 3 complex failed to rescue the phenotype of the IL-6–transgenic mice, probably due to IGF-1 instability (De Benedetti F and Vivarelli M: unpublished observations). Indeed, our previous studies have shown that low circulating IGF-1 levels in IL-6–transgenic mice are due to increased clearance rather than to abnormal liver production (16). Taken together, these findings imply that IGF-1 should not be regarded as a potential candidate to correct the effects of chronic IL-6 overexpression on the skeletal system. In conclusion, our results show that chronic overexpression of IL-6 affects the developing skeleton, causing osteopenia, defective ossification, and growth plate abnormalities. We have demonstrated in vivo uncoupling of osteoclasts from osteoblast activity, and we have 3562 DE BENEDETTI ET AL shown ex vivo increased osteoclastogenesis and inhibition of osteoblast function by IL-6. The molecular mechanisms triggered by IL-6 in osteoblasts and osteoclasts as well as the molecular and cellular mechanisms by which IL-6 affects the growth plate are being investigated in our laboratories. Whatever the mechanisms might be, while IL-6 overexpression alone does not lead to a generalized inflammatory response in our IL-6– transgenic mice, it certainly drives a cascade of events that induce a skeletal phenotype closely resembling growth and skeletal abnormalities found in children with JIA. In this respect, it is worthy of note that IL-6⫺/⫺ mice are protected from severe disease in several models of experimental arthritis (32–35), pointing to IL-6 as a key pathologic mediator in inflammatory arthritis. In addition, a recent study showed that IL-6, but not TNF, played a pivotal role in the induction of bone marrow osteoclastogenesis and osteoclast recruitment in inflamed joints, thereby suggesting that IL-6 is central to the pathogenesis of bone loss in the context of chronic joint inflammation (36). Skeletal abnormalities, as well as evidence of osteoblast/osteoclast uncoupling similar to that found in JIA, are also present in cystic fibrosis and in Crohn’s disease (37–40). In both conditions, some (although indirect) evidence links growth and bone abnormalities with inflammatory activity of the disease (39–44). In cystic fibrosis, IL-6 levels have been found to be associated with defective bone mineral content gain and with deoxypyridinoline levels (40), and in Crohn’s disease, IL-6 has been shown to be the factor responsible for the ability of patients’ sera to inhibit bone mineralization in vitro (44). Increased IL-6 may therefore represent a generalized major mechanism by which chronic inflammation affects the developing skeleton. This would imply that anti–IL-6 therapeutic approaches, which have shown promising antiinflammatory efficacy in rheumatoid arthritis, Crohn’s disease, and systemic JIA (45–47), may also reduce the skeletal defects and prevent the growth retardation present in these diseases. ACKNOWLEDGMENTS We gratefully acknowledge the precious help of Dr. Rita Di Massimo for the editing of the manuscript and of Ms Fanny Cavat for performing microcomputed tomography analysis. REFERENCES 1. Cassidy JT, Hillman LS. 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