Limits...
Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation.

Udagawa N, Horwood NJ, Elliott J, Mackay A, Owens J, Okamura H, Kurimoto M, Chambers TJ, Martin TJ, Gillespie MT - J. Exp. Med. (1997)

Bottom Line: 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.Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not.The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.

View Article: PubMed Central - PubMed

Affiliation: St. Vincent's Institute of Medical Research and The University of Melbourne, Department of Medicine, Victoria, Australia.

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 1alpha,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-gamma (IFN-gamma) 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-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma 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-gamma 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-gamma production.

Show MeSH
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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196233&req=5

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).

Mentions: Total cellular RNA was extracted from cell lines or mouse tissues using guanidine thiocyanate–phenol chloroform and used for reverse transcriptase PCR (RTPCR) essentially as described (27, 28). Differential display PCR (ddPCR) was performed essentially as described (21, 28), except 1 μg of total RNA was reverse transcribed. PCR products were cloned into pCRScript II (Stratagene, La Jolla, CA) or pGEM-T (Promega, Madison, WI). DNA sequence analysis was performed using a T7 sequencingTM kit (Pharmacia Biotech, Uppsala, Sweden). Oligonucleotides were synthesized on an Oligo 1,000M DNA Synthesizer (Beckman Instruments, Inc., Fullerton, CA). The oligonucleotides were the following: for ddPCR, DDMR-2 (5′-CTTGATTGCC-3′) and T12VA (5′-TTTTTTTTTTTT [A,C,G]A-3′); for IL-18 amplification, IL-18-1 (sense strand oligonucleotide 5′-ACTGTACAACCGCAGTAATACGG-3′, nucleotides 286–308 Fig. 1 B) and IL-18-2 (antisense strand oligonucleotide 5′-GGGTATTCTGTTATGGAAATACAGG-3′, nucleotides 804–828; Fig. 1 B), and IL-18-3 (sense strand oligonucleotide 5′-TTGCCAAAAGGAAGATGATG-3′, nucleotides 641–660; Fig. 1 B) served as internal oligonucleotide probe for hybridization studies as described (27, 28); mouse glyceraldehyde3-phosphate dehydrogenase (GAPDH) primers were GAPDH-1, GAPDH-2 (5′-ATGAGGTCCACCACCCTGTT-3′, nucleotides 640–659; 29), and GAPDH-4 as described (30).


Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation.

Udagawa N, Horwood NJ, Elliott J, Mackay A, Owens J, Okamura H, Kurimoto M, Chambers TJ, Martin TJ, Gillespie MT - J. Exp. Med. (1997)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196233&req=5

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).
Mentions: Total cellular RNA was extracted from cell lines or mouse tissues using guanidine thiocyanate–phenol chloroform and used for reverse transcriptase PCR (RTPCR) essentially as described (27, 28). Differential display PCR (ddPCR) was performed essentially as described (21, 28), except 1 μg of total RNA was reverse transcribed. PCR products were cloned into pCRScript II (Stratagene, La Jolla, CA) or pGEM-T (Promega, Madison, WI). DNA sequence analysis was performed using a T7 sequencingTM kit (Pharmacia Biotech, Uppsala, Sweden). Oligonucleotides were synthesized on an Oligo 1,000M DNA Synthesizer (Beckman Instruments, Inc., Fullerton, CA). The oligonucleotides were the following: for ddPCR, DDMR-2 (5′-CTTGATTGCC-3′) and T12VA (5′-TTTTTTTTTTTT [A,C,G]A-3′); for IL-18 amplification, IL-18-1 (sense strand oligonucleotide 5′-ACTGTACAACCGCAGTAATACGG-3′, nucleotides 286–308 Fig. 1 B) and IL-18-2 (antisense strand oligonucleotide 5′-GGGTATTCTGTTATGGAAATACAGG-3′, nucleotides 804–828; Fig. 1 B), and IL-18-3 (sense strand oligonucleotide 5′-TTGCCAAAAGGAAGATGATG-3′, nucleotides 641–660; Fig. 1 B) served as internal oligonucleotide probe for hybridization studies as described (27, 28); mouse glyceraldehyde3-phosphate dehydrogenase (GAPDH) primers were GAPDH-1, GAPDH-2 (5′-ATGAGGTCCACCACCCTGTT-3′, nucleotides 640–659; 29), and GAPDH-4 as described (30).

Bottom Line: 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.Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not.The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.

View Article: PubMed Central - PubMed

Affiliation: St. Vincent's Institute of Medical Research and The University of Melbourne, Department of Medicine, Victoria, Australia.

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 1alpha,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-gamma (IFN-gamma) 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-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma 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-gamma 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-gamma production.

Show MeSH