HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (19) Citing Articles via Google Scholar Google Scholar Articles by Al-Rasheed, A. Articles by Tatakis, D.N. Search for Related Content PubMed PubMed Citation Articles by Al-Rasheed, A. Articles by Tatakis, D.N.

Accelerated Alveolar Bone Loss in Mice Lacking Interleukin-10

¹ Department of Periodontics, School of Dentistry, Loma Linda University, Loma Linda, CA, USA;
² Department of Preventive Dental Science, Periodontics Division, College of Dentistry, King Saud University, Riyadh, Saudi Arabia;
³ Department of Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA, USA;
⁴ Division of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, CA, USA; and
⁵ Section of Periodontology, College of Dentistry, The Ohio State University, 305 West 12th Avenue, PO Box 182357, Columbus, OH 43218-2357, USA;

^* corresponding author, tatakis.1{at}osu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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.

	MATERIALS & METHODS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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
Animal heads were defleshed mechanically, treated with sodium hypochlorite to remove all organic material, and bleached by hydrogen peroxide treatment. Skulls were stained by methylene blue, to help identify the cemento-enamel junction. Jaw images were digitally captured after jaws were positioned on a dissecting microscope stage (Tatakis and Guglielmoni, 2000). All measurements were performed with the use of a computer-assisted image analysis system (Tatakis and Guglielmoni, 2000), and the sole operator performing the measurements was blinded regarding strain and type of animal.

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
Bacteria used were: Porphyromonas gingivalis W83, Bacteroides fragilis ATCC 25285, Bacteroides forsythus ATCC 43037, Bacteroides vulgatus ATCC 8482, and Prevotella intermedia ATCC 25261. Bacterial culture and bacterial extract preparation were performed as previously detailed (Tatakis et al., 2002). Recombinant P. gingivalis GroEL (HSP-60) was partially purified as described (Maeda et al., 1994).

Immunoblotting
Electrophoresis of bacterial extracts and preparation of nitrocellulose membranes for immunoblotting were performed according to published procedures (Tatakis et al., 2002). Blocked membranes were incubated with an appropriate dilution of primary antibody (mouse serum; 1:2000–1:4000), washed, incubated with 1:5000 peroxidase-coupled goat anti-mouse IgG secondary antibody (Zymed Laboratories, Inc., San Francisco, CA, USA), and developed (Tatakis et al., 2002). Negative controls included omission of primary or secondary antibody.

Data Management and Statistical Analysis
Descriptive statistics are presented as mean ± standard deviation (SD). For evaluation of measurement reproducibility, coefficient of variation (%) for replicate measurements was defined by the formula: (standard deviation/mean) x 100. Alveolar bone loss data were analyzed by non-parametric tests (Mann-Whitney U and Kruskal-Wallis). Significance level for rejection of the null hypothesis was set at = 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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).

View larger version (57K): [in this window] [in a new window]   Figure 1. Mandibular lingual aspect of representative IL-10(-/-) (A) and IL-10(+/+) (B) mice of the C57BL/6J strain. The much greater loss of alveolar bone in the IL-10(-/-) mouse (A) compared with the IL-10(+/+) mouse (B) is evident. The jaws depicted were chosen from mice that closely mirror the mean alveolar bone loss value of the respective group. Photographs taken for illustration purposes were not used for measurements.

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
Immunoblotting revealed that both IL-10(-/-) and IL-10(+/+) sera from the 129/SvEv strain exhibited reactivity against a wide range of B. forsythus proteins, although both the patterns of recognized proteins and the strengths of reactivity differed greatly between the 2 types of animals (Fig. 2). IL-10(-/-) sera (Figs. 2B, 2C), in contrast to IL-10(+/+) sera (Figs. 2D, 2E), reacted strongly against B. fragilis, B. vulgatus, and P. intermedia proteins, most prominently in the 45- to 48-kDa range. Both IL-10(-/-) and IL-10(+/+) 129/SvEv sera exhibited very weak, if any, reactivity against P. gingivalis GroEL (HSP-60). These results were consistent among the tested IL-10(-/-) and IL-10(+/+) animals of the 129/SvEv strain, while no reactivity was observed in any of the negative controls (data not shown). The difference in reactivity between IL-10(-/-) and IL-10(+/+) sera was also prominent in the C57BL/6J strain, although the proteins recognized were generally of higher molecular weight (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 2. Immunoblot analysis with serum from representative male IL-10(-/-) 129/SvEv mice aged 7 mos (panels B and C) and age- and sex-matched IL-10(+/+) 129/SvEv mice (panels D and E). Panel A: Stained SDS-PAGE of bacterial crude extracts. On the left margin, the relative position of the molecular weight standards is marked. Each lane was loaded with 20 µg of protein corresponding to extracts of Porphyromonas gingivalis (lane 1), Bacteroides forsythus (lane 2), Bacteroides fragilis (lane 3), Bacteroides vulgatus (lane 4), Prevotella intermedia (lane 5), and partially purified P. gingivalis GroEL (HSP-60) (lane 6). Panels B, C [IL-10(-/-) 129/SvEv sera] and D, E [IL-10(+/+) 129/SvEv sera]: Western blots of the bacterial extracts under identical conditions; primary antibody (mouse sera) at 1:4000 dilution, secondary antibody at 1:5000 dilution; lanes as in panel A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

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, and IL-6 (Cuzzocrea et al., 2001; Puliti et al., 2002)—are found in IL-10(+/+) mice and wild-type mice supplemented with exogenous IL-10, compared with IL-10(-/-) mice and wild-type mice, respectively. Further evidence suggests an association between arthritis and alveolar bone loss, possibly because of common pathogenetic mechanisms (Mercado et al., 2001).

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.

	ACKNOWLEDGMENTS

 
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 August 14, 2002; Last revision March 21, 2003; Accepted May 23, 2003

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Assuma R, Oates T, Cochran D, Amar S, Graves DT (1998). IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis. J Immunol 160:403–409.[Abstract/Free Full Text]

Beamer WG, Donahue LR, Rosen CJ, Baylink DJ (1996). Genetic variability in adult bone density among inbred strains of mice. Bone 18:397–403.[Medline]

Berg DJ, Kuhn R, Rajewsky K, Muller W, Menon S, Davidson N, et al. (1995). Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest 96:2339–2347.

Berg DJ, Davidson N, Kuhn R, Muller W, Menon S, Holland G, et al. (1996). Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses. J Clin Invest 98:1010–1020.[ISI][Medline]

Brown JP, Zachary JF, Teuscher C, Weis JJ, Wooten RM (1999). Dual role of interleukin-10 in murine Lyme disease: regulation of arthritis severity and host defense. Infect Immun 67:5142–5150.[Abstract/Free Full Text]

Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Britti D, De Maio M, et al. (2001). Absence of endogenous interleukin-10 enhances the evolution of murine type-II collagen-induced arthritis. Eur Cytokine Netw 12:568–580.[ISI][Medline]

Gemmell E, Seymour GJ (1998). Cytokine profiles of cells extracted from humans with periodontal diseases. J Dent Res 77:16–26.[Abstract/Free Full Text]

Itoh K, Inoue T, Ito K, Hirohata S (1994). The interplay of interleukin-10 (IL-10) and interleukin-2 (IL-2) in humoral immune responses: IL-10 synergizes with IL-2 to enhance responses of human B lymphocytes in a mechanism which is different from upregulation of CD25 expression. Cell Immunol 157:478–488.[ISI][Medline]

Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W (1993). Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263–274.[ISI][Medline]

Madsen KL, Doyle JS, Tavernini MM, Jewell LD, Rennie RP, Fedorak RN (2000). Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice. Gastroenterology 118:1094–1105.[ISI][Medline]

Maeda H, Miyamoto M, Hongyo H, Nagai A, Kurihara H, Murayama Y (1994). Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: molecular cloning and sequence analysis of its gene and purification of the recombinant protein. FEMS Microbiol Lett 119:129–135.[ISI][Medline]

Mercado FB, Marshall RI, Klestov AC, Bartold PM (2001). Relationship between rheumatoid arthritis and periodontitis. J Periodontol 72:779–787.[ISI][Medline]

Niederman R, Westernoff T, Lee C, Mark LL, Kawashima N, Ullman-Culler M, et al. (2001). Infection-mediated early-onset periodontal disease in P/E-selectin-deficient mice. J Clin Periodontol 28:569–575.[ISI][Medline]

Owens JM, Gallagher AC, Chambers TJ (1996). IL-10 modulates formation of osteoclasts in murine hemopoietic cultures. J Immunol 157:936–940.[Abstract]

Page RC, Schroeder HE (1982). Periodontitis in man and other animals. A comparative review. New York, NY: Karger.

Puliti M, Von Hunolstein C, Verwaerde C, Bistoni F, Orefici G, Tissi L (2002). Regulatory role of interleukin-10 in experimental group B streptococcal arthritis. Infect Immun 70:2862–2868.[Abstract/Free Full Text]

Rousset F, Garcia E, Defrance T, Peronne C, Vezzio N, Hsu DH, et al. (1992). Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc Natl Acad Sci USA 89:1890–1893.[Abstract/Free Full Text]

Sasaki H, Hou L, Belani A, Wang CY, Uchiyama T, Muller R, et al. (2000). IL-10, but not IL-4, suppresses infection-stimulated bone resorption in vivo. J Immunol 165:3626–3630.[Abstract/Free Full Text]

Sellon RK, Tonkonogy S, Schultz M, Dieleman LA, Grenther W, Balish E, et al. (1998). Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect Immun 66:5224–5231.[Abstract/Free Full Text]

Southard KA, Southard TE, Schlechte JA, Meis PA (2000). The relationship between the density of the alveolar processes and that of post-cranial bone. J Dent Res 79:964–969.[Abstract/Free Full Text]

Tatakis DN (1993). Interleukin-1 and bone metabolism: a review. J Periodontol 64:416–431.[ISI][Medline]

Tatakis DN, Guglielmoni P (2000). HLA-B27 transgenic rats are susceptible to accelerated alveolar bone loss. J Periodontol 71:1395–1400.[ISI][Medline]

Tatakis DN, Guglielmoni P, Fletcher HM (2002). Accelerated alveolar bone loss in HLA-B27 transgenic rats: an adult onset condition. J Rheumatol 29:1244–1251.[ISI][Medline]

Wactawski-Wende J (2001). Periodontal diseases and osteoporosis: association and mechanisms. Ann Periodontol 6:197–208.[Medline]

Walmsley M, Katsikis PD, Abney E, Parry S, Williams RO, Maini RN, et al. (1996). Interleukin-10 inhibition of the progression of established collagen-induced arthritis. Arthritis Rheum 39:495–503.[ISI][Medline]

Williams RC (1990). Periodontal disease. N Engl J Med 322:373–382.[ISI][Medline]

This article has been cited by other articles:

				H. Sasaki, N. Suzuki, R. Kent Jr., N. Kawashima, J. Takeda, and P. Stashenko T Cell Response Mediated by Myeloid Cell-Derived IL-12 Is Responsible for Porphyromonas gingivalis-Induced Periodontitis in IL-10-Deficient Mice J. Immunol., May 1, 2008; 180(9): 6193 - 6198. [Abstract] [Full Text] [PDF]

				Y. Houri-Haddad, W.A. Soskolne, A. Halabi, and L. Shapira IL-10 Gene Transfer Attenuates P. gingivalis-induced Inflammation J. Dent. Res., June 1, 2007; 86(6): 560 - 564. [Abstract] [Full Text] [PDF]

				X. Zhang and Y.-T. A. Teng Interleukin-10 Inhibits Gram-Negative-Microbe-Specific Human Receptor Activator of NF-{kappa}B Ligand-Positive CD4+-Th1-Cell- Associated Alveolar Bone Loss In Vivo. Infect. Immun., August 1, 2006; 74(8): 4927 - 4931. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (19) Citing Articles via Google Scholar Google Scholar Articles by Al-Rasheed, A. Articles by Tatakis, D.N. Search for Related Content PubMed PubMed Citation Articles by Al-Rasheed, A. Articles by Tatakis, D.N.