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Accelerated Alveolar Bone Loss in Mice Lacking Interleukin-10
1 Department of Periodontics, School of Dentistry, Loma Linda University, Loma Linda, CA, USA; Correspondence: * corresponding author, tatakis.1{at}osu.edu
Interleukin-10 regulates pro-inflammatory cytokines, including those implicated in alveolar bone resorption. We hypothesized that lack of interleukin-10 leads to increased alveolar bone resorption. Male interleukin-10(–/–) mice, on 129/SvEv and C57BL/6J background, were compared with age-, sex-, and strain-matched interleukin-10(+/+) controls for alveolar bone loss. Immunoblotting was used for analysis of serum reactivity against bacteria associated with colitis and periodontitis. Interleukin-10(–/–) mice had significantly greater alveolar bone loss than interleukin-10(+/+) mice (p = 0.006). The 30–40% greater alveolar bone loss in interleukin-10(–/–) mice was evident in both strains, with C57BL/6J interleukin-10(–/–) mice exhibiting the most bone loss. Immunoblotting revealed distinct interleukin-10(–/–) serum reactivity against Bacteroides vulgatus, B. fragilis, Prevotella intermedia, and, to a lesser extent, against B. forsythus. The results of the present study suggest that lack of interleukin-10 leads to accelerated alveolar bone loss.
Key Words: alveolar bone loss • antibodies • disease models • interleukin-10 • mice • knockout
Interleukin (IL)-10, a cytokine with potent anti-inflammatory properties, has been implicated in the regulation of both cellular and humoral immune responses (Rousset et al., 1992; Itoh et al., 1994; Berg et al., 1995, 1996). IL-10(–/–) mice, i.e., mice lacking the IL-10 gene, were developed for study of the role of IL-10 in immune functions (Kuhn et al., 1993). IL-10(–/–) mice develop chronic colitis if normal gut flora is present (Sellon et al., 1998; Madsen et al., 2000), and their susceptibility to colitis is under genetic control, as demonstrated by the various degrees of disease severity in different mouse strains (Berg et al., 1996). These studies indicate that lack of IL-10 can render a host susceptible to a bacteria-initiated chronic inflammatory condition. Animal studies also indicate a significant contribution of IL-10 in the development and progression of arthritis (Walmsley et al., 1996; Brown et al., 1999; Cuzzocrea et al., 2001; Puliti et al., 2002), a chronic inflammatory condition of the joints characterized by connective tissue destruction. IL-10 has a major role in regulating pro-inflammatory cytokine levels in vivo, e.g., interleukin-1 and tumor necrosis factor production in response to various inflammatory stimuli is elevated in the absence of IL-10 or curtailed by IL-10 administration (Berg et al., 1995; Cuzzocrea et al., 2001; Puliti et al., 2002). Periodontitis is a chronic inflammatory and infectious condition (Williams, 1990), characterized by destruction of the tooth attachment apparatus, including alveolar bone loss. The significant role of IL-10 in regulating pro-inflammatory cytokine levels in vivo (Berg et al., 1995; Cuzzocrea et al., 2001; Puliti et al., 2002), and the demonstrated involvement of such cytokines in alveolar bone resorption (Tatakis, 1993; Assuma et al., 1998), led us to hypothesize that lack of IL-10 would lead to increased alveolar bone resorption. Therefore, IL-10(–/–) mice were examined for naturally occurring alveolar bone loss and their humoral immune response to relevant bacterial species.
Experimental Animals Thirteen IL-10(–/–) and 12 IL-10(+/+) mice were used. The IL-10(–/–) and IL-10(+/+) groups were age- (7 mos) and sex- (male) matched. The IL-10(–/–) group consisted of 6 mice on the 129/SvEv background and 7 mice on the C57BL/6J background. The IL-10(+/+) group consisted of 6 mice from each of the 2 strains. All animals were raised and housed in group cages under identical, conventional, specific mouse pathogen-free conditions at the breeding colony of DNAX Research Institute. The genotype of the mice was confirmed by polymerase chain-reaction with the use of DNA extracted from tail tip digests both prior to the commencement of the experiments and after the animals’ death. All live animal work occurred at DNAX Research Institute, and the IACUC of DNAX approved the study protocol. Animals died from carbon dioxide inhalation and were decapitated. Animal heads were stored frozen (–70°C) until further processing. Whole blood was obtained by cardiac puncture at death. Blood was allowed to clot at 4°C overnight, and serum was collected after centrifugation, aliquoted, and stored at –70°C until being tested.
Alveolar Bone Loss Measurements Alveolar bone loss was measured as exposed molar root surface area (mm2) on the lingual aspect of the right mandible and on both the buccal and palatal aspects of the right maxilla. We averaged the 2 maxillary measurements (buccal, palatal) to calculate mean maxillary bone loss, while animal alveolar bone loss was the sum of the mean maxillary and the mandibular bone loss. Measurement reproducibility was determined by repeated measurements, 1 and 2 wks after the initial measurement, of 6 [3 IL-10(–/–), 3 IL-10(+/+)] randomly chosen pairs (left, right) of mandibles.
Bacteria and Bacterial Extracts
Immunoblotting
Data Management and Statistical Analysis
Alveolar Bone Loss Greater alveolar bone loss was evident in IL-10(–/–) mice (Fig. 1A  ) compared with IL-10(+/+) mice (Fig. 1B). The Table provides detailed descriptive statistics for alveolar bone loss. The alveolar bone loss difference between IL-10(–/–) and IL-10(+/+) mice remained statistically significant when the data from each jaw (maxilla/mandible) were analyzed separately (data not shown).
The alveolar bone loss difference between IL-10(–/–) and IL-10(+/+) mice remained statistically significant for each of the 2 examined strains (Table  ). There was a significant difference in alveolar bone loss between the 2 mouse strains when all animals were analyzed together (data not shown). The significant difference in alveolar bone loss between 129/SvEv and C57BL/6J strains remained when IL-10(+/+) and IL-10(–/–) mice were analyzed separately (Table). The overall mean difference between replicate alveolar bone loss measurements was 0.05 mm2, while the mean difference was 0.05 mm2 and 0.04 mm2 for the IL-10(–/–) and IL-10(+/+) animals, respectively. The overall coefficient of variation for replicate alveolar bone loss measurements was 4.4%. When analyzed individually for IL-10(–/–) and IL-10(+/+) animals, the coefficient of variation was 4.0% and 4.8%, respectively. These coefficients of variation account for both positioning and analysis errors.
Humoral Immune Response
The results of this study indicate that IL-10(–/–) mice and age-/ sex-matched IL-10(+/+) mice maintained under identical conditions exhibit significantly different levels of alveolar bone loss; a host lacking IL-10 exhibits much greater periodontal alveolar bone loss. This is consistent with the reported properties of IL-10. The ability of IL-10 to suppress the synthesis of pro-inflammatory cytokines (Berg et al., 1995, 1996; Cuzzocrea et al., 2001; Puliti et al., 2002) may contribute to the observed acceleration of alveolar bone loss in IL-10(–/–) mice, since the same pro-inflammatory cytokines have been implicated in alveolar bone resorption (Tatakis, 1993; Assuma et al., 1998). The findings of the present study, in conjunction with the recent report by Niederman et al.(2001) on the spontaneous alveolar bone loss in P/E-selectin-deficient mice, indicate that different pathogenetic mechanisms may lead to increased susceptibility to alveolar bone loss.
Lack of IL-10 may also have a direct effect on bone homeostasis, since IL-10 has been shown to be a potent inhibitor of osteoclast formation in vitro (Owens et al., 1996). Recently, IL-10 was shown to suppress infection-stimulated periapical bone resorption in vivo (Sasaki et al., 2000). The results of the present study further underscore the in vivo significance of IL-10 for alveolar bone loss, particularly in locations where inflammatory infiltrates occur. The present findings are consistent with the demonstrated involvement of IL-10 in the development and progression of arthritis (Walmsley et al., 1996; Brown et al., 1999; Cuzzocrea et al., 2001; Puliti et al., 2002), another chronic inflammatory condition characterized by bone destruction. IL-10(–/–) mice develop more severe arthritis than IL-10(+/+) mice in response to bacterial infection (Brown et al., 1999) or collagen injection (Cuzzocrea et al., 2001). In contrast, administration of IL-10 to wild-type mice significantly reduces the severity of collagen-induced (Walmsley et al., 1996) or bacteria-induced (Puliti et al., 2002) arthritis. Significantly reduced levels of pro-inflammatory cytokines—such as tumor necrosis factor, IL-1 The inflammatory-bowel-disease-like colitis that develops in the IL-10(–/–) mice is dependent on the presence of normal gut flora (Sellon et al., 1998; Madsen et al., 2000). It remains to be proven whether the severe alveolar bone loss seen in IL-10(–/–) mice is dependent on the presence of commensal oral flora. In this context, it should be noted that the oral environment of these animals was never manipulated, in contrast to what is required for alveolar bone loss induction in other rodent models (Page and Schroeder, 1982). It is anticipated that IL-10(–/–) mice will be much more susceptible to pathogen-induced periodontal alveolar bone loss; this would make IL-10(–/–) mice an excellent model for study of the virulence of various periodontal pathogens and the significance of IL-10 in the regulation of host responses to such pathogens, as suggested by human data (Gemmell and Seymour, 1998).
The present results also indicate that different mouse strains have various propensities for alveolar bone loss, a finding consistent with the reported genetic variability in adult bone density among mouse strains (Beamer et al., 1996). Among 11 inbred strains, C57BL have the lowest and 129 one of the highest levels of femoral bone mineral density (Beamer et al., 1996). This fact, and the association between long bone status and alveolar bone loss (Southard et al., 2000; Wactawski-Wende, 2001), could account for the approximately 30–40% greater alveolar bone loss in C57BL/6J mice relative to age-, sex-, and IL-10 gene status-matched 129/SvEv mice (Table Because of the reported effects of IL-10 on humoral immune response (Rousset et al., 1992; Itoh et al., 1994), we examined the sera of IL-10(–/–) mice for the presence of antibodies against bacteria normally present in the mouse gut flora, such as B. fragilis and B. vulgatus, and species implicated in periodontal disease. The results demonstrate that the humoral immune response of IL-10(–/–) mice against such bacteria is significantly different from that of IL-10(+/+) control mice. IL-10(–/–) mice exhibited distinct reactivity against B. fragilis, B. vulgatus, P. intermedia, and, to a lesser extent, against B. forsythus. IL-10(–/–) sera reacted strongly against proteins in the 45- to 48-kDa range. The identity of such antigen(s) is currently unknown. The altered humoral response to gut flora in IL-10(–/–) mice may or may not be related to the observed alveolar bone loss. In summary, the results of this study indicate that IL-10(–/–) mice are highly susceptible to spontaneous alveolar bone loss and exhibit altered antibody response against bacteria implicated in colitis and periodontitis. These results suggest that IL-10(–/–) mice could be a useful model for studies on alveolar bone loss pathogenesis and elucidation of the interrelationship between alveolar bone loss and immune regulation.
We thank the following for their generous help: Dr. Francis Roy, Loma Linda University (LLU), CA, for expert technical assistance (Western blotting); Dr. Hiroshi Maeda, Okayama University, Japan, for P. gingivalis GroEL (HSP-60); Dr. Casey Chen, University of Southern California, Los Angeles, for P. intermedia; Mr. Kenneth Godowski, Atrix Laboratories Inc., Fort Collins, CO, for B. forsythus; Dr. Paul McMillan, LLU, for making his histometric equipment available to us; Dr. Grenith J. Zimmerman, LLU, for expert statistical advice; and Mr. Richard Tinker and Mr. Richard Cross, LLU, for graphic and photographic support, respectively. This research is supported by Loma Linda University Schools of Dentistry and Medicine, Ohio State University College of Dentistry, University of California Tobacco Related Disease Research Program grant 10RT-0122 (to H.M.F.), and the DNAX Research Institute of Molecular and Cellular Biology. The DNAX Research Institute is supported by Schering-Plough Corporation. Received for publication August 14, 2002. Revision received March 21, 2003. Accepted for publication May 23, 2003.
Journal of Dental Research, Vol. 82, No. 8,
632-635 (2003) This article has been cited by other articles:
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