Gamma Interferon (IFN-) and IFN- -Inducing Cytokines Interleukin-12 (IL-12) and IL-18 Do Not Augment Infection-Stimulated Bone

Resorption In Vivo
Hajime Sasaki, Khaled Balto, Nobuyuki Kawashima, Jean Eastcott, Katsuaki Hoshino, Shizuo Akira and Philip Stashenko Clin. Diagn. Lab. Immunol. 2004, 11(1):106. DOI: 10.1128/CDLI.11.1.106-110.2004. Downloaded from http://cvi.asm.org/ on January 27, 2014 by guest

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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 2004, p. 106110 1071-412X/04/$08.000 DOI: 10.1128/CDLI.11.1.106110.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved.

Vol. 11, No. 1

Gamma Interferon (IFN-) and IFN--Inducing Cytokines Interleukin-12 (IL-12) and IL-18 Do Not Augment Infection-Stimulated Bone Resorption In Vivo
Hajime Sasaki,1 Khaled Balto,2 Nobuyuki Kawashima,3 Jean Eastcott,4 Katsuaki Hoshino,5 Shizuo Akira,5 and Philip Stashenko1*
Department of Cytokine Biology,1 and Department of Immunology,4 The Forsyth Institute, Boston, Massachusetts; Division of Endodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia2; and Pulp Biology and Endodontics, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo,3 and Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka,5 Japan

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Received 22 August 2003/Returned for modication 15 October 2003/Accepted 29 October 2003

Periapical granulomas are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. In previous studies we have reported that the bone resorption in this model is primarily mediated by macrophage-expressed interleukin-1 (IL-1). The expression and activity of IL-1 is in turn modulated by a network of Th1 and Th2 regulatory cytokines. In the present study, the functional roles of the Th1 cytokine gamma interferon (IFN-) and IFN--inducing cytokines IL-12 and IL-18 were determined in a murine model of periapical bone destruction. IL-12/, IL-18/, and IFN-/ mice were subjected to surgical pulp exposure and infection with a mixture of four endodontic pathogens, and bone destruction was determined by microcomputed tomography on day 21. The results indicated that all IL-12/, IL-18/, and IFN-/ mice had similar infection-stimulated bone resorption in vivo as wild-type control mice. Mice infused with recombinant IL-12 also had resorption similar to controls. IFN-/ mice exhibited signicant elevations in IL-6, IL-10, IL-12, and tumor necrosis factor alpha in lesions compared to wild-type mice, but these modulations had no net effect on IL-1 levels. Recombinant IL-12, IL-18, and IFN- individually failed to consistently modulate macrophage IL-1 production in vitro. We conclude that, at least individually, endogenous IL-12, IL-18, and IFN- do not have a signicant effect on the pathogenesis of infection-stimulated bone resorption in vivo, suggesting possible functional redundancy in proinammatory pathways. The periapical granuloma is an inammatory and immune response that is elicited by anaerobic infection of the dental pulp as a consequence of caries, tooth fracture, and traumatic operative dental procedures. This inammatory process ultimately results in destruction of the alveolar bone surrounding the tooth. Interleukin-1 (IL-1) is a potent bone-resorptive cytokine that is strongly up-regulated following pulpal infection (20). IL-1 mRNA and protein are markedly increased in inltrating macrophages and polymorphonuclear leukocytes (23), and IL-1 levels generally correlate with the extent of bone destruction (15, 22). Most bone-resorptive activity present in periapical granulomas is neutralized by anti-IL-1 antibodies, and bone resorption is signicantly inhibited in vivo with IL-1 receptor antagonist (16, 22). Both proinammatory Th1 and antiinammatory Th2 cytokines are also induced by pulpal infection and may modulate IL-1 expression and activity by macrophages (10). Th1 cytokines (IL-12 and gamma interferon [IFN-]) and bone-resorptive cytokines (IL-1 and tumor necrosis factor alpha [TNF-]) were up-regulated in a linear fashion over 4 weeks following infection, and resorptive cytokines were positively correlated with Th1 cytokine expression. In contrast, Th2 cytokines exhibited increased expression up to 2 weeks after infection, with a decline in levels thereafter. These data suggest that Th1 cytokine-mediated proinammatory pathways generally predominate in inammatory bone lesions and are therefore expected to potentiate inammation and bone resorption adjacent to sites of infection. At the same time, the central Th1 cytokine IFN- as well as the IFN--inducing cytokines IL-12 and IL-18 also possess osteoclast-inhibitory properties, at least in vitro. These three cytokines have been reported to directly or indirectly reduce osteoclast differentiation and bone resorption (7, 8, 18, 21, 25). As a consequence of these opposing activities, the overall role of Th1 cytokines in inammatory bone resorption in vivo is difcult to predict. In this study, we therefore determined the individual function of IL-12, IL-18, and IFN- in the pathogenesis of infection-stimulated bone destruction, using a well-established in vivo periapical lesion model and appropriate knockout mice. The modulatory effect of these three cytokines on endodontic pathogen-stimulated IL-1 expression by macrophages was also assessed in vitro.
MATERIALS AND METHODS Animals. IL-12 knockout (IL-12/), IL-18/, and IFN-/ animals were used in this study. IL-12/ (C57BL/6 background) mice were purchased from Jackson Laboratory, Bar Harbor, Maine. IL-18/ mice (C57BL/6 background)

* Corresponding author. Mailing address: Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115. Phone: (617) 262-5200. Fax: (617) 262-4021. E-mail: pstashenk@forsyth .org. 106

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(19) were kindly provided by S. Akira. IFN-/ mice (BALB/c background) (5) were the generous gift of F. W. van Ginkel and J. R. McGhee, Department of Microbiology, University of Alabama, Birmingham, Ala. IL-18/ and IFN-/ mice were bred in the animal facility of the Forsyth Institute, and all animals were maintained under pathogen-free conditions. Age-matched C57BL/6J and BALB/cJ mice were purchased from Jackson Laboratory and were used as wild-type controls. Induction of periapical lesions. Infection-stimulated periapical bone destruction was induced following the regimen previously described (2, 10, 15). In brief, mice were anesthetized with ketamine HCl (62.5 mg/kg of body weight) and xylazine (12.5 mg/kg). Mandibular rst molar pulps were exposed using a variable-speed electric dental handpiece (Osada Electric, Los Angeles, Calif.) with a no. 1/4 dental round bur, under a surgical microscope (MC-M92; Seiler, St. Louis, Mo.). The exposure site was approximately 1.5 times the diameter of the bur. Exposed pulps were infected with a mixture of four common endodontic pathogens, Prevotella intermedia (ATCC 25611; American Type Culture Collection [ATCC], Manassas, Va.), Fusobacterium nucleatum (ATCC 25586), Peptostreptococcus micros (ATCC 33270), and Streptococcus intermedius (ATCC 27335). A bacterial mixture containing approximately 1010 cells of each bacterial species/ml in methylcellulose (10 mg/ml) was prepared as previously described (15). At the time of pulp exposure (day zero), exposed dental pulps were directly inoculated with 10 l of the bacterial mixture. Mice without pulp exposures and infection were established as negative controls. rIL-12 infusion. Animals were continuously infused with recombinant mouse IL-12 (rIL-12; generous gift of the Genetics Institute, Cambridge, Mass.), 0.3 g (approximately 710 U)/mouse/day in sterile phosphate-buffered saline (PBS), using a microosmotic pump (model 1007D; ALZET Corporation, Palo Alto, Calif.) (17). The osmotic pump was subcutaneously implanted on day zero just after surgical pulp exposure and was replaced on days 7 and 14 under general anesthesia. Infusions of sterile PBS served as a negative control. Tissue sample preparation. Animals were killed on day 21 after pulp exposure, and mandibles were isolated. After removal of soft tissue, one hemi-mandible was xed in fresh 4% paraformaldehyde in PBS for microcomputed tomography (micro-CT). The periapical tissues surrounding the mesial and distal root apices were carefully extracted from the other hemi-mandible, together with surrounding bone in a block specimen under a surgical microscope for cytokine enzymelinked immunosorbent assays (ELISAs). Periapical tissues were rinsed in cold PBS, freed of clots, weighed, and immediately frozen at 70C. Micro-CT analysis. Hemi-mandibles were scanned using a compact fan-beamtype tomograph (Micro-CT 20; Scanco Medical AG, Bassersdorf, Switzerland) providing a 17-m nominal resolution as previously described (1). The most centrally located section that included the distal root of the mandibular rst molar and its root canal apex was chosen from the stack of images for measurement. The cross-sectional area of periapical lesions was dened using Adobe Photoshop 5.5 (Adobe Systems, San Jose, Calif.) and measured with NIH Image 1.62 (Wayne Rasband, National Institutes of Health, Bethesda, Md.). Results were expressed as square millimeters of resorbed area. Protein extraction and cytokine ELISA. For protein extraction, frozen periapical tissue samples were ground using a precooled sterile mortar and pestle, and the tissue fragments were dispersed in 1 ml of lysis buffer as previously described (10, 15). The mixture was incubated at 4C for 1 h, and the supernatant was collected after centrifugation and stored at 70C until assay. For the IL-1 assay, a mouse capture-ELISA system was used (detection range, 7.8 to 500 pg/ml). In brief, 100 l of sample with or without dilution was added to immunomodules (Nunc, Naperville, Ill.) coated with mouse IL-1 capture antibody (R&D Systems, Minneapolis, Minn.). Captured IL-1 was detected with 100 l of biotinylated mouse IL-1-specic polyclonal antibody (R&D Systems), which was reacted with 100 l of streptavidin-horseradish peroxidase (Zymed, San Francisco, Calif.). One hundred microliters of substrate solution (R&D Systems) was added and incubated for 1 h, and the reaction was stopped by adding 50 l of 1 N H2SO4. The optical density of the reaction was read using an ELISA plate reader at 450 nm. Standards for the assay were prepared with rIL-1 (R&D Systems). ELISAs for other mediators in extracts employed commercially available kits (all from BioSource International, Camarillo, Calif.) as follows: IL-4 (sensitivity, 5 pg/ml), IL-6 (3 pg/ml), IL-10 (13 pg/ml), IL-12 (2 pg/ml), IFN- (1 pg/ml) and TNF- (3 pg/ml). All assays using commercial kits were carried out according to the manufacturers instructions. Results were expressed as picograms of cytokine per milligram of periapical tissue. Macrophage cultures. Resident unstimulated peritoneal macrophages were isolated from IL-12/, IL-18/, and IFN-/ mice following the regimen previously described (15). In brief, a total of 5 ml of cold culture medium,

consisting of RPMI 1640 (BioWhittaker, Walkersville, Md.) supplemented with L-glutamine (2 mM), 1% penicillin-streptomycin (Invitrogen, Carlsbad, Calif.), and 10% heat-inactivated fetal bovine serum (Sigma, St. Louis, Mo.), was injected into the peritoneal cavity and collected with constituent peritoneal cells under sterile conditions. After washing three times with cold medium, the cells were resuspended at 106 cells/ml. Aliquots (160 l) were dispensed into 96-well culture plates (Corning Inc., Corning, N.Y.) and incubated for 2 h at 37C in an atmosphere of 5% CO295% air. Nonadherent cells were removed by washing three times with warm medium, and adherent cells (90% macrophages) were subjected to stimulation experiments. Isolated macrophages were preincubated with or without regulatory cytokines for 1 h. Regulatory cytokines included rIL-12 (Genetics Institute), rIL-18 (Medical & Biological Laboratories, Nagoya, Japan), and rIFN- (R&D Systems). Cytokines were used at 0 (control), 0.1, 1, and 10 U/ml. Following preincubation, macrophages were stimulated with each bacterial pathogen for 24 h in 37C in an atmosphere of 5% CO295% air. The four pathogens described above were grown and prepared as previously described, xed in 0.5% FormolPBS, and used at 1.6 106 cells/well (15). Escherichia coli lipopolysaccharide (LPS; serotype 026:B6; Sigma) was used as a positive control (160 ng/well). After 24 h of stimulation, the culture supernatants were collected and stored at 70C until assay. Statistical analysis. Effects of genotype and pulp exposure on bone resorption and cytokine expression were compared using a two-way fractorial analysis of variance. Differences in IL-1 production stimulated by pathogens in vitro were analyzed by Dunnetts two-tailed t test and Duncans multiple range test.

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RESULTS Effect of IL-12, IL-18, and IFN- on periapical bone destruction. As noted, IL-12, IL-18, and IFN- exert opposing regulatory functions with respect to inammatory bone destruction, i.e., they up-regulate inammatory responses but have also been reported to inhibit osteoclast formation. We therefore determined the functional role of these three cytokines in infection-stimulated bone destruction in vivo. Experimental periapical lesions were induced by surgical pulp exposure and infection of the mandibular rst molars of groups of IL-12/, IL-18/, IFN-/, and appropriate wild-type mice in three separate experiments (background strains, C57BL/6J for IL-12/ and IL-18/ and BALB/cJ for IFN-/ animals). The teeth of negative controls of all groups remained uninfected. After 21 days, the extent of periapical bone resorption was determined by micro-CT (1, 2, 15). As shown in Fig. 1, unexposed and uninfected controls exhibited a minimal area of radiolucency surrounding the distal root, representing the intact periodontal ligament space that anchors teeth to the mandible. In contrast, all exposed and infected animals showed signicant increases in periapical bone resorption compared to uninfected controls. Importantly, infected IL-12/, IL-18/, and IFN-/ mice all had periapical bone resorption that was similar in extent to their respective wild-type infected controls. We also attempted to modulate periapical resorption by infusion of rIL-12, which is known to enhance Th1-type responses in vivo. As shown in Fig. 2, rIL-12 failed to up-regulate bone resorption compared to PBS. Taken together, these data indicate that endogenous IL-12, IL-18, and IFN- do not exert a major effect upon the pathogenesis of infection-stimulated bone destruction, at least individually, suggesting that there may be considerable functional redundancy among these cytokines. Cytokine responses in inammatory lesions. IL-1 has previously been shown to be a primary mediator of bone resorption in this model (22). In addition, the elimination of either IL-10 or IL-6 potently increases IL-1 production in periapical

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FIG. 1. Effect of Th1 cytokine deciency on periapical bone destruction. Hemi-mandibles were scanned by micro-CT on day 21, and the area of the normal periodontal ligament space or infection-stimulated bone resorption was measured. Open columns, normal periodontal ligament space; closed columns, periapical bone destruction; vertical bars, standard deviations. No signicant differences were present in knockout mice versus wild-type controls.

lesions in vivo, which correlates with increased bone destruction (2, 15). Therefore, the levels of IL-1 and other regulatory cytokines in inammatory periapical tissues were assessed by ELISA. As shown in Fig. 3, bone-resorptive cytokines were up-regulated in lesions after pulp exposure and infection compared to uninfected controls. In IFN-/ mice, the major bone-resorp-

FIG. 3. Prole of cytokine expression in periapical inammatory lesions. Cytokine concentrations in inammatory tissues were determined by ELISA on day 21, and values were normalized to the weight of periapical tissue. Vertical bar, standard deviation by two-way fractorial analysis of variance. *, P 0.05 for exposure effect; , P 0.05 for genotype effect.

FIG. 2. Effect of IL-12 infusion on periapical bone destruction. The area of the normal periodontal ligament space or infection-stimulated bone resorption was determined by micro-CT. Open column, uninfected control with normal periodontal ligament space; closed columns, periapical bone destruction; vertical bars, standard deviations. No signicant difference was observed between the IL-12- and PBSinfused groups.

tive cytokine, IL-1, was unchanged, whereas less-potent resorptive mediators, TNF- and IL-6, were signicantly increased (P 0.05). IL-12 and IL-10 production was also increased (P 0.05). IL-18 deciency did not affect boneresorptive cytokine expression in the periapical tissues. Both IFN- and IL-10 production were signicantly increased in IL-18/ mice (P 0.05). Thus, although there was a signi-

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FIG. 4. Modulation of pathogen-stimulated macrophage IL-1 production by Th1 cytokines. Resident peritoneal macrophages were harvested from IL-12/, IL-18/, and IFN/ mice and challenged with bacterial pathogens in the presence or absence of recombinant Th1 cytokines. IL-1 concentrations were determined by ELISA, and values were normalized to the volume of culture supernatant. Each plot indicates the bacterial stimulant, and the x axis indicates the modulating cytokine. Vertical bar, standard deviation. Statistical differences were determined by Dunnetts two-tailed t test versus controls preincubated without cytokines. *, P 0.01.

cant modulation of IFN-, IFN--inducing cytokines, and Th2 cytokines in IL-18/ and IFN-/ mice, the expression of IL-1, thought to be the primary mediator of periapical bone destruction, was relatively unaffected. Modulation of IL-1 production by IL-12, IL-18, and IFN- in vitro. The ability of IL-12, IL-18, and IFN- to modulate macrophage IL-1 production in vitro was determined using resident peritoneal macrophages as previously described (15). As shown in Fig. 4, the effects of rIL-12, rIL-18, and rIFN- on macrophage IL-1 production in response to all pathogens were somewhat inconsistent, although IFN- clearly up-regulated IL-1 production at the lowest concentration tested (0.1 U/ml; P 0.01 in response to LPS and P. intermedia). These data suggest that macrophage IL-1 production appears to be IL12-, IL-18-, and IFN- independent, at least when these cytokines are assessed separately. DISCUSSION Anaerobic infections of the dental pulp result in infrabony inammation, cytokine expression, and resorption of the sur-

rounding periapical bone. Macrophages are prominent in the cellular inltrate early after pulpal infection and have been shown to express IL-12 and IL-18, as well as bone-resorptive cytokines IL-1 and TNF- (10, 13). IL-12 induces Th1 cell differentiation from naive T cells and their subsequent IFN- production (24). Although IL-18 alone cannot induce Th1 cell commitment, IL-18 synergizes with IL-12 (11). IL-12 and IL-18 also induce IFN- production in NK cells (4, 13). IFN- activates macrophages, reduces macrophage-suppressive activity, and induces IL-1, NO synthesis, and O 2 (3, 12, 14). There is thus strong evidence that Th1-mediated pathways may be involved in increasing inammation following infection. In the present study, we tested the hypothesis that the central Th1 cytokine, IFN-, and the IFN--inducing cytokines, IL-12 and IL-18, enhance periapical inammation and bone resorption in vivo. Somewhat surprisingly, our ndings demonstrated that IL-12/, IL-18/, and IFN-/ mice did not have signicantly reduced infection-stimulated bone resorption compared to wild-type animals. Furthermore, rIL-12-infused mice failed to signicantly up-regulate bone destruction compared to control mice. The local level of IL-1 in inammatory lesions, previously found to be a primary mediator of infection-stimulated bone resorption (22, 23), was signicantly lower only in IL-12/ mice (data not shown). Taken together, these results suggest that, at least individually, endogenous IL-12, IL-18, and IFN- do not appear to have a major, nonredundant effect on the pathogenesis of infection-stimulated bone resorption in vivo. There are several possible explanations for these ndings. First, it is possible that the levels of these cytokines that are expressed within lesions (Fig. 3) are too low to have a regulatory effect on resorption. However, in a previous study (10) our investigators found that IFN- and IL-12 were induced at similar levels by infection, as observed in the present study (1 pg/mg of tissue), as was the Th2 cytokine IL-10 (1 to 1.5 pg/mg). Despite this modest induction, and in contrast to IFN- and IL-12, endogenous IL-10 has a profound effect in inhibiting bone resorption in this model, i.e., IL-10 knockout mice have a three to ve times greater lesion size than wildtype controls (15). Thus, low levels of T-cell regulatory cytokines clearly can have dramatic effects on inammatory resorption in vivo. Alternatively, in addition to their proinammatory effects, IL-12, IL-18, and IFN- also suppress the receptor-activator of NF-B ligand (RANKL)-induced osteoclast differentiation and mature osteoclast function in vitro. The effect of IFN- is exerted at picomolar concentrations (6) and involves the accelerated degradation of the RANK adaptor TNF receptorassociated kinase 6 (18). Although IL-12 does not act directly on osteoclast precursors, it indirectly reduces RANKL-induced osteoclast differentiation alone or in synergy with IL-18 (7). IL-18, which may also be produced by osteoblasts, enhances granulocyte-macrophage colony-stimulating factor production by T cells (21). Granulocyte-macrophage colony-stimulating factor signaling in osteoclast precursors inhibits RANKL-induced osteoclast differentiation in an IFN--independent manner (8, 21). It is therefore possible that the relative lack of effect seen in deciencies of IL-12, IL-18, or IFN- on inammatory resorption may reect the balance of two opposing

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2. Balto, K., H. Sasaki, and P. Stashenko. 2001. Interleukin-6 deciency increases inammatory bone destruction. Infect. Immun. 69:744750. 3. Boraschi, D., S. Censini, and A. Tagliabue. 1984. Interferon-gamma reduces macrophage-suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production. J. Immunol. 133:764768. 4. Chan, S. H., B. Perussia, J. W. Gupta, M. Kobayashi, M. Pospisil, H. A. Young, S. F. Wolf, D. Young, S. C. Clark, and G. Trinchieri. 1991. Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173:869879. 5. Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, and T. A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science 259:17391742. 6. Gowen, M., G. E. Nedwin, and G. R. Mundy. 1986. 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processes: the loss of osteoclast inhibitory activities versus the reduction in proinammatory signals. A third possibility is that the functional redundancy of these mediators may obscure their individual roles in inammatory bone loss. Although the differences were not statistically signicant, a small decrease (5 to 10%) in the extent of resorption was in fact seen in all three cytokine-decient strains (Fig. 1). This consistent trend suggests that a real biological effect might be exerted in vivo but that very large numbers of animals and/or a deciency of two or even all three mediators might be required to observe a signicant reduction in bone resorption. A direct test of this possibility will require the generation of double or triple cytokine knockout animals. In addition, there are direct pathways, particularly those mediated by Toll-like receptors, which activate macrophage proinammatory cytokine expression independent of Th1 cytokines. In this regard, our investigators have found that mice decient in Toll-like receptor-4 have reduced expression of IL-1 and TNF-, and they also have less bone resorption than wild-type animals following anaerobic infection (9). Although the background strains were identical in the IL-12 and IL-18 experiments (C57BL/6) (Fig. 1), a larger absolute resorptive response was observed in the IL-18 mice. It should be noted that these represent independent experiments and that similar interexperimental variability in the size of lesions in infected teeth has been observed previously (H. Sasaki, unpublished data), emphasizing the need for an appropriate wild-type control assessed concurrently. A likely explanation is that such differences in lesion size are related to the viability and/or activity of the bacterial inoculum used, perhaps being in log phase of growth in studies in which larger lesions are induced. These results with IFN- and its inducing cytokines are in sharp contrast to our previous ndings with Th2 cytokines, several of which are potent suppressors of inammatory resorption as noted above. Both IL-10-decient and IL-6-decient mice exhibited up-regulated local IL-1 production and increased infection-stimulated bone resorption in vivo compared to the wild type (2, 15). In the case of IL-10 deciency, the increase in locally produced IL-1 production was dramatic (10-fold) and correlated with severe bone resorption. These Th2 mediators thus play signicant and nonredundant roles in suppressing bone inammation and resorption in vivo. Interestingly, another Th2 cytokine, IL-4, had no effect on resorption in this model, indicating functional heterogeneity among this cytokine group (15). Taken together, these ndings suggest that Th2 cytokines may represent more attractive targets for immunomodulation than Th1 and Th1-cytokine-inducing cytokines in preventing inammatory bone loss.
ACKNOWLEDGMENTS We thank Ralph Kent, Jr. for his assistance in statistical analyses and Ralph Mu ller and Thomas Kohler for their assistance in micro-CT scanning. This work was supported by grants DE-09018 and DE-13747 from the National Institutes of Health.
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