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The Journal of Immunology, 2000, 165: 3626-3630.
Copyright © 2000 by The American Association of Immunologists

IL-10, But Not IL-4, Suppresses Infection-Stimulated Bone Resorption In Vivo1

Hajime Sasaki*, Linda Hou*, Anita Belani*, Cun-Yu Wang{dagger}, Toru Uchiyama{ddagger}, Ralph Müller{ddagger} and Philip Stashenko2,*

* Department of Cytokine Biology, The Forsyth Institute, Boston, MA 02115; {dagger} Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI 48109; and {ddagger} Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1{alpha} in the murine model. The production and activity of IL-1{alpha} is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10-/- mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4-/- exhibited no increased resorption. IL-10-/- had markedly elevated IL-1{alpha} production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4-/- exhibited decreased IL-1{alpha} production (p < 0.05). IL-10 also suppressed IL-1{alpha} production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1{alpha} expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The periapical lesion constitutes an inflammatory and immune response against bacterial pathogens that infect and destroy the dental pulp subsequent to dental caries, tooth fracture, and operative dental procedures. This immune response appears to be similar to the response to bacterial infections elsewhere in vivo, with the additional feature that the alveolar bone surrounding the dental root apex is resorbed.

Several cytokines, including IL-1, TNF, IL-6, and IL-11, have been reported to stimulate bone resorption (1, 2, 3, 4, 5). In previous studies in the periapical model, the expression of IL-1{alpha} mRNA and protein was markedly increased in infiltrating macrophages and polymorphonuclear leukocytes in inflammatory lesions. In functional studies, most bone resorptive activity was attributable to the activity of IL-1{alpha} (6, 7, 8).

IL-1{alpha} production and activity are potentially regulated by T cell-derived cytokines (9). Mediators produced by Th1 cells up-regulate inflammation and IL-1 expression, whereas Th2 cell-derived cytokines are generally inhibitory. The Th2-type cytokines IL-4 and IL-10 have been reported to down-regulate the production both of IL-1 (10, 11, 12, 13, 14) and Th1-type cytokines (15, 16). IL-4 and IL-10 also suppress bone resorption in vitro (17, 18, 19, 20). In murine periapical lesions, both IL-4 and IL-10 expression was increased up to 2 wk after infection, declining thereafter (21). There was a positive correlation between Th1 cytokines IFN-{gamma} and IL-12 and bone resorptive cytokine expression and a lack of correlation with IL-4 and IL-10 (21). However, the intrinsic role of these cytokines in vivo is not known.

In this study, the effect of IL-4 and IL-10 on infection-stimulated bone destruction was examined in IL-4-deficient (IL-4-/-), IL-10-deficient (IL-10-/-), and wild-type mice using an in vivo model of periapical inflammation. The regulation of endodontic pathogen-stimulated IL-1{alpha} production by IL-4 and IL-10 in macrophages was also assessed in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Genetically engineered 7-wk-old male IL-4-/- and IL-10-/- mice on backgrounds of BALB/c (for IL-4-/-) and C57BL/10J (IL-10-/-) were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in the Forsyth Institute Animal Facility (Boston, MA) under pathogen-free conditions. Age-matched wild-type BALB/c and C57BL/10J mice were used as controls.

Periapical lesion induction

Infection-stimulated periapical bone destruction was induced as previously described (22). In brief, mice were anesthetized with 62.5 mg/kg ketamine HCl and 12.5 mg/kg xylazine in sterile PBS by intraperitoneal injection. All four first molar pulps were exposed using a no. 1/4 dental round bur and a variable speed electric handpiece (Osada Electric, Los Angeles, CA) under a surgical microscope (MC-M92; Seiler, St. Louis. MO). The exposure site was ~1.5-fold the diameter of the bur.

Exposed pulps were infected with a mixture of four common endodontic pathogens, including Prevotella intermedia (American Type Culture Collection (ATCC)3 25611; ATCC, Manassas, VA), Fusobacterium nucleatum (ATCC 25586), Peptostreptococcus micros (ATCC 33270), and Streptococcus intermedius (ATCC 27335). Organisms were grown on tryptic soy broth with yeast agar plates, and subsequently in mycoplasma liquid medium under anaerobic conditions (80% N2, 10% H2, and 10% C02). The cells were harvested by centrifugation at 7000 x g for 15 min and resuspended in prereduced anaerobically sterilized Ringer’s solution under nitrogen influx. The final concentration of each organism was determined spectrophotometrically, and the four species of microorganism were mixed to yield ~1010 cells of each bacterial species/ml in 10 µg/ml methylcellulose. At the time of pulp exposure (day 0), animals were infected with 10 µl of the bacterial mixture inoculated directly into the exposed pulp. This regimen results in reproducible infection of the root canal with pathogens in this model (23). Mice without pulp exposure and infection were established as negative controls in each strain.

Macrophage cultures

Resident peritoneal cells were isolated from IL-10-/- mice. A total of 5 ml of cold culture medium, consisting of RPMI 1640 (Mediatech, Herndon, VA) supplemented with 2 mM L-glutamine, 1% penicillin-streptomycin (Life Technologies, Gaithersburg, MD), and 10% heat-inactivated FBS (Sigma, St. Louis, MO), was injected into the peritoneal cavity and collected with the cells under sterile conditions. After washing three times with cold medium, the cells were resuspended at 106 cells/ml. Aliquots (160 µl) were dispensed into 96-well culture plates (Corning, Corning, NY), and incubated for 2 h at 37°C in an atmosphere of 5% CO2/95% air. Nonadherent cells were removed by washing three times with warm medium, and the number of adherent cells was determined. Adherent cells comprised 67 ± 7% (n = 3) of the total cells plated.

For in vitro experiments, isolated macrophages were preincubated with or without regulatory cytokines for 1 h, following which they were stimulated with the four pathogens for 24 h in 37°C in an atmosphere of 5% CO2/95% air. Pathogens were grown as described above, fixed with 0.5% formal saline for 24 h, washed three times in sterile PBS, resuspended in culture medium, and used to stimulate macrophages at 1.6 x 106 cells/well. Escherichia coli LPS (serotype 026:B6, Sigma) was used as a positive control (160 ng/well). Mouse rIL-4 and mouse rIL-10 (both from R&D Systems, Minneapolis, MN) were used at 0 (control), 0.1, 1, and 10 U/ml. After 24 h, the culture supernatants were collected and stored at -70°C until assay.

Tissue sample preparation

Animals were killed on day 21 after pulp exposure. After removal of soft tissue, one hemi-mandible was fixed in fresh 4% paraformaldehyde in PBS. For the other hemi-mandible and maxillae, the periapical tissues surrounding the mesial and distal root apices were carefully extracted, together with surrounding bone in a block specimen under a surgical microscope. Periapical tissues were rinsed in PBS, freed of clots, weighed, and immediately frozen at -70°C.

Micro-computed tomography analysis

Hemi-mandibles were scanned as previously described (24) using a compact fan-beam-type tomograph (µCT 20, Scanco Medical, Bassersdorf, Switzerland) providing a 17-µm nominal resolution. The most centrally located section, which included the crown and distal root of the mandibular first molar and that exhibited a patent root canal apex was selected for quantitation. The cross-sectional area of periapical lesions was selected with Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA) and measured with NIH Image 1.61 (Wayne Rasband, National Institutes of Health, Bethesda, MD).

Protein preparations and ELISA

For protein extraction, frozen periapical tissue samples were ground using a precooled sterile mortar and pestle, and the tissue fragments were dispersed in 800 µl lysis buffer consisting of 100 µg/ml BSA (fraction V, Sigma), 100 µg/ml Zwittergent-12 (Boehringer Mannheim, Indianapolis, IN), 50 µg/ml gentamicin (Life Technologies), 10 mM HEPES buffer (Life Technologies), 1 µg/ml aprotinin (Sigma), 1 µg/ml leupeptin (Sigma), and 0.1 µM EDTA (Sigma) in RPMI 1640 (Mediatech) as previously described (6). The mixture was placed on ice and was subjected to a 20-s sonication. The supernatant was collected after centrifugation and stored at -70°C until assay.

Assays for cytokines in extracts employed commercially available ELISA kits obtained from the following sources: IL-1{alpha} (Endogen, Cambridge, MA; sensitivity 6 pg/ml), IL-6 (8 pg/ml), IL-12 (2 pg/ml), IFN-{gamma} (1 pg/ml) and TNF-{alpha} (3 pg/ml; all from BioSource International, Camarillo, CA). All assays were conducted in accordance with the manufacturer’s instructions. The concentration of each cytokine was calculated from a standard curve that was constructed using recombinant cytokines provided with each kit. Results were expressed as pg cytokine/mg periapical tissue.

Statistical analysis

Differences in bone resorption and cytokine expression in periapical lesions were analyzed by the nonpaired Student’s t test. Differences in IL-1{alpha} production stimulated by pathogens in vitro were analyzed by Dunnett’s two-tailed t test and Duncun’s multiple range test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IL-4 and IL-10 gene knockouts on periapical bone destruction

Th2-type cytokines, including IL-4 and IL-10, suppress inflammatory responses, including the expression of IL-1, and have been shown to inhibit bone resorption in vitro (10, 11, 12, 13, 14, 17, 18, 19, 20). Therefore, we determined the effect of deficiencies of these cytokines on infection-stimulated bone destruction in vivo. The mandibular first molars of groups of IL-4-/- (n = 8), IL-10-/-(n = 10), and wild-type mice (n = 8 for IL-4-/- background strain BALB/c, n = 10 for IL-10-/- background strain C57BL/10J) were subjected to pulp exposure and were infected with a mixture of four bacterial pathogens. The teeth of negative controls of all strains remained unexposed and uninfected (n = 3 each). After 21 days, the extent of periapical bone destruction was determined by micro-computed tomography analysis. As shown in Fig. 1Go, unexposed and uninfected controls exhibited no resorption; the indicated radiolucent area represents the normal periodontal ligament that anchors teeth to the bone. In contrast, all exposed and infected animals showed increased bone resorption. Of importance, IL-10-/- mice exhibited ~5-fold increased periapical bone resorption, compared with wild-type controls (p < 0.001), and often even involved the neighboring uninfected second molar (Fig. 2Go). Somewhat surprisingly, IL-4-/- mice exhibited no increased resorption compared with wild type. These data suggest that endogenously expressed IL-10, but not IL-4, is an important inhibitor of infection-stimulated bone destruction in vivo.



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  		FIGURE 1. Micro-computed tomography images of periapical bone resorption. A, IL-4 wild type, nonexposed, noninfected. B, IL-4 wild type, exposed, infected. C, IL-4-/-, exposed, infected. D, IL-10 wild type, nonexposed, noninfected. E, IL-10 wild type, exposed, infected. F, IL-10-/-, exposed, infected. FM, First molar; SM, second molar; INC, incisal periodontal ligament space and incisor. The arrow heads indicate the limit of measured periapical bone resorption.

 


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  		FIGURE 2. Effect of IL-4 and IL-10 deficiency on periapical bone destruction. Vertical bars, SD. {square}, Normal periodontal ligament space. , Periapical bone destruction including periodontal ligament space. Differences were analyzed by Student’s t test. *, p < 0.001.

 
Cytokine responses in inflammatory lesions

IL-1{alpha} has previously been shown to be the primary mediator of bone resorption in this model, whereas TNF-{alpha} has no significant resorptive effect (6). Therefore, the levels of IL-1{alpha} and other regulatory cytokines in inflammatory periapical tissues were assessed by ELISA. As shown in Fig. 3Go, the levels of IL-1{alpha} were markedly increased (>10-fold) in response to infection in the periapical tissues of IL-10-/- mice compared with wild-type (p < 0.01). IL-6 was also increased (p < 0.05), whereas the levels of TNF-{alpha} and IL-12 (data not shown) were similar to wild type. Only IFN-{gamma} was higher in wild-type mice (p < 0.05, data not shown). In contrast, for IL-4-/- mice, a significant decrease (p < 0.01) in IL-1{alpha} was noted. There were also no differences in the other mediators tested. Therefore, these data demonstrate a direct correlation between the local expression of IL-1 in inflammatory tissue, and the extent of bone resorption.



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  		FIGURE 3. Bone resorptive cytokine expression in periapical inflammatory lesions. Cytokine concentrations were determined by ELISA on day 21, and values were normalized to the weight of inflamed periapical tissue. Vertical bar, SD. Statistical differences were determined by Student’s t test. *, p < 0.05; **, p < 0.001.

 
Macrophage IL-1{alpha} responses to pathogens

Macrophages are prominent in inflammatory periapical tissues, and produce large quantities of IL-1 (7, 8). Therefore, we tested the ability of the four infecting pathogens to induce IL-1{alpha} by macrophages in vitro. Resident peritoneal macrophages were harvested, and were stimulated with pathogens. After 24 h, culture supernatants were harvested and analyzed for IL-1{alpha} content by ELISA. As shown in Fig. 4Go, stimulation of macrophages with all pathogens induced substantial IL-1{alpha} expression, compared with control cultures. The levels of IL-1{alpha} induced by Gram-negative bacteria or LPS were higher than those induced by Gram-positive bacteria. F. nucleatum especially induced significantly greater amounts of IL-1{alpha} than were induced by other pathogens (p < 0.05).



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  		FIGURE 4. IL-1{alpha} production by pathogen-stimulated macrophages in vitro. IL-1{alpha} concentrations were determined by ELISA after 24-hour bacterial stimulation of IL-10-/- macrophages. Values were normalized to the volume of culture supernatant. Vertical bar, SD. Statistical differences were determined by Duncun’s multiple range test. *, p < 0.05.

 
Modulation of IL-1{alpha} production by IL-4 and IL-10

The ability of IL-4 and IL-10 to modulate IL-1{alpha} production by macrophages in vitro was determined. As shown in Fig. 5Go, rIL-10 suppressed IL-1{alpha} production by macrophages in response to all pathogens, in a dose-dependent fashion (p < 0.05). In contrast, rIL-4 did not show a significant suppressive effect on pathogen-stimulated IL-1{alpha} production. Therefore, these findings confirm and extend the in vivo findings above with respect to the suppressive effect of IL-10.



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  		FIGURE 5. Modulation of pathogen-stimulated IL-1{alpha} production by IL-4 and IL-10. IL-1{alpha} concentrations were determined by ELISA after 1 h preincubation with or without cytokines and 24-h bacterial stimulation of IL-10-/- macrophages. Values were normalized to the volume of culture supernatant. Each plot indicates the bacterial stimulant, and the x-axis indicates the modulating cytokines. Vertical bar, SD. Statistical differences were determined by Dunnett’s two-tailed t test vs control, preincubated without cytokines. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we tested the hypothesis that a lack of endogenous expression of the Th2-type cytokines IL-4 and IL-10 results in increased periapical inflammation and bone resorption following mixed anaerobic infection in vivo. Our data demonstrate that, surprisingly, IL-10-/-, but not IL-4-/-, mice have significantly increased infection-stimulated resorption compared with wild-type controls. The bone destruction in IL-10-/- mice correlated with 10-fold higher levels of locally produced IL-1{alpha}, previously reported to be the primary mediator of bone resorption in the murine model (6, 25). In contrast, IL-4-/- mice had decreased local IL-1{alpha} expression compared with wild-type controls. Taken together, these findings demonstrate that endogenous IL-10, but not IL-4, suppresses infection-stimulated inflammatory responses, including bone resorption, and suggests that there is functional heterogeneity among the Th2-type cytokines with regard to their regulation of osseous inflammation in vivo.

Similar functional discordance between IL-4 and IL-10 has been reported previously in other systems. For example, IL-10, but not IL-4, plays a critical role in the suppression of experimental autoimmune encephalomyelitis (26). IL-10 attenuates excessive inflammation and weight loss in Pseudomonas lung infections, and improves survival without affecting bacterial burden (27). In contrast, IL-4-deficient mice exhibit enhanced bacterial clearance, reduced inflammation, and improved survival in Staphylococcus aureus-induced septic arthritis (28).

The elevated IL-1{alpha} expression in IL-10-/- mice was not unexpected, given previous reports that IL-1 production in vitro (29, 30) and serum monokine levels in vivo are increased by IL-10 neutralization (31). Therefore, we confirmed the suppressive effect of both cytokines on pathogen-stimulated IL-1{alpha} production by resident peritoneal macrophages in vitro. The IL-10-/- strain was used as a source of peritoneal macrophages to circumvent the effect of endogenous IL-10 on modulating recombinant cytokines. IL-10 was found to strongly suppress the levels of IL-1{alpha} in a dose-dependent fashion, whereas IL-4 had only weak and variable inhibitory effects. These data are consistent with findings that IL-10 is a more potent suppressor of inflammatory cytokines than IL-4 in vitro (10, 11, 12, 13).

Whereas both IL-4 and IL-10 reduce steady-state levels of IL-1 mRNA, there are reported differences in their suppressive mechanisms (14, 32, 33, 34, 35, 36, 37, 38). IL-10 inhibits IL-1 at the transcriptional level (39), likely by preventing LPS-stimulated activation of NF-{kappa}B in monocytes (14), which is involved in the induction of many inflammatory cytokine genes. In contrast, IL-4 has weak effects on transcription (35, 36), but shows little modulation of NF-{kappa}B (14). Both cytokines may also inhibit IL-1 via posttranscriptional mechanisms (14, 33, 35, 38). IL-4 enhances LPS-stimulated IL-1 mRNA degradation (14, 35), a process dependent on de novo protein synthesis (33). IL-10 also decreases mRNA stability through an AU-rich cluster present in the 3' untranslated region of a number of inflammatory cytokine genes (38). The destabilizing effect of IL-10 was relatively selective, because the stability of mRNAs was not modulated by IL-4. In the present study, rIL-4 showed a modest suppressive effect on LPS and S. intermedius-stimulated IL-1{alpha} production, but did not inhibit IL-1{alpha} stimulated by Gram-negative bacteria. In contrast, IL-10 showed a more potent suppressive effect for all pathogens except S. intermedius. Finally, IL-4 and IL-10 also increase IL-1R antagonist (34, 40, 41). Taken together, these data suggest that the anti-inflammatory suppressive mechanisms induced by IL-10 on bone are more effective than those mediated by IL-4.

The interaction of IL-10 with its receptor leads to the activation of STAT1, STAT3, and STAT5 (42). STAT3 activation by IL-10 is an important factor in the deactivation of LPS-stimulated macrophages, because LPS-induced inflammatory cytokine production was augmented in mutant mice with a cell type-specific disruption of the STAT3 gene in macrophages and neutrophils (43). In the present study, strong IL-10 suppression was preferentially exerted on IL-1{alpha} production induced by LPS and Gram-negative bacteria in vitro, consistent with a STAT3-mediated effect. However, there was also obvious inhibition by rIL-10 of Gram-positive bacterial stimulation of IL-1{alpha}. The dramatic (10-fold) increase in IL-1{alpha} levels in IL-10-/- mice suggests that Gram-negative bacteria were likely the most important pathogens in the mixed anaerobic innoculum in vivo.

Both IL-4 and IL-10 have been reported to inhibit IL-1- and TNF-{alpha}-stimulated bone resorption in vitro (17, 18, 19, 20). IL-1 stimulates resorption both directly and indirectly via expression of cyclooxygenease-2 by osteoblasts (44, 45). Both mediators inhibit bone resorption by suppressing cyclooxygenease-2-dependent prostaglandin E2 synthesis (45, 46). Both IL-4 (17, 18, 19, 47, 48) and IL-10 (20, 49) also inhibit recruitment of osteoclast precursors and their differentiation to mature multinucleated osteoclasts. There was little difference reported in the potency of resorption inhibition between IL-4 and IL-10 (46, 50), suggesting that the differential effects of these mediators on infection-stimulated resorption in the present study is not exerted on bone cells.

In IL-10-/- mice, IL-6 levels were also significantly increased in local inflammatory tissues (Fig. 3Go), whereas the levels of IL-4 were unchanged. Although often considered to be a proinflammatory mediator, recent data suggests that the predominant effects of IL-6 may in fact be Th2-like and anti-inflammatory (51). IL-6 is induced by IL-1 (52, 53, 54), but can act as a feedback inhibitor by suppressing IL-1 transcription (55, 56), and inducing IL-1R antagonist (57, 58). IL-6-/- also exhibit periapical bone destruction that is increased, but less dramatically than we observed in IL-10-/- mice (K. Balto and P. Stashenko, unpublished observations). Therefore, increased expression of IL-6 may represent a compensatory mechanism that is incompletely effective in reducing inflammation in the absence of IL-10.


   Acknowledgments
 
We thank Drs. Szu-I Aurora Liao and Ralph Kent, Jr. for their statistical advice, and Dr. Ricardo Battaglino for helpful discussion.


   Footnotes
 
1 This work was supported by Grant DE-09018 from the National Institutes of Health, and the M. E. Müller Professorship of Bioengineering at Harvard Medical School. Back

2 Address correspondence and reprint requests to Dr. Philip Stashenko, Department of Cytokine Biology, Forsyth Institute, 140 Fenway, Boston, MA 02115. Back

3 Abbreviation used in this paper: ATCC, American Type Culture Collection. Back

Received for publication May 30, 2000. Accepted for publication July 12, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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