Peripheral blood monocytes from healthy donors were collected using Ficoll-Conray (Imuuno-Biological Laboratories, Gunma, Japan) gradient centrifugation. Negative selection of monocytes was performed using MACS microbeads (Miltenyi Biotec, Auburn, CA, USA) according to the protocol supplied by the manufacturer.

The purified monocytes were separated into two subsets, CD16⁺ and CD16^- monocytes, using CD16 MicroBeads (Miltenyi Biotec). Flow cytometry analysis using FITC-conjugated mouse anti-CD14 mAb (MY4; Bechman Coulter, Fullerton, CA, USA) and phycoerythin-conjugated mouse anti-CD16 mAb (3G8; BD Biosciences, San Jose, CA, USA) showed that the purities of the CD16⁺ and CD16^- monocytes were more than 90% and 92%, respectively.

For the other experiment, monocytes were purified using CD14 MicroBeads (Miltenyi Biotec), and then stained either with FITC-conjugated mouse anti-CD33 mAb (MY9; Bechman Coulter) or phycoerythin-conjugated mouse anti-CD16 mAb (3G8). Cell sorting of the stained cells was performed using a FACS Vantage cytometer (BD Biosciences) or a MoFlo cell sorter (Dako, Glostrup, Denmark).

Purified CD16⁺ and CD16^- monocytes (5 × 10⁴ cells/well) were incubated in 96-well plates in αMEM (Sigma, St Louis, MO, USA) with heat-inactivated 10% fetal bovine serum (FBS) (Sigma) or with Ultra-Low IgG FBS (IgG < 5 μg/ml; Invitrogen, Carlsbad, CA, USA), and where indicated with M-CSF + RANKL (Peprotech, Rocky Hill, NJ, USA).

For the other experiments, varied numbers of CD16⁺ monocytes (1 × 10³, 2.5 × 10³, 5 × 10³) were mixed with CD16^- monocytes (5 × 10⁴ cells/well), and were cultured in 96-well plates in αMEM with heat-inactivated 10% FBS. The medium was replaced with fresh medium 3 days later, and after incubation for 7 days the cells were stained for tartrate-resistant acid phosphatase (TRAP) expression using a commercial kit (Hokudo, Sapporo, Japan). The number of TRAP-positive multinucleated cells (MNC) in three randomly selected fields examined at 100× magnification or the total number of TRAP-positive MNC per well was counted under light microscopy.

Monocytes were seeded onto plates coated with calcium phosphate thin films (Biocoat Osteologic; BD Biosciences) and were incubated with RANKL (40 ng/ml) + M-CSF (25 ng/ml) for 7 days. The cells were then lysed in bleach solution (6% NaOCl, 5.2% NaCl). The resorption lacunae were examined under light microscopy.

Purified monocytes were cultured in 96-well plates where indicated either with RANKL or M-CSF for 24 hours. Concentrations of TNFα and IL-6 in the culture supernatant were measured with an ELISA kit (BioSourse International, Camarillo, CA, USA). For experiments of matrix metalloproteinase (MMP)-9 and TRAP-5b, culture supernatants were collected on day 7 and the concentrations of these enzymes were measured using an MMP-9 ELISA kit (Amersham Biosciences, Piscataway, NJ, USA) or a TRAP-5b ELISA kit (Suomen, Turku, Finland).

Monocytes (1 × 10⁶ cells/well) were cultured in six-well plates with M-CSF alone or with M-CSF + RANKL for 3 days. Total RNA was extracted using RNeasy Micro (Qiagen, Valencia, CA, USA). The RNA was then treated with DNase I (Qiagen). The oligo(dT)-primed cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). The amount of cDNA for amplification was adjusted by the amount of RNA measured by an optical density meter and also by β-actin or GAPDH PCR products. One microliter of cDNA was amplified in a 50 μl final volume containing 25 pmol appropriate primer pair, 10 pmol each of the four deoxynucleotide triphosphates, and 5 units FastStart Taq DNA Polymerase (Roche, Manheim, Germany) in a thermal cycler (PTC-200; MJ GeneWorks, Waltham, MA, USA).

The PCR conditions were 25–40 cycles of denaturation (95°C for 30 s), annealing (60–62°C for 1 min) and extension (72°C for 1 min). The sequences of the primers are presented in Table 1. The PCR products were separated by electrophoresis through 2% agarose gel.

Purified monocytes were cultured for 3 days in the presence of 40 ng/ml M-CSF with or without 25 ng/ml RANKL. Cells were lysed in RIPA Lysis buffer (upstate, Lake Placid, NY, USA) containing protease inhibitors (Roche) for 15 min at 4°C. A total of 20 μg protein was boiled in the presence of 6 × sodium dodecyl sulfate sample buffer, and was separated on 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gel (ATTO, Tokyo, Japan). Proteins were then electrotransferred to a polyvinylidene fluoride microporous membrane (Millipore, Billerica, MA, USA) in a semidry system. Membranes were incubated in 10% skim milk prepared in phosphate-buffered saline (PBS) containing 0.1% Tween 20, and were subjected to immunoblotting. Antibodies used were goat anti-RANK antibody (Techne Corporation, Minneapolis, MN, USA), goat anti-c-fms antibody (R&D systems, Minneapolis, MN, USA), and mouse anti-β-actin mAb (AC-15; Sigma). Peroxidase-conjugated rabbit anti-goat IgG antibody (Dako) or peroxidase-conjugated rabbit anti-mouse IgG antibody (Dako) was used as the second antibody. The signals were visualized by chemiluminescence reagent (ECL; Amersham Biocsiences, Little Chalfont, UK).

The following mAbs were used for analysis of c-fms expression: Alexa 647-conjugated anti-CD14 mAb (UCHM1; Serotec, Oxford, UK), FITC-conjugated anti-CD16 mAb (3G8; Bechman Coulter) and phycoerythin-conjugated anti-c-fms mAb (61708; R&D systems). Alexa 647-conjugated mouse IgG2a (Serotec), FITC-conjugated mouse IgG₁ (BD Biosciences) and phycoerythin-conjugated mouse IgG₁ (Bechman Coulter) were used as isotype controls. Peripheral blood monocytes (1 × 10⁵ cells) were incubated with 1 μg human IgG for 15 minutes, and were then stained with three fluorochrome-labeled mAbs for 45 minutes on ice. The stained cells were analyzed with a FACS Calibur (BD Biosciences).

Monocytes (8 × 10⁴ cells/well) were allowed to adhere on 96-well plates overnight or were cultured with M-CSF and RANKL for 2–4 days. The cells were fixed in acetone and then stained with anti-αvβ3 mAb (LM609; Chemicon, Temecula, CA, USA) or mouse IgG₁ (11711; R&D Systems) as an isotype-matched control. Alexa fluor546-conjugated goat anti-mouse IgG₁ antibody (Molecular Probes, Eugene, OR, USA) was used as the second antibody. TOTO-3 (Molecular Probes) was used for nuclear staining.

Purified monocytes were cultured in the presence of 25 ng/ml M-CSF for 3 days, and were either left unstimulated or were stimulated with 40 ng/ml RANKL at 37°C. Stimulations were stopped by adding an equal volume of PhosFlow Fix Buffer I solution (BD Biosciences) to the cell culture. After incubation for 10 minutes at 37°C, the cells were permeabilized by washing twice at room temperature in PhosFlow Perm/Wash Buffer I (BD Biosciences). A total of 1 × 10⁵ cells was then Fc blocked with 1 μg human IgG for 15 minutes, and was stained with Alexa Fluor 647-conjugated mAb either to phospho-p38 MAPK (T180/Y182) or to phospho-ERK1/2 (T202/Y204) (BD Biosciences) for 30 minutes at room temperature. Alexa Fluor 647-conjugated mouse IgG₁ (BD Biosciences) was used as an isotype control. The cells were washed in PhosFlow Perm/Wash Buffer I, and were analyzed by flow cytometry, as already described.

RNA oligonucleotides (iGENE, Tsukuba, Japan) were designed based on the algorithm that incorporates single nucleotide polymorphism and homology screening to ensure a target-specific RNA interference effect. The following sense and antisense oligonucleotides were used: integrin β3, 5'-GCU UCA AUG AGG AAG UGA AGA AGC A-AG and 3'-UA-CGA AGU UAC UCC UUC ACU UCU UCG U; randomized control, 5'-CGA UUC GCU AGA CCG GCU UCA UUG C-AG and 3'-UA-GCU AAG CGA UCU GGC CGA AGU AAC G; and lamin, 5'-GAG GAA CUG GAC UUC CAG AAG AAC A-AG and 3'-UA-CUC CUU GAC CUG AAG GUC UUC UUG U.

CD16^- monocytes (8 × 10⁴ cells/well) were incubated in 96-well plates in optimem (Invitrogen). After 1 hour, siRNAs were transfected into the cells using oligofectamine (Qiagen) based on the method recommended by the manufacturer. After 2 hours, the cells were washed once with PBS, followed by the addition of αMEM supplemented with 10% FBS, M-CSF and RANKL. After a 2-day incubation, the β3 mRNA expression was analyzed by RT-PCR with different PCR cycles, as described earlier. Immunofluorescent staining for the αvβ3 heterodimer was also performed as described above, and numbers of αvβ3-positive cells were counted in randomly selected three fields at 100× magnification. Seven days after the transfection of siRNAs, the number of TRAP-positive MNC in five fields examined at 100× magnification was counted under light microscopy.

CD16^- monocytes were incubated in 96-well plates with M-CSF + RANKL for 2 days. A medium containing either cyclic RGDfV peptide (Arg-Gly-Asp-D-Phe-Val) (Calbiochem, San Diego, CA, USA) or dimethyl sulfoxide was then added. After incubation for a further 5 days, the number of TRAP-positive MNC in five fields examined at 100× magnification was counted under light microscopy.

Synovial tissue samples were obtained during total knee joint replacement surgery from four RA patients. Signed consent forms were obtained before the operation. The experimental protocol was approved by the ethics committee of the Tokyo Medical and Dental University. RA was diagnosed according to the American College of Rheumatology criteria 14.

Double immunofluorescent staining for CD68 and CD16 antigens was conducted on optimal cutting temperature-embedded sections of frozen synovial samples. Eight-micrometer-thick cryostat sections of RA synovium were fixed in acetone for 3 minutes and were then rehydrated in PBS for 5 minutes. The samples were incubated in 5 μg/ml proteinase K (Roche), 50 mM ethylenediamine tetraacetic acid, 100 mM Tris–HCl, pH 8.0, for 15 minutes at room temperature followed by a wash in PBS. The samples were then blocked with 10% goat serum in PBS for 60 minutes at room temperature, and were incubated with anti-CD16 mAb (3G8; Immunotech, Marseille, France) or mouse IgG₁ (11711) as an isotype-matched control in 1% bovine serum albumin/PBS for 60 minutes at room temperature. The samples were then washed three times in PBS, for 5 minutes each, and incubated with Alexa fluor546-conjugated goat anti-mouse IgG₁ antibody (Molecular Probes) in 1% bovine serum albumin/PBS for 60 minutes at room temperature. The samples were then sequentially stained for CD68 antigen in a manner similar to that used for CD16 staining. The samples were stained with anti-CD68 mAb (PGM1; Immunotech) or mouse IgG₃ (6A3; MBL, Nagoya, Japan) followed by labeling with Alexa fluor488-conjugated goat anti-mouse IgG₃ antibody (Molecular Probes). The samples were examined by confocal laser scanning microscope (Olympus, Tokyo, Japan).

Data are expressed as the mean ± standard error of the mean. A nonpaired Student's t test was used for comparison, using the StatView program (Abacus Concepts, Berkeley, CA, USA). P < 0.05 was considered statistically significant.

To identify the monocyte subset that differentiates into osteoclasts, we examined osteoclast formation from CD16⁺ and CD16^- human peripheral blood monocytes. The monocyte subsets were purified using magnetic beads. Incubation with M-CSF alone did not induce osteoclast formation from either subset (Figure 1a). Culture with M-CSF + RANKL induced a significant number of TRAP-positive MNC from the CD16^- subset, whereas only few CD16⁺ monocytes differentiated into TRAP-positive MNC (Figure 1a,b). We then assessed the bone resorptive ability by culturing cells on calcium phosphate-coated plates with M-CSF + RANKL. Resorption lacunae were detected only in the CD16^- subset (Figure 1c), indicating the TRAP-positive CD16^--derived MNC possessed the osteoclast phenotype.

Similar results were obtained using purified monocytes by FACS sorting (purities: CD16⁺, 96%; CD16^-, 97%). The number of TRAP-positive MNC induced were 36 ± 3 cells/well and 348 ± 13 cells/well from CD16⁺ and CD16^- monocytes, respectively. To exclude the possibility that the anti-CD16 antibody used for isolation of CD16⁺ monocytes inhibits osteoclast formation, we separated the two subsets, CD33^low monocytes and CD33^high monocytes, using anti-CD33 mAb and a fluorescent cell sorter, since it was reported that CD33^low monocytes correspond to CD16⁺, and that CD33^high correspond to CD16^- monocytes 15. On average, the CD33^low population contained CD16^- (10.2%)/CD16⁺ (89.8%) monocytes, and the CD33^high population contained CD16^- (86.3%)/CD16⁺ (13.7%) monocytes.

Culture with M-CSF + RANKL induced TRAP-positive MNC from CD33^high monocytes, whereas no or few CD33^low monocytes differentiated into TRAP-positive MNC (CD33^low vs CD33^high, 2 ± 1 vs 192 ± 71 cells/well; n = 3). TRAP-5b and MMP-9 in the culture supernatants, both of which are known to be produced by osteoclasts, were measured by ELISA. The concentrations of both enzymes were significantly higher in the culture supernatant of CD16^- monocytes than in that of CD16⁺ monocytes (Figure 1d). These results suggest that the CD16^- peripheral blood monocyte subset, but not the CD16⁺ subset, differentiate into osteoclasts by incubation with RANKL + M-CSF.

To examine whether CD16⁺ monocytes affect osteoclastogenesis from CD16^- monocytes, varied numbers of CD16⁺ monocytes were mixed with CD16^- monocytes (5 × 10⁴ cells/well), and were cultured for 7 days in the presence of M-CSF + RANKL. The number of TRAP-positive MNC was not altered by the presence of CD16⁺ monocytes (Figure 2). The results indicated that CD16⁺ monocytes did not hamper or enhance the osteoclastogenesis from CD16^- monocytes.

To compare the biological response of CD16⁺ and CD16^- subsets with either RANKL or M-CSF stimulation, we measured the amount of TNFα and IL-6 production after exposure of cells to various concentrations of RANKL or M-CSF with an ELISA. Without RANKL the CD16⁺ subset produced a significant amount of TNFα and IL-6, whereas the CD16^- subset produced undetectable levels (Figure 3a). RANKL stimulation increased TNFα production from both subsets in a dose-dependent manner, although the CD16⁺ subset produced more TNFα than did the CD16^- subset. RANKL stimulation also enhanced IL-6 production from the CD16⁺ subset, but not from the CD16^- subset. M-CSF stimulation increased TNFα and IL-6 production from both subsets, although the CD16⁺ subset produced more than the CD16^- subset (Figure 3b).

These results suggest that CD16⁺ monocytes also respond both to RANKL and M-CSF stimulation, although such stimulation does not result in differentiation into osteoclasts. CD16⁺ monocytes were also noted to express higher amounts of inflammatory cytokines compared with CD16^- monocytes with or without RANKL or M-CSF stimulation.

Diverse molecules are involved in RANKL/RANK and its costimulatory signal transduction pathways 16. The different response to RANKL + M-CSF stimulation between the CD16⁺ monocyte subset and the CD16^- monocytes subset might be explained by the expression profiles of these molecules. We therefore examined the mRNA levels of the following molecules: receptor activator of NF-κB (RANK), the receptor for RANKL; c-fms, the receptor for M-CSF; tumor necrosis factor receptor-associated factor 6 (TRAF-6), the adaptor protein for RANK; c-Fos and nuclear factor of activated T cells c1 (NFATc1), transcription factors that are essential for osteoclastogenesis; DNAX-activation protein 12 (DAP12) and Fc receptor γ chain (FcRγ), adaptor proteins known to deliver costimulatory signals in RANKL-induced osteoclastogenesis; signal regulatory protein β1 (SIRP-β1), triggering receptor expressed on myeloid cells 2 (TREM-2) and osteoclast-associated receptor (OSCAR), transmembrane receptors that associate with either DAP12 or FcRγ; and αv and β3, integrins known to be expressed as the αvβ3 heterodimer on osteoclasts.

The mRNA levels of RANK, c-fms, TRAF-6, DAP12 and SIRP-β1 under the baseline condition (no stimulation) varied between the donors; however, we did not find consistent differences in the mRNA levels of these molecules between the CD16⁺ monocyte subset and the CD16^- monocyte subset among three to six donors (Figure 4a). The mRNA levels of other molecules, apart from integrin β3, were similar between the two subsets under the no-stimulation condition. Although the mRNA levels of RANK, c-fms, DAP12, FcRγ, TREM-2 and OSCAR increased in response to M-CSF alone or M-CSF + RANKL in both subsets, the expression levels were not significantly different between the two subsets. Expressions of TRAF-6, c-Fos and SIRP-β1 mRNA did not change following stimulation with M-CSF + RANKL. Of note, the expression of NFATc1 mRNA was enhanced by M-CSF + RANKL treatment only in the CD16^- subset. Furthermore, expression of integrin αv in both subsets was enhanced by M-CSF with or without RANKL; however, the expression level was greater in the CD16^- subset. It was noted that integrin-β3 mRNA was detected only in the CD16^- subset and was increased by M-CSF + RANKL stimulation, but not by M-CSF alone. The protein expression of RANK under the baseline condition was weakly detected in both subsets, and the levels were varied between donors by western immunoblotting.

The protein expression of c-fms was weakly detected in unstimulated CD16⁺ monocytes, but not in CD16^- monocytes (Figure 4b). Flow cytometry analysis of c-fms in fresh monocytes, however, showed that both subsets express the molecule on the cell surface (Figure 4c). Expressions of both RANK and c-fms were upregulated by M-CSF alone and by M-CSF + RANKL, and we did not find consistent differences in the protein levels of these molecules between the two monocyte subsets. The profiles of expression levels of molecules involved in RANKL/RANK and its costimulatory pathways are similar between the two subsets, except for NFATc1, integrin αv and integrin β3. We therefore assumed that the distinct induction of NFATc1, integrin αv and integrin β3 in response to RANKL stimulation among the two monocyte subsets might explain the differences in their abilities to differentiate into osteoclasts.

The integrin-β3 subunit binds to integrin αv only and is expressed as the heterodimeric protein αvβ3 on monocytes and osteoclasts 17. We examined the expression of αvβ3 on CD16⁺ and CD16^- monocytes by immunofluorescent staining. Neither unstimulated nor M-CSF-stimulated monocyte subsets expressed αvβ3 (Figure 4d and data not shown). After 48 and 72 hours of treatment with M-CSF + RANKL, αvβ3-positive mononuclear cells were observed in CD16^- monocyte cultures but not in CD16⁺ monocyte cultures. At 96 hours, both αvβ3-positive mononuclear cells and multinucleated cells were present in the CD16^- monocyte culture. The results indicated that αvβ3 was selectively expressed on CD16^- monocytes in the presence of M-CSF + RANKL, and the expression was revealed before the cells differentiate into typical multinucleated osteoclasts.

Since ERK and p38 MAPK are essential in RANKL-induced osteoclastogenesis 181920, we next examined whether these kinases were activated differently in CD16⁺ monocytes and in CD16^- monocytes. Purified monocytes were precultured with 25 ng/ml M-CSF for 3 days to enhance RANK expression, and were then treated with RANKL. The RANKL treatment induced phosphorylation of both ERK and p38 MAPK in CD16^- monocytes at 5 minutes postexposure, although the p38 MAPK phosphorylation was weak. Both phosphorylations declined to a basal level within 20 minutes (Figure 5). In contrast, ERK and p38 MAPK were not detectably phosphorylated in CD16⁺ monocytes with RANKL.

The integrin-β3 cytoplasmic domain is essential for activation of intracellular signals from αvβ3 heterodimers 17. We therefore examined the involvement of αvβ3 in RANKL + M-CSF-induced osteoclastogenesis in human CD16^- monocytes using siRNA targeting the integrin-β3 subunit. The integrin-β3 siRNA or control randomized siRNA were transfected into CD16^- monocytes. At 48 hours post-transfection, we determined the integrin-β3 mRNA level and αvβ3 heterodimer protein expression. The integrin-β3 mRNA level was reduced in the integrin-β3 siRNA-transfected monocytes compared with control siRNA-transfected monocytes (Figure 6a). The αvβ3 heterodimer expression was evaluated by immunofluorescent staining. The number of αvβ3-positive cells was significantly decreased in integrin-β3 siRNA-transfected monocytes compared with that in control siRNA (Figure 6b).

After 7 days of incubation, the number of TRAP-positive MNC was counted. Transfection with integrin-β3 siRNA significantly reduced the number of TRAP-positive MNC in a dose-dependent manner compared with control siRNA transfection (Figure 6c). In addition, the use of siRNA directed toward a different site of integrin-β3 mRNA also inhibited osteoclast formation from CD16^- monocytes (data not shown). On the other hand, siRNA that targeted lamin, which was used as a negative control, did not inhibit the induction of osteoclasts (data not shown). These results indicate the importance of integrin β3 in RANKL-induced osteoclast formation from CD16^- peripheral blood monocytes.

Integrin αvβ3 recognizes a common tripeptide sequence, RGD (Arg-Gly-Asp), which is present in bone matrix proteins such as vitronectin and fibronectin 21. Cyclic RGDfV peptide (Arg-Gly-Asp-D-Phe-Val) inhibits binding of the RGD-containing molecules to αvβ3 22. To investigate the role of ligand binding to the αvβ3 heterodimer in the osteoclastogenesis, we examined whether cyclic RGDfV peptide inhibits the formation of osteoclasts. Cyclic RGDfV peptide significantly reduced the number of TRAP-positive MNC in a dose-dependent manner (Figure 6d). The results imply possible involvement of ligand bindings to αvβ3 in the osteoclastogenesis.

In the next step, we determined whether integrin-β3-siRNA-induced inhibition of the osteoclastogenesis reflects downregulation of NFATc1, which is a key transcription factor in osteoclastogenesis 23. For this purpose, we compared NFATc1 mRNA levels between integrin β3 and control siRNA-transfected monocytes. Interestingly, integrin-β3 knockdown did not alter the NFATc1 mRNA level (Figure 7), suggesting that signal transduction mediated by integrin β3 does not affect the expression of NFATc1.

RA synovial macrophages are derived from peripheral blood monocytes, and their recruitment into the synovium is facilitated by various adhesion molecules and chemokines 24. To analyze CD16 expression on synovial macrophages, RA synovial tissues were double-stained for CD16 and a macrophage marker, CD68. CD16^-/CD68⁺ macrophages were widespread in the synovium. Although less frequent, CD16⁺/CD68⁺ macrophages were also observed both in the synovial intima and subintima (Figure 8). The presence of two subsets of macrophages, CD16⁺ and CD16^-, in RA synovium indicates that both CD16⁺ and CD16^- peripheral blood monocytes are recruited into the synovium.