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Cited by 378Bone Metabolism and DiseasesRheumatoid Arthritis Research and TherapiesOsteomyelitis and Bone Disorders Research

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Interleukin-18 (Interferon-γ–inducing Factor) Is Produced by Osteoblasts and Acts Via Granulocyte/Macrophage Colony-stimulating Factor and Not Via Interferon-γ to Inhibit Osteoclast Formation

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Udagawa, Nobuyuki, Horwood, Nicole J., Elliott, Jan, Mackay, Alan, Owens, Jane, Okamura, Haruki, Kurimoto, Masashi, Chambers, Timothy J., Martin, T.  John, Gillespie, Matthew T.·1997·The Journal of Experimental Medicine
DOI 10.1084/jem.185.6.1005PMC PMC2196233
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Abstract

We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1α,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-γ (IFN-γ) and granulocyte/macrophage colony-stimulating factor (GM–CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM–CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-γ did not. In cocultures with osteoblasts and spleen cells from IFN-γ receptor type II–deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-γ production: IFN-γ had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-γ inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM–CSF production and not via IFN-γ production.

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Figures

Figure 1 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 (GenBankTM 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).
Figure 1 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 (GenBankTM 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).CC BY-NC-SA · PubMed Central
Figure 2 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.
Figure 2 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.CC BY-NC-SA · PubMed Central
Figure 3 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.
Figure 3 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.CC BY-NC-SA · PubMed Central
Figure 4 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.
Figure 4 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.CC BY-NC-SA · PubMed Central
Figure 5 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.
Figure 5 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.CC BY-NC-SA · PubMed Central
Figure 6 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.
Figure 6 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.CC BY-NC-SA · PubMed Central
Figure 7 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.
Figure 7 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.CC BY-NC-SA · PubMed Central
Figure 8 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.
Figure 8 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.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Udagawa, N., Horwood, N. J., Elliott, J., Mackay, A., Owens, J., Okamura, H., Kurimoto, M., Chambers, T. J., Martin, T. J., & Gillespie, M. T.
Affiliations
Hyogo Medical University (JP)
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
1997-03-17
Volume
185
Issue
6
Pages
1005-1012
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.185.6.1005
PMC
PMC2196233
OpenAlex
W2119656862
Cited by
378
References
35
Citations by year
2026: 1 · 2025: 2 · 2024: 5 · 2023: 7 · 2022: 2 · 2021: 6 · 2020: 7 · 2019: 3 · 2018: 3 · 2017: 4 · 2016: 7 · 2015: 3 · 2014: 10 · 2013: 9 · 2012: 13
FWCI
9.02
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • St George's Online Research Archive (St George's University of London) (acceptedVersion)
  • Minerva Access (University of Melbourne) (submittedVersion, cc-by-nc-sa)
  • UEA Digital Repository (University of East Anglia) (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Health Sciences › Medicine › Rheumatology › Rheumatoid Arthritis Research and Therapies
  • Health Sciences › Medicine › Rheumatology › Osteomyelitis and Bone Disorders Research
Keywords
IL-18, osteoblasts, osteoclastogenesis, osteoclast-like multinucleated cells, GM-CSF, interferon-gamma, bone remodeling, osteoclastogenesis inhibition
MeSH terms
Interferon gamma Receptor, Animals, Animals, Newborn, Base Sequence, Bone Marrow Cells, Cell Differentiation, Interferon-gamma, Male, Mice, Inbred C57BL, Molecular Sequence Data, Osteoblasts, Osteoclasts, Recombinant Proteins, RNA, Messenger, Transcription, Genetic, Polymerase Chain Reaction, Granulocyte-Macrophage Colony-Stimulating Factor, Cytokines, Interleukin-11, Receptors, Interferon, Mice, Knockout, Coculture Techniques, Interleukin-18, Mice

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