OCL formation in cocultures of mouse bone marrow and osteoblastic cells in the presence of IL-18 (A) or IFN-γ (B). Mouse bone marrow and primary osteoblastic cells were cocultured with 1α,25(OH)₂ D₃ (10⁻⁸ M) and PGE₂ (10⁻⁷ M) in the presence of increasing concentrations of IL-18 (A) or IFN-γ (B). For negative and positive controls, cocultures were performed in the absence and presence of 1α,25(OH)₂ D₃ and PGE₂, respectively. After culture for 7 d, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures, and are representative of three similar experiments.

Effect of IL-18 and IFN-γ on OCL formation in cocultures of mouse bone marrow and osteoblastic cells during the coculture period in the absence and presence of 1α,25(OH)₂ D₃ (10⁻⁸ M) and PGE₂ (10⁻⁷ M). IL-18 (10 ng/ml) and IFN-γ (50 U/ml) were present over the entire culture period (days 0–6) or during the first 3 d (0–3) or the last 3 d (3–6). Media change occurred at day 3 of the culture. After culture for 6 d, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. This experiment was repeated on two further occasions with similar results.

OCL formation in cocultures of spleen cells and osteoblastic cells derived from IFN-γ receptor type II knockout (IFN-γ R^−/−) mice. IL-18 (10 ng/ml) or IFN-γ (50 U/ml) were present over the entire culture period. For negative and positive controls, cocultures were performed in the absence and presence of 1α,25(OH)₂ D₃ (10⁻⁸ M) and PGE₂ (10⁻⁷ M), respectively. After culture for 7 d, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. This experiment was repeated twice.

Effect of neutralizing antibodies against GM–CSF to rescue OCL formation in cocultures of mouse bone marrow and osteoblastic cells treated with GM–CSF (0.1 ng/ml), IL-18 (10 ng/ml), or IFN-γ (50 U/ml). Cocultures were incubated in the presence or absence of GM– CSF, IL-18, or IFN-γ and the effect of neutralizing antibodies to GM– CSF (1 μg/ml) were determined. For negative and positive controls, cocultures were performed in the absence and presence of 1α,25(OH)₂ D₃ (10⁻⁸ M) and PGE₂ (10⁻⁷ M), respectively. After culture for 7 d, TRAPpositive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. Similar results were obtained with three repeat experiments.

Identification of IL-18. (A) An example of a ddPCR gel. Lanes correspond to RNA from the different sources: (lane 1) hydrocortisone-treated tsJ10 cells, (lane 2) hydrocortisone-treated tsJ14 cells, (lane 3) 1α,25(OH)₂ D₃- and PGE₂treated tsJ2 cells, and (lane 4) 1α,25(OH)₂ D₃- and PGE₂treated tsJ14 cells. The PCR fragment identified as IL-18 is indicated by the arrow on the left. Indicated by the arrow on the right is a PCR fragment corresponding to a hitherto uncharacterized mRNA species, which is expressed in greater abundance in the OCL-supportive cell lines. The osteoclast-supporting activity (OSA) of these cell lines is indicated below the gel: plus (supportive) or minus (nonsupportive). (B) Nucleotide sequence of mouse IL-18 (GenBank^TM accession number D49949). The region corresponding to the differentially expressed PCR fragment isolated from (A) is between nucleotides 636–830. Sequences underlined correspond to oligonucleotides specific to IL-18 used for RT-PCR analysis and detection of RT-PCR products (IL-18-1, IL-18-3, and IL-18-2 from 5′ to 3′). Nucleotides in capitals corrrespond to the coding region of IL-18, whereas those in lower case correspond to the 5′ and 3′ untranslated sequences. (C) Semiquantitative RT-PCR analysis of IL-18 mRNA. PCR products for RNA isolated from different sources was reversed transcribed with oligo (dT) and PCR performed with the primers IL-18-1 and IL-18-2 for 23 cycles, which was in the log-linear range of amplification. Lanes correspond to RNA from (1) hydrocortisone-treated tsJ10 cells, (2) hydrocortisone-treated tsJ14 cells, (3) 1α,25(OH)₂ D₃- and PGE₂-treated tsJ2 cells, and (4) 1α,25(OH)₂ D₃- and PGE₂-treated tsJ14 cells. Resultant PCR products were electrophoresed, transferred to a nylon membrane, and hybridized with a γ-³²P–labeled internal detection oligonuleotide for IL-18 (IL-18-3). Similar amplifications for GAPDH with GAPDH-2 and GAPDH-4 for 20 cycles were performed and products detected with γ-³²P–labeled GAPDH-1 as previously described (30). The osteoclast-supporting activity (OSA) of these cell lines is indicated below the gel: plus (supportive) or minus (nonsupportive).

Effect of IL-18 (10 ng/ml) on OCL formation in cocultures of mouse bone marrow and osteoblastic cells in the presence of 1α,25(OH)₂ D₃ (10⁻⁸ M), PGE₂ (10⁻⁷ M), PTH (200 ng/ml), IL-11 (20 ng/ml), and IL-1 (100 ng/ml). After culture for 7 d, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures and are representative of three similar experiments.

Effect of neutralizing antibodies against IL-18 or IFN-γ in rescuing OCL formation in cocultures of mouse bone marrow and osteoblastic cells treated with IL-18 or IFN-γ. Cocultures were incubated in the presence or absence of IL-18 (10 ng/ml) and IFN-γ (50 U/ml) and the effect of antibodies against IL-18 (A) or IFN-γ (B) were determined. For negative and positive controls, cocultures were performed in the absence and presence of 1α,25(OH)₂ D₃ (10⁻⁸ M) and PGE₂ (10⁻⁷ M), respectively. After culture for 7 d, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. This experiment was repeated twice.

OCL formation in cocultures of normal C57/BL6 mousederived spleen cells with osteoblastic cells derived from IFN-γ R^−/− mice and cocultures of normal C57BL/J6 mouse-derived osteoblastic cells with spleen cells derived from IFN-γ R^−/− mice. Cocultures were performed in the presence of 1α,25(OH)₂ D₃ (10⁻⁸ M) and PGE₂ (10⁻⁷ M) and treated with IL-18 (10 ng/ml) or IFN-γ (50 U/ml). For negative and positive controls, cocultures were performed in the absence and presence of 1α,25(OH)₂ D₃ and PGE₂, respectively. After culture for 7 d, TRAPpositive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. This experiment was repeated twice.