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Endocrinology Vol. 144, No. 4 1194-1201
Copyright © 2003 by The Endocrine Society

ARTICLE

Interleukin-18 Is a Novel Mitogen of Osteogenic and Chondrogenic Cells

J. Cornish, M. T. Gillespie, K. E. Callon, N. J. Horwood, J. M. Moseley and I. R. Reid

Department of Medicine, University of Auckland, Auckland 1001, New Zealand; and St. Vincent’s Institute of Medical Research (M.T.G., N.J.H., J.M.M.), Melbourne 3065, Australia

Address all correspondence and requests for reprints to: Dr. Jillian Cornish, Department of Medicine, University of Auckland, Private Bag 92019, Auckland 1001, New Zealand. E-mail: j.cornish{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-18 was identified due to its ability to induce interferon-{gamma} (IFN{gamma}) production by T cells. It is a pleiotropic factor that shares structural features with IL-1 and functional activities with IL-12. IL-18 has a role in T cell development, where it has been demonstrated to act cooperatively with IL-12 to regulate IFN{gamma}. In bone, IL-18 is mainly produced by macrophages, but is also expressed by osteoblasts and inhibits osteoclast formation through granulocyte-macrophage colony-stimulating factor (GM-CSF) and not IFN{gamma} production by T cells. We have investigated the effects of IL-18 on mature osteoclast activity and for potential actions on osteoblasts or chondrocytes.

The effects of IL-18 on mature osteoclast activity were determined using two assays: isolated mature osteoclast cell culture and neonatal murine calvarial organ culture. IL-18 did not affect bone resorption in either assay system. The actions of IL-18 on osteogenic cells (primary cell cultures of fetal rat and neonatal mouse osteoblasts, as well as neonatal mouse calvarial organ culture) and primary chondrocytes (canine) were assessed by proliferation assays (quantification of cell numbers and thymidine incorporation). In each assay system, IL-18 acted as a mitogen to the osteogenic and chondrogenic cells. Since IL-18 signal transduction may involve IFN{gamma} or GM-CSF, we assessed their involvement in the IL-18 response. IL-18 did not induce IFN{gamma} production by primary osteoblasts, but, of greater significance, IFN{gamma} had the opposing action to IL-18 in that it inhibited the primary osteoblast cell proliferation. Although IL-18 rapidly induced GM-CSF production by primary osteoblasts, IL-18 was still mitogenic in osteoblast preparations established from GM-CSF-deficient mice. Combined, these studies indicate that IL-18 may have an autocrine/paracrine mitogen role for both osteogenic and chondrogenic cells, independent of the production of IFN{gamma} or GM-CSF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE GROWTH is controlled by systemic hormones as well as by an increasing number of factors produced locally. Bone growth involves the complex coupling of the actions of osteoblasts and osteoclasts, which occurs by a contact-dependent mechanism and also involves paracrine and autocrine factors. It has recently been demonstrated that IL-18 suppresses the development of osteoclasts in vitro via granulocyte-macrophage colony-stimulating factor (GM-CSF; Ref. 1). The mechanism proposed for this action is that IL-18 binds to its receptor on T cells in the bone microenvironment to activate GM-CSF production, and the secreted GM-CSF, in turn, binds to its receptor on osteoclastic precursors to inhibit proliferation/differentiation (2).

IL-18 is a recently discovered cytokine originally named for its ability to induce interferon-{gamma} (IFN{gamma}) production by T cells (3). However, it has since been shown to be more than an inducer of IFN{gamma} (4). IL-18 shares structural features with the IL-1 family of proteins and functional properties with IL-12 (5). IL-18 is a pleiotropic factor with many proinflammatory functions, such as inducing IFN{gamma}, stimulating the proliferation of activated T cells and the differentiation of T helper type 1 cells (where IL-18 acts in synergy with IL-12), enhancing natural killer (NK) cell cytotoxicity (6), up-regulating intercellular adhesion molecules (7), and augmenting the production of GM-CSF (8). Both of the latter functions are independent of IFN{gamma} production. The inhibitory effect of IL-18 on osteoclast generation is also independent of IFN{gamma} (1).

Murine and human IL-18 gene sequences encode 192- and 193-amino-acid precursor proteins, respectively (9). These 24-kDa precursor molecules lack a signal peptide and have limited biological activity. The active mature 18.3-kDa molecule is cleaved from the precursor molecule by the action of IL-1ß-converting enzyme, also known as caspase 1 (10, 11). Caspases belong to a protease family that plays a pivotal role in inflammation and apoptotic cell death (12). Yet another caspase, caspase 3, has a regulatory effect on IL-18, where it cleaves both precursor and mature forms of IL-18 into biologically inactive degraded products and may constitute a potential down-regulator of IL-18 (13).

The IL-18 receptor comprises two molecules, both of which belong to the IL-1 receptor family: IL-18R, a ligand-binding chain, and accessory protein-like (AcPL), an accessory chain (14, 15). IL-18R binds to IL-18 with low affinity, but the complex of the IL-18R and AcPL binds IL-18 with high affinity. IL-18 receptor genes are located in the region of chromosome 2, shared with other IL-1 receptor genes (16).

Antigen-presenting cells represent the major source of IL-18 production (13), but IL-18 expression has been demonstrated in a wide variety of cells, including osteoblasts and chondrocytes (1, 17). This study was designed to investigate further effects of IL-18 on skeletal cells, other than the cytokine’s ability to inhibit osteoclastogenesis, and whether these effects were dependent on GM-CSF or IFN{gamma} activity. We have used two culture systems that demonstrate the function of mature osteoclasts, namely, a bone organ culture and isolated mature osteoclast cultures, as well as various primary cultures of osteoblasts and chondrocytes to address these questions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
For the bone organ culture experiments ARC Swiss mice were used. Chondrocytes were isolated from cartilage removed from dogs purchased from the Dog Pound (Auckland, New Zealand). For osteoblast-like cell cultures, fetal Wistar rats were used, or cells were isolated from C57/BL6J mice (wild-type, purchased from Monash University Animal Services Center, Clayton, Australia) or GM-CSF-deficient mice (provided by Dr. Ashley Dunn, Ludwig Institute, Australia; Ref. 18). All experiments were conducted in accordance with the University of Auckland (New Zealand) and the National Health and Medical Research Council (Australia) guidelines and were approved by the institutional ethics committees.

Materials
IL-18 was provided by Dr. Kurimoto (Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan). EDTA and collagenase were obtained from Sigma-Aldrich (St. Louis, MO). Trypsin-EDTA, MEM, DMEM, medium 199, and fetal bovine serum (FBS) were obtained from Life Technologies, Inc. (Grand Island, NY). BSA was obtained from Immuno Chemical Products (Auckland, New Zealand). Pronase was purchased from Roche Molecular Biochemicals (Mannheim, Germany). [3H]Thymidine and [3H]phenylalanine were obtained from Amersham International (Little Chalfont, UK).

Mature isolated osteoclast culture
Rat osteoclasts were isolated from 1-d-old neonatal rats as previously described (19). Briefly, long bones were excised, chopped, and homogenized in acidified medium. The osteoclast-rich cell suspension (low numbers of stromal cells are also present) was collected and placed over bovine bone slices in 96-well plates and incubated for 35 min to allow the mature osteoclasts to settle. The bone slices were washed and placed in 6-well plates containing medium with cytokine or vehicle and were incubated for 20 h. Bone slices were fixed with 2.5% glutaraldehyde/PBS and stained for tartrate-resistant acid phosphatase (TRAP) using Sigma-Aldrich kit 387-A. The number of TRAP-positive multinucleated cells were quantified, and then the cells were removed by gentle scrubbing, stained with toluidine blue, and assessed for pits excavated by the osteoclasts using reflected light microscopy and metallurgic lenses.

Bone organ culture
Bone resorption studies were carried out in neonatal mouse calvariae as described previously (20). Two-day-old mice were injected s.c. with 45CaCl2. Four days later the hemicalvariae were excised and cultured on small steel grids in 35-mm petri dishes containing medium 199 supplemented with 0.1% BSA. After a 24-h preculture period, fresh medium was added containing cytokine or vehicle. Calvariae were incubated for an additional 48 h. [3H]Thymidine was added in the last 4 h of the incubation period. Bone resorption was measured by calculating the amount of 45Ca released into the medium. DNA synthesis was assessed by measuring thymidine incorporation (21).

Primary osteoblast-like cell culture
Osteoblasts were isolated by sequential collagenase digestion of 20-d-old fetal rat calvariae as previously described (22). Digests 3–5 were pooled and grown in 75-cm2 flasks in DMEM containing 10% FBS. Cells were grown to confluence, and then proliferation studies were performed. Cells were trypsinized and seeded into 24-well plates. Cells were serum-starved for 24 h in MEM/0.1% BSA, and then the medium was replaced, and cytokine or vehicle was added. The plates were incubated for an additional 24 h, with a pulse of [3H]thymidine added 4 h before the end of the incubation period. Plates were assessed for thymidine incorporation or cell number.

The osteoblast-like character of these cells has been established by demonstration of high levels of alkaline phosphatase activity and osteocalcin production (23) and a sensitive adenylate cyclase response to PTH and prostaglandin E2 (24). The osteoblast-like cells produced in this culture system are early in the osteoblast differentiation pathway and are often termed preosteoblastic.

Primary chondrocyte-like cell culture
Cartilage slices were removed from the articular surfaces of knee joints of adult dogs. The slices were finely chopped and incubated at 37 C first with pronase (0.8%, wt/vol) for 90 min, followed by collagenase (0.1%, wt/vol) for 18 h to complete the digestion. The isolated cells were washed and seeded into 75-cm2 flasks containing DMEM supplemented with 10% FBS and 50 µg/ml ascorbic acid. Chondrocytes were grown to confluence and seeded into 24-well plates, and proliferation assays were performed as detailed for the osteoblast-like cell culture.

RNA extraction, cDNA synthesis, and PCR
Total cellular RNA was extracted from newborn mouse calvarial cells using guanidine thiocyanate-phenol chloroform and used in RT-PCR essentially as previously described (25). Oligonucleotides (Table 1Go) were synthesized on an Oligo1000M DNA Synthesizer (Beckman, Fullerton, CA). The following oligonucleotides were used to amplify murine mRNA species: GM-CSF (GM-CSF-1 and GM-CSF-2), GM-CSF R (GM-CSF-R-1 and GM-CSF-R-2), IFN{gamma} (IFN{gamma}-1 and IFN{gamma}-2), IL-18 (IL-18-1 and IL-18-2), IL-18R (IL-18-R-4 and IL-18-R-5), IL-18AcPL (IL-18AcPL-1 and IL-18AcPL-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GAPDH-2 and GAPDH-4). The specificity of the products was confirmed by Southern blot detection using a 32P-labeled internal oligonucleotide probe (GM-CSF-3, IFN{gamma}-3, IL-18-3, IL-18-R-6, IL-18AcPL-2, and GAPDH-1) as previously described (26) or by nucleic acid sequencing of amplified products. Bound probe was detected by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA) (26).


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  		Table 1. Oligonucleotides to amplify and detect cytokine and receptor mRNAs

 
Statistical analysis
Data are presented as the mean ± SEM. The significance of differences between groups was determined by t test. The comparisons to be made in each experiment were specified a priori, so no adjustment for multiple comparisons was necessary. Where several experiments have been shown in one figure, the data are expressed as the ratio of results in treatment groups to those in the control group, and the P values shown were calculated using data from individual experiments before data were pooled.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-18 does not inhibit mature osteoclast activity
IL-18 has been previously shown to inhibit osteoclast formation. We investigated whether IL-18 affected mature osteoclast activity using two different culture systems: 1) mature isolated osteoclasts, and 2) calvarial organ culture. IL-18 did not have any effect on mature osteoclast activity as measured by the number of bone-resorptive pits per TRAP-positive multinucleated cell (Fig. 1AGo). As a positive control, salmon calcitonin was analyzed in this culture system, and the peptide acted as a potent inhibitor of bone resorption (50% effective dose, 2.3 x 10-13 M; data not shown). We also demonstrated that IL-18 does not inhibit bone resorption in organ culture, as measured by calcium release from neonatal mouse calvaria (Fig. 1BGo). Bone resorption in this culture system predominantly reflects mature osteoclast activity rather than osteoclastogenesis due to the minimal amount of bone marrow in the calvarial tissue (27). However, IL-18 was found to significantly increase thymidine incorporation compared with that in controls in this organ culture (Fig. 1CGo). We have previously shown that increased thymidine incorporation in this model system reflects the proliferation of cells of the osteoblast lineage (28). As such, the effects of IL-18 on osteoblast and chondrocyte proliferation were assessed in primary cell cultures.



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  		Figure 1. Effects of IL-18 on neonatal rat mature osteoclast activity (A), bone resorption in neonatal murine calvarial organ culture (B), and DNA synthesis in organ culture (C). Data are expressed as the mean ± SEM number of bone resorptive pits per TRAP-positive multinucleated cell (A), percent calcium release (B), and [3H]thymidine incorporation (C). *, Significantly different from control (P < 0.03).

 
IL-18 effects on chondrocyte proliferation
The proliferative effects of IL-18 on primary canine chondrocyte-like cells were assessed by the measurement of cell numbers and DNA synthesis. Treatment with IL-18 for 24 h produced a dose-dependent increase in cell numbers (Fig. 2AGo) and thymidine incorporation (Fig. 2BGo).



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  		Figure 2. Dose-dependent effect of IL-18 on primary canine chondrocyte cell number (A) and [3H]thymidine incorporation (B). Data are the mean ± SEM. *, Significantly different from control (P < 0.03).

 
IL-18 effects on osteoblast proliferation
The effects of IL-18 on primary fetal rat osteoblast-like cells were assessed by measurements of cell numbers, DNA, and protein synthesis. Treatment with IL-18 for 24 h produced dose-dependent proliferative effects with increases in cell number (Fig. 3AGo) and DNA synthesis, as assessed by the measurement of [3H]thymidine incorporation into cultured osteoblast-like cells (Fig. 3BGo). The magnitude of this response was comparable to that of classic osteoblast growth factors, such as TGFß and epidermal growth factor (29). The effect of IL-18 on protein synthesis, as assessed by the measurement of [3H]phenylalanine incorporation into cultured osteoblast-like cells, produced a dose-dependent increase (Fig. 3CGo). This increase in protein synthesis probably reflects the increased number of cells, because the percent increases in these two indexes were almost identical. Combined, these data demonstrate that IL-18 acts as a mitogen for osteogenic cells.



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  		Figure 3. Dose-dependent effect of IL-18 on primary fetal rat osteoblast cell number (A), [3H]thymidine incorporation (B), and [3H]phenylalanine incorporation (C). Data are the mean ± SEM. *, Significantly different from control (P < 0.04).

 
IL-18 mitogenic effect on primary osteoblasts is independent of both IFN{gamma} and GM-CSF
IL-18 is known to signal through GM-CSF or IFN{gamma}. To determine whether the enhanced osteoblast-like cell proliferation in response to IL-18 was a direct or an indirect action of IL-18, we examined the expression of GM-CSF, IFN{gamma}, and receptor components in primary murine osteoblasts in response to IL-18 treatment. Primary murine osteoblasts were treated with IL-18 (10 ng/ml) over a 24-h time course (0.5, 1, 2, 4, 8, 12, and 24 h), RNA was extracted, and regulation of target genes was assessed by RT-PCR. The most striking regulation was observed with GM-CSF mRNA (Fig. 4Go). GM-CSF mRNA levels increased within 1 h of exposure to IL-18, and a 4-fold elevation in mRNA levels continued through to 24 h. A concomitant decrease in GM-CSF receptor mRNA was observed (Fig. 4Go). Although no detectable alteration in IL-18 R mRNA was observed, the mRNAs for IL-18 and IL-18AcPL gradually decreased over the 24-h time course (Fig. 4Go). IFN{gamma} mRNA was not detectable (data not shown). The finding that IFN{gamma} was not expressed by the primary osteoblasts, even in response to IL-18, indicated that it was unlikely that IFN{gamma} was implicated in the osteoblast mitogenic response to IL-18. Since the primary osteoblast GM-CSF mRNA levels were elevated in response to IL-18, GM-CSF may be involved in the IL-18-induced mitogenic response. To test this possibility we studied the mitogenic response of IL-18 on osteoblast-like cells from GM-CSF-deficient mice. IL-18 significantly enhanced osteoblast proliferation whether the osteoblasts were obtained from GM-CSF-deficient (Fig. 5AGo) or wild-type (Fig. 5BGo) mice. This suggests that the IL-18 mitogenic effect on these osteoblast-like cells was GM-CSF independent.



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  		Figure 4. Semiquantitative RT-PCR analysis of IL-18, IL-18AcPL, IL-18 R, GM-CSF, and GM-CSF R mRNA. Primary neonatal murine osteoblasts were treated with IL-18 (10 ng/ml) as indicated by the time course (0.5, 1, 2, 4, 8, 12, and 24 h), and total RNA was extracted. Total RNA samples were reverse transcribed with oligo(deoxythymidine) and subjected to PCR for IL-18 (30 cycles), IL-18AcPL (28 cycles), IL-18 R (34 cycles), GM-CSF (35 cycles), GM-CSF R (32 cycles), and GAPDH (20 cycles), which were in the log-linear range of amplification. Resultant PCR products were electrophoresed, transferred to nylon membrane, and hybridized with {gamma}-32P-labeled internal detection oligonucleotides specific to each of the cDNA products. RT-PCR analysis was repeated in triplicate.

 


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  		Figure 5. Effect of IL-18 on neonatal murine osteoblast numbers from wild-type (A) and GM-CSF deficient (B) mice. Data are the mean ± SEM. *, Significantly different from control (P < 0.03).

 
In contrast to IL-18, IFN{gamma} was not mitogenic to the primary osteoblasts, but, rather, IFN{gamma} decreased cell numbers (Fig. 6AGo) and thymidine incorporation (Fig. 6BGo) of primary rat osteoblast cultures. This opposing action of IFN{gamma} compared with that of IL-18 combined with the inability to detect IFN{gamma} production by these osteoblasts would exclude IFN{gamma} from this osteoblast mitogenic response to IL-18.



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  		Figure 6. Effect of IFN{gamma} on fetal rat osteoblast cell number (A) and [3H]thymidine incorporation (B). Data are the mean ± SEM. *, Significantly different from control (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that IL-18 has direct effects on skeletal cells. IL-18 was a potent mitogen of primary osteoblast- and chondrocyte-like cells. This cytokine has been previously shown to be expressed by osteoblasts (1) and chondrocytes (17); thus, IL-18 is probably acting in a paracrine/autocrine manner in skeletal tissue. The mitogenic potential of IL-18 was independent of IFN{gamma} or GM-CSF.

IL-18 was initially described as an IFN{gamma}-inducing factor (30); however, it clearly has more functions than inducing IFN{gamma} production alone, and many biological effects of IL-18 are now known to be independent of IFN{gamma}’s actions (4), including IL-18 inhibition of osteoclast formation (1). Our findings show that IFN{gamma} is not expressed by primary osteoblast-like cells, and indeed, this cytokine has an inhibitory, rather than a proliferative, effect on primary osteoblasts, which confirms previous work performed in osteoblast-like cell lines (31). IL-18 acts to enhance not only IFN{gamma}, but also GM-CSF, in a number of cell systems, such as peripheral blood monocytes (9). We have previously established that IL-18 inhibits osteoclast formation through stimulating the T cell production of GM-CSF (2). GM-CSF is a known autocrine mitogenic factor for osteoblasts (32), inducing a dose-dependent increase in human osteoblast proliferation (33). However, we were able to exclude GM-CSF in the IL-18 action by using osteoblasts isolated from GM-CSF-deficient mice. In such cultures IL-18 was still mitogenic. These findings imply that IL-18 is likely to be acting in an autocrine/paracrine manner for chondrogenic and osteogenic cells. The fact that the mitogenic action of IL-18 is independent of IFN{gamma} or GM-CSF suggests that IL-18 is acting directly through the IL-18 receptor complex. Consistent with this proposition, both IL-18R and AcPL components were expressed by primary osteoblasts.

The finding that primary osteoblasts express both components of the IL-18 receptor, as also found in T and NK cells, implies that the osteoblasts are target cells responding to IL-18, and that IL-18 may have a physiological function on osteogenic cells. Further, the phosphorylation of MAPK in bone marrow stromal cells and osteoblasts in response to IL-18 supports this proposal (34). This observation is consistent with those in T and NK cells (35, 36), and it is possible that the phosphorylation of MAPK by IL-18 is involved in the proliferation of osteoblasts. However, as activation of nuclear factor-{kappa}B through the IL-1 receptor-associated kinase signaling pathway is considered a major pathway in the IL-18 signaling of T and NK cells (37), further studies are necessary to clarify how the proliferation of osteoblasts is regulated by IL-18.

Osteoprotegerin (OPG) has also been implicated as an IL-18 target gene in bone marrow stromal ST2 cells, osteoblastic MC3T3-E1 cells, and mouse calvarial osteoblasts (34). The expression of OPG mRNA was transiently, but modestly, increased, reaching a maximal level 3 h after IL-18 exposure. It is unlikely that the enhancement of osteoblastic OPG production would account for any activity to inhibit osteoclast formation, because the presence of T cells was obligatory for this IL-18 effect; IL-18 had no effect in cultures where osteoblasts were present, but T cells were absent (2).

The role of IL-18 on the osteoclast appears to be restricted to limiting osteoclast formation, as, in this current study, we found that IL-18 had no effect on the activity of mature isolated osteoclasts. In addition, we showed that IL-18 did not affect bone resorption, as measured by 45Ca release from neonatal mouse calvarial organ cultures. In this organ culture system, bone resorption reflects predominantly mature osteoclast activity, although some osteoclasts may form from committed osteoclast precursors. Thus, it appears that IL-18 does not act directly upon hemopoietic cells or differentiated osteoclasts to affect osteoclast formation or activity. Further, IL-18 acts in synergy with IL-12 to inhibit osteoclast formation through the production of an as yet to be identified secreted factor from T cells (25). The ability of IL-18 to limit osteoclast formation in vitro lends support to the idea that IL-18 may be a useful therapy to limit cancer-induced bone loss. In proof of concept experiments, IL-18 inhibits osteolytic bone metastases by human lung cancer cells or human breast cancer cells in nude mouse models of cancer metastasis in bone (38, 39).

IL-18 has a key role in T cell development, activation, and IFN{gamma} secretion by type 1 helper T cells and NK cells, and evidence is accruing that IL-18 has a proinflammatory role in rheumatoid arthritis and is implicated in diabetes (40, 41, 42). T cells are present in the bone microenvironment, and their impact on skeletal maintenance is only now being recognized. Their numbers and activities are modified in response to changes in estrogen levels, as a result of inflammatory conditions of bone such as the onset and development of rheumatoid arthritis and accompanying cancer metastasis in bone (43, 44). As a consequence of these conditions, T cells may produce activators of bone resorption such as receptor activator of nuclear factor-{kappa}B ligand (RANKL) or participate in inhibitory pathways of osteoclast formation such as that used by IL-18 (2, 45, 46). It is therefore not surprising that T cell-derived factors may not only regulate osteoclast formation, but may also influence osteoblast and chondrocyte activities, such as we describe here for IL-18.

In light of these stimulatory effects of IL-18 on osteogenic cells and considering that IL-18 has been previously demonstrated to have an angiogenic role (47) and to inhibit osteoclast development, we postulate that IL-18 may be yet another locally produced factor that can regulate bone growth. IL-18 may be important in bone development by increasing bone and cartilage formation and inhibiting bone resorption. Preliminary work in one of our laboratories indicated that the IL-18 knockout mouse has reduced trabecular bone volume, which is consistent with the anabolic effect of this cytokine demonstrated in the present work (Sims, N. A., D. Mirosavljevic, M. J. Smyth, and M. T. Gillespie, in preparation). Consequently, IL-18 may be a physiological regulator of bone growth.


   Acknowledgments
 
We thank Assoc. Prof. C. A. Poole for kindly providing cartilage samples.


   Footnotes
 
This work was supported by program grants from the Medical Research Council of New Zealand and the National Health and Medical Research Council of Australia (003211).

Abbreviations: AcPL, Accessory protein-like; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN{gamma}, interferon-{gamma}; NK, natural killer; OPG, osteoprotegerin; TRAP, tartrate-resistant acid phosphatase.

Received October 25, 2002.

Accepted for publication December 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Udagawa N, Horwood NJ, Elliott J, Mackay A, Owens J, Okamura H, Kurimoto M, Chambers TJ, Martin TJ, Gillespie MT 1997 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. J Exp Med 185:1005–1012[Abstract/Free Full Text]
  2. Horwood NJ, Udagawa N, Elliott J, Grail D, Okamura H, Kurimoto M, Dunn AR, Martin T, Gillespie MT 1998 Interleukin 18 inhibits osteoclast formation via T cell production of granulocyte macrophage colony-stimulating factor. J Clin Invest 101:595–603[Medline]
  3. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K, Akita K, Namba M, Tanabe F, Konishi K, Fukuda S, Kurimoto M 1995 Cloning of a new cytokine that induces IFN-{gamma} production by T cells. Nature 378:88–90[CrossRef][Medline]
  4. Dinarello CA, Novick D, Puren AJ, Fantuzzi G, Shapiro L, Muhl H, Yoon DY, Reznikov LL, Kim SH, Rubinstein M 1998 Overview of interleukin-18: more than an interferon-{gamma} inducing factor. J Leukocyte Biol 63:658–664[Abstract]
  5. Bazan J, Timans J, Kastelein R 1996 A newly defined interleukin-1? Nature 379:591[CrossRef][Medline]
  6. Micallef M, Ohtsuki T, Kohno K, Tanabe F, Ushio S, Namba M, Tanimoto T, Torigoe K, Fujii M, Ikeda M, Fukuda S, Kurimoto M 1996 Interferon-{gamma}-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-{gamma} production. Eur J Immunol 26:1647–1651[Medline]
  7. Kohka H, Yoshino T, Iwagaki H, Sakuma I, Tanimoto T, Matsuo Y, Kurimoto M, Orita K, Akagi T, Tanaka N 1998 Interleukin-18/interferon-{gamma}-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells. J Leukocyte Biol 64:519–527[Abstract]
  8. Conti B, Jahng J, Tinti C, Son J, Joh T 1997 Induction of interferon-{gamma} inducing factor in the adrenal cortex. J Biol Chem 272:2035–2037[Abstract/Free Full Text]
  9. Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S, Ikeda M, Okamura H, Kurimoto M 1996 Cloning of the cDNA for human IFN-{gamma}-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 156:4274–4279[Abstract]
  10. Gu Y, Kuida K, Tsutsui H, Ku G, Hsiao K, Fleming MA, Hayashi N, Higashino K, Okamura H, Nakanishi K, Kurimoto M, Tanimoto T, Flavell RA, Sato V, Harding MW, Livingston DJ, Su MS 1997 Activation of interferon-{gamma} inducing factor mediated by interleukin-1ß converting enzyme. Science 275:206–209[Abstract/Free Full Text]
  11. Ghayur T, Banerjee S, Hugunin M, Butler D, Herzog L, Carter A, Quintal L, Sekut L, Talanian R, Paskind M, Wong W, Kamen R, Tracey D, Allen H 1997 Caspase-1 processes IFN-{gamma}-inducing factor and regulates LPS-induced IFN-{gamma} production. Nature 386:619–623[CrossRef][Medline]
  12. Henkart PA 1996 ICE family proteases: mediators of all apoptotic cell death? Immunity 4:195–201[CrossRef][Medline]
  13. Akita K, Ohtsuki T, Nukada Y, Tanimoto T, Namba M, Okura T, Takakura-Yamamoto R, Torigoe K, Gu Y, Su MS, Fujii M, Satoh-Itoh M, Yamamoto K, Kohno K, Ikeda M, Kurimoto M 1997 Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells. J Biol Chem 272:26595–26603[Abstract/Free Full Text]
  14. Torigoe K, Ushio S, Okura T, Kobayashi S, Taniai M, Kunikata T, Murakami T, Sanou O, Kojima H, Fujii M, Ohta T, Ikeda M, Ikegami H, Kurimoto M 1997 Purification and characterization of the human interleukin-18 receptor. J Biol Chem 272:25737–25742[Abstract/Free Full Text]
  15. Born TL, Thomassen E, Bird TA, Sims JE 1998 Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signaling. J Biol Chem 273:29445–29450[Abstract/Free Full Text]
  16. Dale M, Nicklin MJ 1999 Interleukin-1 receptor cluster: gene organization of IL1R2, IL1R1, IL1RL2 (IL-1Rrp2), IL1RL1 (T1/ST2), and IL18R1 (IL-1Rrp) on human chromosome 2q. Genomics 57:177–179[CrossRef][Medline]
  17. Olee T, Hashimoto S, Quach J, Lotz M 1999 IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses. J Immunol 162:1096–1100[Abstract/Free Full Text]
  18. Stanley E, Lieschke GJ, Grail D, Metcalf D, Hodgson G, Gall JA, Maher DW, Cebon J, Sinickas V, Dunn AR 1994 Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc Natl Acad Sci USA 91:5592–5596[Abstract/Free Full Text]
  19. Cornish J, Callon KE, Bava U, Kamona SA, Cooper GJ, Reid IR 2001 Effects of calcitonin, amylin, and calcitonin gene-related peptide on osteoclast development. Bone 29:162–168[Medline]
  20. Reid LR, Lowe C, Cornish J, Skinner SJ, Hilton DJ, Willson TA, Gearing DP, Martin TJ 1990 Leukemia inhibitory factor: a novel bone-active cytokine. Endocrinology 126:1416–1420[Abstract]
  21. Lowe C, Cornish J, Martin TJ, Reid IR 1991 Effects of leukemia inhibitory factor on bone resorption and DNA synthesis in neonatal mouse calvaria. Calcif Tissue Int 49:394–397[Medline]
  22. Naot D, Callon KE, Grey A, Cooper GJ, Reid IR, Cornish J 2001 A potential role for adrenomedullin as a local regulator of bone growth. Endocrinology 142:1849–1857[Abstract/Free Full Text]
  23. Groot C, Danes J, van der Meer J, Herrmann-Erlee M 1985 Osteocalcin antigenicity in cultured osteoblast-like cells after stimulation with 1,25-vitamin D3. Cell Biol Int Rep 9:528[CrossRef][Medline]
  24. Hermann-Erlee M, van der Meer J, The effect of PTH and PGE2 on growth and differentiation of primary fetal rat osteoblast-like cells, on UMR106 osteosarcoma cells, and on a SV40 transformed "osteoblast-like" cell line [Abstract]. 9th International Conference of Calcium Regulatory Hormones and Bone Metabolism, Montréal, Canada, 1986, p 409
  25. Horwood NJ, Elliott J, Martin TJ, Gillespie MT 2001 IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro. J Immunol 166:4915–4921[Abstract/Free Full Text]
  26. Southby J, O’Keeffe LM, Martin TJ, Gillespie MT 1995 Alternative promoter usage and mRNA splicing pathways for parathyroid hormone-related protein in normal tissues and tumours. Br J Cancer 72:702–707[Medline]
  27. Pfeilschifter J, Seyedin SM, Mundy GR 1988 Transforming growth factor ß inhibits bone resorption in fetal rat long bone cultures. J Clin Invest 82:680–685[Medline]
  28. Cornish J, Callon KE, Edgar SG, Reid IR 1997 Leukemia inhibitory factor is mitogenic to osteoblasts. Bone 21:243–247[Medline]
  29. Cornish J, Callon KE, Lin CQ, Xiao CL, Gamble GD, Cooper GJ, Reid IR 1999 Comparison of the effects of calcitonin gene-related peptide and amylin on osteoblasts. J Bone Miner Res 14:1302–1309[CrossRef][Medline]
  30. Nakamura K, Okamura H, Wada M, Nagata K, Tamura T 1989 Endotoxin-induced serum factor that stimulates {gamma} interferon production. Infect Immun 57:590–595[Abstract/Free Full Text]
  31. Yoshihara R, Shiozawa S, Imai Y, Fujita T 1990 Tumor necrosis factor {alpha} and interferon {gamma} inhibit proliferation and alkaline phosphatase activity of human osteoblastic SaOS-2 cell line. Lymphokine Res 9:59–66[Medline]
  32. Modrowski D, Lomri A, Marie PJ 1998 Glycosaminoglycans bind granulocyte-macrophage colony-stimulating factor and modulate its mitogenic activity and signaling in human osteoblastic cells. J Cell Physiol 177:187–195[CrossRef][Medline]
  33. Modrowski D, Lomri A, Marie PJ 1997 Endogenous GM-CSF is involved as an autocrine growth factor for human osteoblastic cells. J Cell Physiol 170:35–46[CrossRef][Medline]
  34. Makiishi-Shimobayashi C, Tsujimura T, Iwasaki T, Yamada N, Sugihara A, Okamura H, Hayashi S, Terada N 2001 Interleukin-18 up-regulates osteoprotegerin expression in stromal/osteoblastic cells. Biochem Biophys Res Commun 281:361–366[CrossRef][Medline]
  35. Tsuji-Takayama K, Matsumoto S, Koide K, Takeuchi M, Ikeda M, Ohta T, Kurimoto M 1997 Interleukin-18 induces activation and association of p56(lck) and MAPK in a murine TH1 clone. Biochem Biophys Res Commun 237:126–130[CrossRef][Medline]
  36. Kalina U, Kauschat D, Koyama N, Nuernberger H, Ballas K, Koschmieder S, Bug G, Hofmann WK, Hoelzer D, Ottmann OG 2000 IL-18 activates STAT3 in the natural killer cell line 92, augments cytotoxic activity, and mediates IFN-{gamma} production by the stress kinase p38 and by the extracellular regulated kinases p44erk-1 and p42erk-21. J Immunol 165:1307–1313[Abstract/Free Full Text]
  37. Kanakaraj P, Ngo K, Wu Y, Angulo A, Ghazal P, Harris CA, Siekierka JJ, Peterson PA, Fung-Leung WP 1999 Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice. J Exp Med 189:1129–1138[Abstract/Free Full Text]
  38. Nakata A, Tsujimura T, Sugihara A, Okamura H, Iwasaki T, Shinkai K, Iwata N, Kakishita E, Akedo H, Terada N 1999 Inhibition by interleukin 18 of osteolytic bone metastasis by human breast cancer cells. Anticancer Res 19:4131–4138[Medline]
  39. Iwasaki T, Yamashita K, Tsujimura T, Kashiwamura S, Tsutsui H, Kaisho T, Sugihara A, Yamada N, Mukai M, Yoneda T, Okamura H, Akedo H, Terada N 2002 Interleukin-18 inhibits osteolytic bone metastasis by human lung cancer cells possibly through suppression of osteoclastic bone-resorption in nude mice. J Immunother 25:S52–S60
  40. Gillespie MT, Horwood NJ 1998 Interleukin-18: perspectives on the newest interleukin. Cytokine Growth Factor Rev 9:109–116[CrossRef][Medline]
  41. Swain SL 2001 Interleukin 18: tipping the balance towards a T helper cell 1 response. J Exp Med 194:F11–F14
  42. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H 2001 Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol 19:423–474[CrossRef][Medline]
  43. Cenci S, Weitzmann MN, Roggia C, Namba N, Novack D, Woodring J, Pacifici R 2000 Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-{alpha}. J Clin Invest 106:1229–1237[Medline]
  44. Romas E, Gillespie MT, Martin TJ 2002 Involvement of receptor activator of NF{kappa}B ligand and tumor necrosis factor-{alpha} in bone destruction in rheumatoid arthritis. Bone 30:340–346[Medline]
  45. Horwood NJ, Kartsogiannis V, Quinn JM, Romas E, Martin TJ, Gillespie MT 1999 Activated T lymphocytes support osteoclast formation in vitro. Biochem Biophys Res Commun 265:144–150[CrossRef][Medline]
  46. Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, Capparelli C, Li J, Elliott R, McCabe S, Wong T, Campagnuolo G, Moran E, Bogoch ER, Van G, Nguyen LT, Ohashi PS, Lacey DL, Fish E, Boyle WJ, Penninger JM 1999 Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402:304–309[CrossRef][Medline]
  47. Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE 2001 Evidence of IL-18 as a novel angiogenic mediator. J Immunol 167:1644–1653[Abstract/Free Full Text]



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