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Keywords:

  • CHEMOKINES;
  • CCL5;
  • OSTEAL MACROPHAGES;
  • OSTEOIMMUNOLOGY

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Chemokines play crucial roles in the recruitment of specific hematopoietic cell types, and some of them have been suggested to be involved in the regulation of bone remodeling. Because we have previously observed that chemokine (C-C motif) ligand 2 (Ccl2) and Ccl5 are direct target genes of noncanonical Wnt signaling in osteoblasts, we analyzed the skeletal phenotypes of Ccl2-deficient and Ccl5-deficient mice. In line with previous studies, Ccl2-deficient mice display a moderate reduction of osteoclastogenesis at the age of 6 months. In contrast, 6-month-old Ccl5-deficient mice display osteopenia associated with decreased bone formation and increased osteoclastogenesis. Moreover, unlike in wild-type and Ccl2-deficient mice, large areas of their trabecular and endocortical bone surfaces are not covered by osteoblasts or bone-lining cells, and this is associated with a severe reduction of endosteal bone formation. Although this phenotype diminishes with age, it is important that we could further identify a reduced number of osteal macrophages in 6-month-old Ccl5-deficient mice, because this cell type has previously been reported to promote endosteal bone formation. Because Ccl5-deficient mice also display increased osteoclastogenesis, we finally addressed the question of whether osteal macrophages could differentiate into osteoclasts and/or secrete inhibitors of osteoclastogenesis. For that purpose we isolated these cells by CD11b affinity purification from calvarial cultures and characterized them ex vivo. Here we found that they are unable to differentiate into osteoblasts or osteoclasts, but that their conditioned medium mediates an antiosteoclastogenic effect, possibly caused by interleukin-18 (IL-18), an inhibitor of osteoclastogenesis expressed by osteal macrophages. Taken together, our data provide in vivo evidence supporting the previously suggested role of Ccl5 in bone remodeling. Moreover, to the best of our knowledge, Ccl5-deficient mice represent the first model with a spontaneous partial deficiency of osteal macrophages, a recently identified cell type, whose impact on bone remodeling is just beginning to be understood. © 2013 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Osteoimmunology is an emerging research area focusing on the interactions between bone and immune cells.[1, 2] More specifically, because hematopoietic cells in the bone marrow are in close proximity to osteoblasts and osteoclasts, and because osteoclasts develop by fusion of myeloid precursor cells, it is not surprising that these interactions are critically involved in the regulation of bone remodeling. In fact, whereas macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) are not only major activators of osteoclastogenesis, but also serve functions in the immune system, specific immune cell–derived cytokines have been demonstrated to influence bone cell differentiation and/or function. These include members of the interleukin-1 (IL-1) family of cytokines, IL-6, tumor necrosis factor α/β (TNFα/β), or interferon γ (IFNγ),[3-10] but also certain chemokines and their receptors. For instance, mice lacking the chemokine receptor Ccr2 display high bone mass due to decreased osteoclastogenesis, which was molecularly explained by an impaired response of Ccr2-deficient preosteoclasts to chemokine (C-C motif) ligand 2 (Ccl2) and Ccl7, which normally induce RANK production.[11] Moreover, Ccl5 has been shown to promote chemotaxis and survival of osteoblasts in vitro, and it was hypothesized that its expression by osteoclasts is required to target osteoblasts to sites of bone resorption.[12]

Our interest in this area of research came from recent findings showing that chemokine-encoding genes are direct targets of noncanonical Wnt signaling in osteoblasts.[13, 14] In fact, based on the previously established role of low-density lipoprotein receptor (Lrp5), a coreceptor for Wnt ligands, in bone formation,[15] we addressed the following question: which of the 10 known Wnt receptors of the Frizzled (Fzd) family could be essential for osteoblast function. Using genomewide expression analysis we identified Fzd9 as a candidate and subsequently analyzed the skeletal phenotype of Fzd9-deficient mice, in which we observed osteopenia due to decreased bone formation. Our molecular analysis of Fzd9-deficient osteoblasts further revealed that canonical Wnt signaling was not affected by the absence of Fzd9, in contrast to noncanonical pathways, which can be induced by administration of Wnt5a. Importantly, when we analyzed these effects on the level of gene expression, we found that Fzd9-deficient osteoblasts displayed lower expression of several chemokine-encoding genes, and that these genes were induced by Wnt5a administration to wild-type cells for 6 hours.[13] Taken together, these results demonstrated that chemokines, such as Ccl2 and Ccl5, are direct targets of Wnt signaling in osteoblasts, which raised the question of whether they are involved in the regulation of bone remodeling in vivo.

Here we show that mice lacking Ccl2 display a moderate reduction of osteoclastogenesis, whereas Ccl5 deficiency results in increased osteoclastogenesis and a transient reduction of bone formation. Remarkably, 6-month-old Ccl5-deficient mice display large areas of cell-free trabecular bone surfaces, thus underscoring its potential relevance in osteoprogenitor recruitment.

Subjects and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Mice

Ccl2-deficient and Ccl5-deficient mice were obtained from the Jackson Laboratory (#005090, #004434; Bar Harbor, ME, USA) and first crossed with C57Bl6 mice to obtain heterozygous mice. Offspring from heterozygous matings was genotyped according to the instructions from the Jackson Laboratory, and wild-type and homozygous mutant littermates were used for skeletal analysis. All mice received two injections of calcein 9 and 2 days before euthanasia.

Skeletal analysis

After their initial analysis by X-ray (Faxitron X-ray Corp., Wheeling, IL, USA), the vertebral bodies L1 to L4 and one tibia of each animal were dissected and fixed in 4% buffered formalin for 24 hours. After fixation, the samples were dehydrated in ascending alcohol concentrations and embedded nondecalcified into methyl methacrylate for sectioning. Four micrometer (4-µm)-sections were either stained with toluidine blue or by the von Kossa/van Giessen procedure as described.[16] Static and cellular histomorphometry was carried out on toluidine blue–stained sections using the OsteoMeasure system (Osteometrics, Decatur, GA, USA) following the guidelines of the American Society of Bone and Mineral Research.[17] All parameters of static, cellular, and dynamic histomorphometry were measured in two vertebral bodies (L3 and L4) for each animal, and the mean value was used for statistical analysis. Dynamic histomorphometry for determination of the bone formation rate was performed on two nonconsecutive stained 12-μm sections for each animal.

Immunoassays

Serum concentrations of osteocalcin were determined by radioimmunoassay (Immutopics, San Clemente, CA, USA; #50-1300). Medium concentrations of osteoprotegerin (Opg), IL-1ra, Il-33, Il-1β, and Il-18 were determined by ELISA (R&D Systems, Minneapolis, MN, USA; #MOP00; #MRA00, #M3300, #MLB00C, and #7625, respectively). Immunofluorescence was performed on decalcified tibia sections with antibodies against F4/80 (rat anti-mouse; Abcam, Cambridge, MA, USA; #ab 16911), osteocalcin (rabbit anti-mouse; Enzo Life Sciences, Loerrach, Germany; #ALX-201-333-C100) and IL-18 (rabbit anti-mouse; Abcam; #ab71495) according to standard protocols. For immunocytochemistry, primary osteoblasts were seeded onto chamber slides and differentiated for 10 days as described in the Cell Culture section. To block endogenous peroxidase activity and nonspecific antibody binding, sections were incubated with 3% hydrogen peroxide for 15 minutes and with 5% normal goat serum (NGS)/Tris-buffered saline (TBS) (Dako, Carpinteria, CA, USA; #X0907) for 30 minutes. A monoclonal rat anti-mouse F4/80 antibody (Abcam; #ab16911) was applied overnight at 4°C at a 100-fold dilution. Biotinylated rabbit anti-rat immunoglobulin G (IgG) (Dako; #E0468) was used as a secondary antibody at a 200-fold dilution, followed by incubation with streptavidin/horseradish peroxidase (HRP) (Dako; #P0397), also at a 200-fold dilution. Peroxidase activity was detected using diaminobenzidine (DAB) as a chromogenic substrate, and the slides were counterstained with hematoxylin/eosin (Merck).

Fluorescence-activated cell sorting analysis

Bone marrow was flushed out as described in the Cell Culture section and flow cytometry was conducted after blocking unspecific binding sites with 0.5% fetal bovine serum (FBS) in phosphate-buffered saline (PBS). As fluorescently labeled primary antibodies we used anti-CD11b (CD11b, PE; BD Biosciences, San Jose, CA, USA), anti-F4/80 (F4/80, APC; eBioscience, San Diego, CA, USA), anti-CD4 (CD4, APC-H7; BD Biosciences), and anti-CD8 (CD8, FITC; BD Biosciences). Additionally, we used matched fluorescently conjugated isotype controls. Flow cytometry was accomplished with the fluorescence-activated cell sorting (FACS)-Canto II flow cytometer (BD Bioscience). Data were processed with CellQuest-Pro software (BD Biosciences).

Cell culture

Long bones (femurs and tibias) were aseptically removed from 12-week-old mice, and the bone marrow was flushed out using α modified essential medium (α-MEM) containing 10% FBS (HyClone, Waltham, MA, USA). These cells were separated with magnetic-activated cell sorting (MACS) technology using a mature hematopoietic lineage cell depletion kit supplemented with mouse CD11b microbeads (Miltenyi Biotec, Auburn, CA, USA) following the manufacturer's instructions. CD11b-positive and CD11b-negative cells were seeded alone or together in 12-well plates at a density of 1 × 106 cells/well. To induce osteoblastic differentiation the medium was supplemented with 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate for 10 days. Alizarin red staining and quantification of mineralization was performed with alizarin red S solution (40 mM, pH 4.2) followed by dissolving the cell-bound alizarin red S in 10% acetic acid. Alternatively, osteoblasts were isolated from the calvariae of 5-day-old mice following digestion with 0.1% collagenase and 0.2% dispase for 45 minutes. CD11b-positive and CD11b-negative cells were again separated by CD11b microbeads and differentiated in the presence 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate for 10 days. For osteoclastogenesis assays, bone marrow cells (or osteal macrophages) were differentiated with 10 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2 vitamin D3], 20 nM M-CSF and 40 nM RANKL for 10 days. Formation of multinuclear osteoclasts was assessed following tartrate-resistant acid phosphatase (TRAP) activity staining using Naphtol ASMX-Phosphate (Sigma-Aldrich, St. Louis, MO, USA) as a substrate. Resorptive activity was determined by differentiating cells on dentin chips, that were subsequently stained with 0.2% toluidine blue for 1 minute. Recombinant Ccl5 and IL-18 were obtained from R&D Systems (#487-MR-025, B002-5). To induce macrophage differentiation, bone marrow cells were differentiated with 10 nM 1,25(OH)2 vitamin D3, 20 nM M-CSF for 6 days in the presence or absence of Ccl5 (200 ng/mL). To monitor apoptosis we applied a commercially available system to measure caspase-3 activities (Caspase-Glo 3/7 Assay; Promega, Madison, WI, USA).

Expression analysis

RNA was isolated using QuiaShredder and RNeasyMiniKit (Quiagen, Valencia, CA, USA) according to the manufacturer's instructions. The concentration and quality of RNA were determined by using a NanoDrop ND-1000 system (NanoDrop Technology, Wilmington, DE, USA). For qRT-PCR, 100 ng RNA was reverse-transcribed using Super ScriptIII (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. qRT-PCR was performed using a StepOnePlus system (Applied Biosystems, Grand Island, NY, USA) and predesigned gene expression assays (TaqMan; Applied Biosystems) for Bglap, Ibsp, Emr1, Csfr1, Il18, or Sirpa. Gapdh expression was monitored as an internal control. The relative quantification was performed according to the delta-delta cycle threshold (ΔΔCT) method, and results were expressed in the linear form using formula 2–ΔΔCT.

Statistical analysis

All data are presented as mean ± SD. Statistical analysis was performed by two-tailed unpaired Student's t test. Values of p <0.05 were considered as statistically significant (indicated by asterisks).

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Differential influence on bone remodeling caused by Ccl2 or Ccl5 deficiency

We first analyzed the skeletal phenotypes of 6-month-old mice lacking either Ccl2 or Ccl5. Nondecalcified histology of the spine followed by static histomorphometry revealed a slight increase of the trabecular bone volume in Ccl2-deficient mice, which essentially confirms published data.[11] In contrast, Ccl5-deficient mice displayed a significant reduction of the trabecular bone volume compared to wild-type littermates (Fig. 1A), which led us to perform cellular histomorphometry to explain the observed phenotype. Although osteoblast number was not affected in either mouse model, osteoclast number was found to be significantly decreased in Ccl2-deficient mice, but significantly increased in Ccl5-deficient mice (Fig. 1B). In addition, only Ccl5-deficient mice displayed a significant reduction of bone formation, as assessed by dynamic histomorphometry and by measuring serum osteocalcin levels (Fig. 1C). Besides these alterations of bone remodeling we made another potentially important observation specifically in Ccl5-deficient mice. In fact, only in these mice we found that more than 20% of the trabecular bone surface was not covered by osteoblasts or bone-lining cells; ie, there was direct contact between bone marrow cells and the bone matrix (Fig. 1D).

image

Figure 1. Histomorphometric analysis of spine sections from 6-month-old wild-type, Ccl2-deficient and Ccl5-deficient mice. (A) Von Kossa/van Giessen staining of spine sections and quantification of the trabecular bone volume per tissue volume (BV/TV). (B) Quantification of osteoblast and osteoclast numbers per bone perimeter (Ob.N/B.Pm and Oc.N/B.Pm). (C) Quantification of the trabecular bone formation rate per bone surface (BFR/BS) and serum levels of osteocalcin. (D) Toluidine blue staining showing the existence of cell-free trabecular bone surfaces in Ccl5-deficient mice. Quantification is given below. All bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences compared to wild-type controls.

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We further analyzed tibia sections, where we did not observe significant differences in terms of trabecular bone mass between the three groups of mice (Fig. 2A). In contrast, there was again a striking pathology specifically in Ccl5-deficient mice. In fact, the majority of the endocortical bone matrix of these mice was not covered by osteoblasts or bone-lining cells, unlike was the case in wild-type and Ccl2-deficient mice (Fig. 2B). Likewise, endosteal bone formation was dramatically reduced in 6-month-old Ccl5-deficient mice, thereby suggesting a physiologically relevant role of this chemokine for the recruitment of osteoblast progenitors to the bone matrix (Fig. 2C). However, because it was puzzling to explain the absence of cortical thinning in 6-month-old Ccl5-deficient mice, we further addressed the question of whether this phenotype would be more pronounced with age. For that purpose we additionally analyzed wild-type and Ccl5-deficient littermates at 3 and 12 months of age, and we made at least three important observations (Table 1). First, the appearance of cell-free bone surfaces precedes the reduction of bone formation in Ccl5-deficient mice. Second, the severe defect of endocortical bone formation in these mice is transient and diminishes with age. And third, although 12-month-old Ccl5-deficient mice only display a moderate skeletal phenotype, their osteoclast number is still increased compared to wild-type littermates.

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Figure 2. Histomorphometric analysis of tibia sections from 6-month-old wild-type, Ccl2-deficient and Ccl5-deficient mice. (A) Von Kossa/van Giessen staining of tibia sections and quantification of the trabecular bone volume (BV/TV). (B) Toluidine blue staining of endocortical bone surfaces and quantification of the cell-free areas. (C) Fluorescent microscopy showing a reduction of calcein-labeled endocortical bone surfaces in Ccl5-deficient mice. Quantification of the endosteal bone formation rate is given on the right. All bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences compared to wild-type controls.

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Table 1. Histomorphometric Parameters of Wild-Type and Ccl5-Deficient Mice at 3, 6, and 12 Months of Age
 3 Months6 Months12 Months
Ccl5+/+Ccl5−/−Ccl5+/+Ccl5−/−Ccl5+/+Ccl5−/−
  1. Values represent mean ± SD from 6 animals each. Asterisks indicate statistically significant differences (p < 0.05).

  2. Ccl5 = chemokine (C-C motif) ligand 5; BV/TV = bone volume fraction; Ob.N/B.Pm = number of osteoclasts per bone perimeter; Oc.N/B.Pm = number of osteoblasts per bone perimeter; Tb = trabecular; BFR/BS = bone formation rate per bone surface; cell-free BS = cell-free bone surface.

BV/TV (%)14.7 ± 1.414.1 ± 2.814.6 ± 1.411.2 ± 0.8*9.8 ± 2.29.6 ± 0.6
Ob.N/B.Pm (mm−1)14.1 ± 2.313.7 ± 0.912.9 ± 1.813.2 ± 2.114.7 ± 1.614.4 ± 1.7
Oc.N/B.Pm (mm−1)3.5 ± 0.94.9 ± 1.32.9 ± 0.44.5 ± 0.4*3.9 ± 0.95.7 ± 0.9*
Tb.BFR/BS (µm3/µm2/y)82 ± 881 ± 1196 ± 974 ± 13*87 ± 2181 ± 10
Tb cell-free BS (%)4.4 ± 2.129.0 ± 10.9*1.6 ± 0.423.2 ± 4.8*2.9 ± 1.115.3 ± 3.9*
Endocortical BFR/BS (µm3/µm2/year)110 ± 292 ± 18120 ± 1112 ± 11*108 ± 14102 ± 11
Endocortical cell-free BS (%)2.5 ± 0.936.9 ± 12.7*16.7 ± 6.082.4 ± 9.8*4.0 ± 2.75.5 ± 3.4

Impaired hematopoietic cell differentiation in the bone marrow of Ccl5-deficient mice

To monitor changes in the bone marrow composition between 6-month-old wild-type and Ccl5-deficient littermates, we next applied FACS technology (Fig. 3A). Although we did not detect differences in CD4-positive and CD8-positive T cell populations, we observed a shift in terms of macrophage differentiation. More specifically, the number of F4/80-positive macrophages was significantly decreased in Ccl5-deficient mice, whereas the number of Cd11b-positive progenitor cells was increased (Fig. 3B). Because it has been reported that an F4/80-positive cell type (termed osteal macrophage) is required to promote bone formation especially at endocortical bone surfaces,[18] we next performed immunohistochemistry with antibodies against F4/80 and osteocalcin. Although we consistently found F4/80-positive cells overlying osteocalcin-positive osteoblasts in tibia sections from wild-type mice, both cell types were essentially absent from the endocortical bone surfaces of 6-month-old Ccl5-deficient mice (Fig. 3C).

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Figure 3. Impaired macrophage differentiation in Ccl5-deficient mice. (A) FACS analysis of bone marrow from 6-month-old wild-type and Ccl5-deficient mice using the indicated antibodies. (B) Quantification of CD4+, CD8+, F4/80+, and CD11b+ cells. Bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences compared to wild-type controls. (C) Immunohistochemistry using antibodies against F4/80 (stained in green) and osteocalcin (stained in red) demonstrates reduced number of osteal macrophages and osteoblasts in Ccl5-deficient mice. (D) Representative alizarin red staining of bone marrow cells from wild-type and Ccl5-deficient mice differentiated in the presence of ascorbic acid and β-glycerophosphate for 10 days. (E) Schematic presentation of the strategy applied to address the interaction of CD11b+ hematopoietic and CD11b mesenchymal cells. Bone marrow cells from wild-type and Ccl5-deficient mice were separated by CD11b-immunoaffinity and mixed as indicated. (F) Matrix mineralization in the four different cultures of bone marrow cells following osteogenic differentiation in the presence of ascorbic acid and β-glycerophosphate for 10 days. Bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences compared to non-mixed cultures.

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Because these results suggested that the transient defect of endosteal bone formation caused by Ccl5-deficiency is indirectly caused by impaired macrophage differentiation, we isolated bone marrow cells from wild-type and Ccl5-deficient mice and assessed their osteogenic capacity ex vivo. Although the first set of experiments revealed that Ccl5-deficient bone marrow cells formed less mineralized matrix compared to wild-type cultures (Fig. 3D), we next fractionated the bone marrow cells by Cd11b immunoaffinity to separate CD11b-negative osteoprogenitor cells from CD11b-positive macrophage precursors. We then mixed the two fractions from wild-type and Ccl5-deficient mice and differentiated them into osteoblasts by adding ascorbic acid and β-glycerophosphate (Fig. 3E). When we monitored matrix mineralization after 10 days we found that wild-type CD11b-negative cells mineralized to a significantly lesser extent, when they were mixed with CD11b-positive cells from Ccl5-deficient mice (Fig. 3F). Likewise, the reduced mineralization of Ccl5-deficient CD11b-negative cells was nearly corrected, when the CD11b-positive cells were derived from wild-type mice. Taken together, these results suggested that Ccl5-deficiency results in a defect of macrophage differentiation, which can potentially explain the impaired bone formation in Ccl5-deficient mice.

Osteal macrophages are not osteoclast progenitors, but inhibit osteoclastogenesis

Because Ccl5-deficient mice also displayed a higher number of osteoclasts, we next addressed the question of whether this could also be related to a macrophage differentiation defect. As it has been reported that osteal macrophages are copurified with osteoblasts by collagenase digestion of calvariae from newborn mice,[18] we again applied CD11b immunoaffinity to separate the two cell populations. The success of this approach was verified by F4/80 immunostaining of the cultured cells (Fig. 4A) and by qRT-PCR for osteoblast and macrophage markers following 10 days of differentiation in the presence of ascorbic acid and β-glycerophosphate (Fig. 4B). Likewise, Alizarin red staining was performed to demonstrate that, unlike the CD11-negative calvarial osteoblasts, the CD11-positive osteal macrophages were unable to form a mineralized matrix, as expected (Fig. 4C). Because one possibility to explain the increased osteoclastogenesis in Ccl5-deficient mice was that osteal macrophages could themselves differentiate into osteoclasts, we cultured the CD11-positive calvarial cells in the presence of M-CSf and RANKL for 10 days and then monitored formation of TRAP-positive multinucleated cells (Fig. 4D) and dentin resorption (Fig. 4E). In contrast to CD11-positive bone marrow cells plated at an identical density and cultured in the same way, the calvaria-derived osteal macrophages were unable to differentiate into functional osteoclasts, which led us to address the question of whether these cells would produce antiosteoclastogenic molecules.

image

Figure 4. Isolation and characterization of osteal macrophages. (A) F4/80 staining following sorting of calvarial cells by CD11b-immunoaffinity reveals absence of macrophages in the osteoblast fraction (Obl), while all cells stained positive in the osteal macrophage fraction (Omac). (B) qRT-PCR expression analysis for the indicated genes following culture of both cell populations in the presence of ascorbic acid and β-glycerophosphate for 10 days. Bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences between the two groups. (C) Alizarin red staining and subsequent quantification of cultures differentiated in the same way. (D) TRAP activity staining of CD11b+ bone marrow cells (BM Ocl) and CD11b+ calvarial cells (Omac) differentiated in the presence of M-CSF and RANKL for 10 days. Quantification of TRAP-positive multinucleated cells is given below. (E) Toluidine blue staining for dentin resorption after culturing the same cells on dentin chips under the same conditions. Quantification of the resorbed area is given below. All values represent mean ± SD (n = 6).

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For that purpose we collected conditioned medium (CM) of CD11b-negative calvarial osteoblasts and CD11-positive osteal macrophages after 10 days of incubation with ascorbic acid and β-glycerophosphate. This medium was then mixed with osteoclast differentiation medium and applied to bone marrow cells. Using TRAP activity staining after 10 days of M-CSF/Rankl-induced differentiation we observed a significant reduction of osteoclastogenesis not only with CM from osteoblasts, but also with CM from osteal macrophages (Fig. 5A, B). Interestingly, these inhibitory effects occurred despite the fact that Opg levels were much lower in CM from osteal macrophages compared to osteoblast CM (Fig. 5C). Because we and others have previously shown that members of the IL-1 family of cytokines play crucial roles in regulating osteoclastogenesis,[4-6, 19-21] we next addressed the question, whether IL-1β, IL-1ra, IL-18, or IL-33 would be specifically expressed by osteal macrophages (Fig. 5D). Here we found that IL-1β, a positive regulator of osteoclast differentiation was present in the CM of osteoblasts and osteal macrophages, whereas the levels of IL-1ra and IL-33, two negative regulators of osteoclastogenesis, were significantly higher in osteoblast CM. In contrast, IL-18 concentrations were increased in CM from osteal macrophages, which was considered potentially interesting, because IL-18 does not only inhibit osteoclast differentiation, but also promote osteoblast proliferation, at least in vitro.[5, 22] To analyze whether IL-18 is expressed by osteal macrophages in vivo, we applied immunohistochemistry. Here we observed that IL-18 is primarily produced by F4/80-positive cells located at the endocortical bone surfaces, suggesting predominant expression by osteal macrophages (Fig. 5E).

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Figure 5. Inhibition of osteoclastogenesis by CM from osteoblasts and osteal macrophages. (A) TRAP activity staining of bone marrow cells differentiated in the presence of M-CSF and RANKL for 10 days without CM (control) or with CM from osteoblasts and osteal macrophages. (B) Quantification of the TRAP-positive multinucleated cells differentiated in the presence of osteoblast or osteal macrophage CM, as indicated. Bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences compared to controls. (C) Concentrations of Opg in CM from osteoblasts and osteal macrophages. (D) Concentrations of IL-1 family members in CM from osteoblasts and osteal macrophages. All bars represent mean ± SD (n = 4). Asterisks indicate statistically significant differences between the two cell types. (E) Immunohistochemistry using antibodies against F4/80 (stained in green) and IL-18 (stained in red) demonstrates that cells expressing both antigens (stained in yellow) are primarily located at the endocortical bone surfaces.

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Il-18 reduces osteoclastogenesis in wild-type and Ccl5-deficient bone marrow cultures

We next determined the concentration of the above-listed osteoclastogenesis regulators in conditioned medium of cultured bone marrow cells from wild-type and Ccl5-deficient mice. Here we found no significant differences for Opg, IL-1β, IL-1ra, and IL-33 (Fig. 6A), but importantly IL-18 levels were reduced in Ccl5-deficient cultures (Fig. 6B). That Il18 expression is also affected by Ccl5-deficiency in vivo, was confirmed by qRT-PCR, where we observed significantly lower expression levels in Ccl5-deficient bone marrow. To analyze the possibility that reduced IL-18 production is causative for the increased osteoclastogenesis in Ccl5-deficient mice, we differentiated bone marrow cells from wild-type and Ccl5-deficient mice in the presence of M-CSf and RANKL with or without Ccl5 and/or IL-18. Here we observed that the number of TRAP-positive multinucleated cells was significantly higher in Ccl5-deficient cultures and this difference was unaffected by the addition of Ccl5 (Fig. 6C). In contrast, when IL-18 was present during the course of differentiation, osteoclastogenesis was significantly reduced in wild-type cultures, but even more in Ccl5-deficient cultures, and the difference between the two genotypes was eliminated.

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Figure 6. Decreased IL-18 production in the absence of Ccl5. (A) Concentrations of Opg, IL-1β, IL-1ra, and IL-33 in CM from cultured bone marrow cells of wild-type and Ccl5-deficient mice. (B) Concentrations of IL-18 in the same samples (left) and qRT-PCR for Il18 expression in the bone marrow of wild-type and Ccl5-deficient mice (right). All bars represent mean ± SD (n = 4). Asterisks indicate statistically significant differences between the two genotypes. (C) Quantification of the TRAP-positive multinucleated cells in cultures from wild-type (white bars) and Ccl5-deficient mice (black bars) differentiated in the presence or absence of Ccl5 or Il-18 as indicated. All bars represent mean ± SD (n = 4). Asterisks indicate statistically significant differences compared to wild-type cultures differentiated under control conditions.

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Ccl5 promotes macrophage differentiation in vitro

To analyze if Ccl5 would directly regulate Il18 expression in macrophages or their progenitors, we differentiated bone marrow cells in the presence of M-CSF for 3 or 6 days and then treated the cultures with Ccl5 for 6 hours. As demonstrated by qRT-PCR expression analysis, this short-term administration did not induce or repress Il18 transcription (Fig. 7A). In contrast, when Ccl5 was present during the whole course of differentiation Il18 expression was significantly increased compared to cultures differentiated in the absence of Ccl5, which was also confirmed by ELISA (Fig. 7B). To address the possibility that Ccl5 has a general influence on macrophage differentiation, we further determined the expression of three established macrophage markers and found that they were all increased when the cells were differentiated in the presence of Ccl5 (Fig. 7C). Because others have previously demonstrated that Ccl5 has an antiapoptotic effect for macrophages during viral infection,[23] we further applied a caspase-3 activity assay, but here we did not observe differences between cultures differentiated in the absence or presence of Ccl5 (Fig. 7D). Taken together, these findings suggest that Il18 is not a direct transcriptional target of Ccl5, but that Ccl5 has a positive influence on macrophage differentiation.

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Figure 7. Ccl5 enhances macrophage differentiation in vitro. (A) qRT-PCR for Il18 expression following short-term administration (6 hours) of Ccl5 to bone marrow cells differentiated in the presence of M-CSF for 3 or 6 days as indicated. (B) qRT-PCR for Il18 expression in bone marrow cells differentiated in the presence of M-CSF for 6 days with or without Ccl5 as indicated (left). The IL18 concentration in conditioned media from the same cultures is shown on the right. (C) qRT-PCR for expression of the indicated genes in the same cultures. (D) Caspase-3 activities in bone marrow cells differentiated in the presence of M-CSF for 6 days with or without Ccl5. All bars represent mean ± SD (n = 4). Asterisks indicate statistically significant differences compared to control cultures.

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Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Ccl5, also known as RANTES, belongs to the large family of chemokines, which are primarily known for their role in recruiting various immune cells to sites of inflammation.[24] Although Ccl5 was originally described as a T-cell–specific gene, it is now known to be expressed in a variety of cell types, including eosinophils, macrophages, fibroblasts, renal tubular cells, or endothelial cells.[25] Ccl5 has been shown to act as a chemoattractant for various cell types, such as monocytes, T cells, or eosinophils, and to promote T cell proliferation.[26] With respect to bone remodeling, Ccl5 has been shown to induce chemotaxis of osteoclasts and osteoblasts in vitro, thereby suggesting a critical role in bone remodeling.[12, 27] To define the physiological relevance of Ccl5 in vivo, Ccl5-deficient mice have been generated, and although they do not display an obvious phenotype, they were found less prone to ear and footpad swelling in a cutaneous delayed hypersensitivity assay.[28]

In the present work we have focused on the assessment of the skeletal phenotype in Ccl5-deficient mice. In contrast to wild-type and Ccl2-deficient mice, whose skeletal phenotype has previously been analyzed by others,[11] we observed three major defects at 6 months of age. First, Ccl5-deficient mice display decreased bone formation, especially at endocortical sites, a phenotype diminishing with age, which is possibly explained by functional redundancy with other chemokines. Second, osteoclastogenesis is increased compared to wild-type and Ccl2-deficient mice, which argues against a physiologically relevant role of Ccl5 in osteoclast recruitment. And third, there is a striking increase of the cell-free trabecular and endocortical bone surface in Ccl5-deficient mice, which is a fairly unique skeletal phenotype, although it is transient. Taken together, these data provide in vivo evidence confirming, at least in part, the previously suspected role of Ccl5 in bone remodeling. Moreover, to the best of our knowledge, the impairment of bone remodeling is the only phenotype of Ccl5-deficient mice that arises spontaneously without an immunological trigger.

By focusing on 6-month-old wild-type and Ccl5-deficient mice we made another observation with potential relevance for the field of osteoimmunology. In fact, we observed reduced numbers of F4/80-positive cells in the bone marrow of Ccl5-deficient mice and a partial lack of osteal macrophages, a cell type that has been introduced only recently.[18, 28] More specifically, osteal macrophages have been reported to form a canopy-like structure over cuboidal osteoblasts, especially at endocortical bone surfaces. Importantly, depletion of this cell type in a mouse model of macrophage-specific Fas-induced apoptosis resulted in a simultaneous loss of bone-forming osteoblasts, thereby confirming in vitro data showing that osteal macrophages promote matrix mineralization of calvarial osteoblast cultures.[18] Consistent with these findings we observed that F4/80-positive cells were indeed present in close association with osteocalcin-positive cells at endocortical bone surfaces of wild-type mice, while in the case of Ccl5-deficiency both cell types were depleted, thus resulting in a transient decrease of endosteal bone formation. As such, these observations fully confirm the relevance of osteal macrophages for proper bone formation, and to the best of our knowledge, Ccl5-deficient mice provide the first model with a spontaneous deficiency of this particular cell type, although they only display a transient phenotype. Although we were able to provide in vitro evidence supporting the deduced hypothesis that the transiently decreased bone formation in Ccl5-deficient mice is a consequence of partial osteal macrophage deficiency, we would like to state, however, that this conclusion still needs to be supported in vivo. To do so, it might be useful to transfer wild-type hematopoietic stem cells into Ccl5-deficient mice, but given the transient nature of the Ccl5-deficient phenotype we did not perform such experiments yet.

Because we further observed increased osteoclastogenesis in Ccl5-deficient mice, we finally addressed the question, whether osteal macrophages could differentiate into osteoclasts and/or secrete inhibitors of osteoclast differentiation. For that purpose we isolated osteal macrophages by CD11b immunoaffinity from calvariae of newborn mice, as described by others.[18] Although we could rule out the possibility that these cells can transdifferentiate into functional osteoclasts, we did observe that their CM inhibited osteoclastogenesis of bone marrow progenitor cells to the same extent as CM from osteoblasts. Interestingly, when we determined the CM concentrations of known inhibitors of osteoclastogenesis, we found that Opg, IL1-ra, and IL-33 were only present at low concentration in the CM of osteal macrophages, thus suggesting that other antiosteoclastogenic cytokines are specifically expressed by these cells. One likely candidate for such a function is IL-18, for which we did not only observe higher levels in CM from osteal macrophages as compared to CM of osteoblasts, but also lower expression in Ccl5-deficient bone marrow cells. Moreover, although our findings showing a negative influence of IL-18 on osteoclastogenesis are essentially confirming results from others, it is reasonable to speculate that reduced IL-18 production in Ccl5-deficient mice is causative for their increased osteoclastogenesis phenotype, because the ex vivo difference between wild-type and Ccl5-deficient cultures was normalized by IL-18, and not by Ccl5.

Regardless of these latter findings, however, we truly believe that it was important to characterize the skeletal phenotype of Ccl5-deficient mice, not only because we found Ccl5 expression induced by noncanonical Wnt signaling in osteoblasts, but even more, because there was in vitro evidence for a role of Ccl5 in bone remodeling, which has not been addressed in vivo so far.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

This study was supported by the Focus Program SPP1468 “Osteoimmunology – IMMUNOBONE – A program to unravel the mutual interactions between the immune system and bone” of the Deutsche Forschungsgemeinschaft.

Authors' roles: Study design: KW, JA, GT, MA, and TS; Study conduct: KW, FTB, JA, AJ, MS, and BC; Data analysis: KW, JA, GT, MA, and TS; Drafting manuscript: KW, JA, MA, and TS; Revising manuscript: KW, JA, MA, and TS.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
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