Immunization Enhances Inflammation and Tissue Destruction in Response to Porphyromonas gingivalis
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感染与免疫杂志 2006年第4期
Department of Periodontology and Oral Biology, School of Dental Medicine, Boston University, Boston, Massachusetts
ABSTRACT
It is well established that host-bacterium interactions play a critical role in the initiation and progression of periodontal diseases. By the use of inhibitors, it has been shown that mediators associated with the innate immune response significantly contribute to the disease process. Less is known regarding the role of the acquired immune response. To investigate mechanisms by which the acquired immune response to Porphyromonas gingivalis could affect connective tissue, we used a well-documented calvarial model to study host-bacterium interactions. Injection of P. gingivalis stimulated gamma interferon, interleukin 6, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 expression as determined by real-time PCR. Prior immunization against P. gingivalis significantly enhanced the mRNA levels of these cytokines and chemokines. Similarly, immunization significantly increased and prolonged the formation of a polymorphonuclear leukocyte and mononuclear cell infiltrate (P < 0.05). In addition, the area of connective tissue destruction, osteoclastogenesis, bone loss, mRNA expression of proapoptotic genes, and degree of fibroblast apoptosis were increased in immunized mice (P < 0.05). These results indicate that activation of the acquired immunity by P. gingivalis increases the inflammatory and destructive responses which occur in part through up-regulating the innate immune response and enhancing osteoclastogenesis and fibroblast apoptosis.
INTRODUCTION
Periodontal diseases are highly prevalent chronic inflammatory disorders induced by a bacterial biofilm that forms on teeth (25, 42). The destructive form of periodontal disease, periodontitis, affects approximately 15 to 20% of the U.S. population and is one of the most significant causes of tooth loss in adults. Periodontitis is characterized by irreversible destruction of both soft tissue and bone and is one of the most prevalent forms of bone pathology in humans. Periodontal tissue destruction appears to result from a complex interplay between the host response and specific plaque microorganisms, such as Porphyromonas gingivalis (15, 37). P. gingivalis is a gram-negative anaerobic bacterium that induces a host response that leads to soft and hard tissue loss in periodontitis (17, 20).
Both the innate and acquired arms of the immune response are believed to have roles in periodontal disease progression (15, 38). We have previously demonstrated that interleukin 1 (IL-1) and tumor necrosis factor (TNF) cytokines produced by the innate immune response are significant contributors to bone destruction in a nonhuman primate model (4, 8). Specific inhibitors of IL-1/TNF activity reduced recruitment of inflammatory cells in close proximity to bone, the number of osteoclasts formed, bone loss, and loss of attachment. Inhibition of IL-1 alone significantly reduced inflammation, connective tissue attachment loss, and bone resorption induced by periodontal pathogens (9). Similarly, inhibition of prostaglandins has been shown to significantly reduce inflammation and bone loss in animal models of periodontal disease (28, 43). Conversely, the pivotal role in periodontal bone loss of CD4+ T cells of the acquired immune system was demonstrated in studies of lymphocyte-immunodeficient mice challenged by oral infection with Porphyromonas gingivalis (5) or Actinobacillus actinomycetemcomitans (39). Furthermore, osteoprotegerin ligand, a bone-resorbing factor produced by activated lymphocytes, was a key modulator in a murine model of periodontal bone loss (39).
The inflammatory response in periodontal disease is thought to contribute to the loss of bone, connective tissue, and critical matrix-producing cells (15, 42). It is noteworthy that loss of fibroblasts is one of the most distinctive cellular changes that occur in progressing periodontal disease (45). Fibroblastic cells in patients with periodontitis have the highest rate of apoptosis among the various cells in the gingiva and are observed predominantly in areas where inflammatory cells have been recruited (26, 40). This association between leukocytes and fibroblast apoptosis was subsequently confirmed by the observation that P. gingivalis stimulates fibroblast apoptosis in vivo by induction of TNF activity of the innate immune response (18). The clinical significance of fibroblast apoptosis has recently been suggested by linking it to loss of attachment, an early feature of periodontitis that precedes loss of bone (11).
The purpose of experiments described here was to investigate the degree to which the addition of the acquired immune response augments cytokine expression, the destruction of soft tissue and bone, and apoptosis of fibroblastic cells induced by inoculation of P. gingivalis. These experiments provide insight into mechanisms by which host-bacterium interactions result in tissue loss in conditions such as periodontal disease.
MATERIALS AND METHODS
Animals. Eight-week-old CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were immunized and subsequently challenged with P. gingivalis as described below. Anesthesia was achieved with ketamine (80 mg/kg of body weight) and xylazine (10 mg/kg) in sterile phosphate-buffered saline (PBS) delivered intraperitoneally (i.p.). All animal protocols were approved by the Institutional Animal Care and Use Committee of Boston University Medical Center.
Bacterial inoculation and experimental design. We have previously demonstrated that P. gingivalis strain 381 reproducibly induces formation of an inflammatory infiltrate, proapoptotic gene expression, and fibroblast apoptosis when inoculated into connective tissue (19, 29). Broth-grown P. gingivalis strain 381 in logarithmic growth phase was collected and suspended in sterile PBS. For immunization, bacteria were fixed with 1% paraformaldehyde for 4 hours just prior to injection. An inoculum of 2.5 x 108 bacteria in 50 μl of sterile PBS was injected subcutaneously into the dorsal dermis of animals once weekly for three consecutive weeks. Animals undergoing this protocol represent the immunized group in this study, and we have previously shown that this protocol results in activation of the acquired immune response 1 week after the third inoculation (7). Control animals were sham immunized with an equal volume of sterile PBS according to the same scheme; these animals are the sham-immunized (i.e., nonimmunized) group in this study. Four weeks after the first inoculation, both the immunized and the sham-immunized animals were inoculated with live P. gingivalis in the scalp as described previously (18, 27). Briefly, mice were inoculated by supraperiosteal injection at the midline of the scalp between the ears with P. gingivalis (2 x 108 bacteria), and animals were subsequently euthanized 0, 5, or 8 days later for histologic analysis and at 0, 3, or 5 days for mRNA analysis. In some cases, vehicle alone, sterile PBS, was inoculated into the scalp.
Preparation of histologic specimens and RNA isolation. Calvaria with intact soft tissue were prepared for fixation, embedding in paraffin, and sectioning as previously described (19). For mRNA analysis, the soft tissue was immediately frozen in liquid nitrogen and pulverized, and total RNA was extracted twice with Trizol reagent (Life Technologies Inc., Rockville, MD) according to the manufacturer's instructions.
mRNA expression. The mRNA levels of cytokines, chemokines, and proapoptotic factors were assessed by real-time PCR using primers and probe sets purchased from Applied Biosystems (Foster City, CA) as previously described (2). Briefly, 2 μg of total RNA was used from each sample and Taqman reagents were used for first-strand cDNA synthesis and amplification. Results were normalized with an 18S ribosomal primer and probe set. Each experiment was performed three times, and the results from the three separate experiments were combined in order to derive mean values that could be tested for statistical significance. The mRNA levels of the experimental groups were compared to baseline levels which were established with RNA isolated from the scalp of animals that received no injections of P. gingivalis or vehicle. The mRNA values of proinflammatory and proapoptotic factors were normalized to the baseline value, which was set at 1, and data were expressed as increases in stimulation.
Histomorphometric analysis. To assess the inflammatory response, 5-μm hematoxylin-and-eosin-stained sections were examined at x1,000 magnification at the center of the inflammatory lesion and three flanking fields on each side so that a total of seven fields were examined per specimen. The number of polymorphonuclear leukocytes (PMNs) and mononuclear cells was determined in each field, and the results were expressed as mean values per field. The degree of tissue destruction 5 days after inoculation was determined by measuring the area that contained cells which were clearly necrotic. We have previously shown that this area is well defined at the 5-day time point (29).
Osteoclast counts and bone destruction. Osteoclasts were identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells lining the bone between coronal and occipital sutures. The area of bone loss was determined with van Geisson-stained slides using Image ProPlus software (Media Cybernetics, Silver Spring, MD), as previously described (19). Bone loss was assessed by subtracting each value from the value for the baseline group which did not receive injection of bacteria.
Apoptosis assay. Apoptotic cells were detected by an in situ terminal deoxynucleotidyltransferase-mediated dUTP (TdT)-biotin nick end labeling (TUNEL) assay using a TACS 2 TdT kit with nuclear fast red counterstain (Trevigen, Gaithersburg, MD). The number of TUNEL-positive fibroblastic cells between the occipital and coronal sutures was counted at x1,000 magnification. Apoptotic fibroblasts were distinguished by their characteristic appearance. In selected experiments, the number of apoptotic fibroblasts was confirmed by identifying cells that were both TUNEL positive and strongly vimentin positive as we have previously described (29).
Data analysis. Histologic sections were examined under blind conditions by one examiner, and measurements were independently confirmed by a second examiner. The mouse was the unit of measurement, and for each data point, there were at least six mice. Data are presented as means ± standard errors of the means (SEMs). Statistical analysis between immunized and sham-immunized groups for a given parameter was established by Student's t test, with P values of <0.05 considered statistically significant. The real-time PCR data from three separate experiments were combined, and results were expressed as means ± SEMs. Statistical analysis between immunized and sham-immunized groups for a given molecular mediator was established by Student's t test, with P values of <0.05 considered statistically significant.
RESULTS
Immunization enhances and prolongs the inflammatory response to bacterial infection. The effect of P. gingivalis on cytokine mRNA levels was established at 3 and 5 days after inoculation (Fig. 1). In the sham-immunized animals, P. gingivalis did not increase cytokine mRNA levels. In contrast, the mRNA levels of IL-6 and gamma interferon (IFN-) were significantly higher in immunized animals challenged with P. gingivalis at both time points (P < 0.05) than in sham-immunized animals (Fig. 1A and B). The levels of chemokines that recruit PMNs and mononuclear cells, monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2), were also examined (Fig. 1C and D). The mRNA levels of both chemokines were significantly higher (P < 0.05) at both time points in immunized mice than in sham-immunized mice.
The impact of immunization on the recruitment of PMNs and mononuclear cells was assessed. The numbers of PMNs were severalfold higher in immunized mice than in sham-immunized mice at both day 5 (Fig. 2A) and day 8 (Fig. 2B). Similarly, the numbers of mononuclear cells were severalfold higher in immunized mice than in sham-immunized mice at day 5 (Fig. 2C) and day 8 (Fig. 2D), and the values were statistically significant at both time points (P < 0.05). Immunized animals that were inoculated with PBS (vehicle alone) served as a negative control; these animals showed no measurable inflammatory cell recruitment.
Immunization enhances destructive events following bacterial infection. Since infection with P. gingivalis leads to bone resorption, the TRAP stain was used to quantify the number of osteoclasts in the histologic sections (Fig. 3). The immunized group had nearly twice the number of osteoclasts compared to sham-immunized mice, and the values were statistically significant (P < 0.05). The amount of bone loss followed a similar pattern (Fig. 3C). Immunized animals had more than twice the area of bone destruction that the sham-immunized animals had, and these values were also significant (P < 0.05). Immunized animals inoculated with PBS vehicle alone showed no measurable bone destruction (data not shown).
To study the impact of the acquired immune response on loss of connective tissue, the area of tissue destruction was measured. Connective tissue destruction was markedly enhanced by immunization (Fig. 4A). There was a 20-fold increased area of soft tissue destruction in the immunized animals 5 days following challenge with P. gingivalis compared to the sham-immunized animals. Immunized animals inoculated with PBS vehicle alone showed no measurable soft tissue destruction (data not shown).
A significant sequela of infection is the loss of cells through apoptosis (30). To determine whether the acquired immune response would significantly enhance apoptosis triggered by P. gingivalis inoculation, the TUNEL assay was carried out. When the mice were immunized, there was a 5.6-fold increase in fibroblast apoptosis on day 5 (Fig. 4B) and a 3.3-fold increase on day 8 (Fig. 4C), and both of these values were significant (P < 0.05). We also examined the mRNA levels of proapoptotic genes to establish how immunization might augment cell apoptosis. TNF-, caspase 3, and Fas ligand (FasL) mRNA levels were measured by real-time PCR (Fig. 5). The immunized group had significantly higher levels of expression of these three genes on both days 3 and 5 compared to the sham-immunized group (P < 0.05).
DISCUSSION
Periodontal pathogens initiate a cascade of events, which include destruction of connective tissue and loss of fibroblasts. Investigation of periodontal disease mechanisms have traditionally focused on the progressive loss of connective tissue integrity which results from the host inflammatory response to infection (16, 31, 43, 46). To date, most studies investigating destructive mechanisms of periodontal pathogens or their products, such as lipopolysaccharide, have focused on the innate immune response. However, there is increasing evidence that the acquired immune response may play an active role in this process (5, 38). The purpose of experiments described here was to investigate further the impact of adding the acquired immune response to P. gingivalis-host interactions in a model where many of the variables could be controlled. The murine calvarial model used in this study is well suited for examining host-bacterium interactions in vivo because it facilitates examination of host-bacterium interactions that affect both soft and hard tissue and allows for delivery of a bacterial inoculum of known size that induces a well-defined spatiotemporal sequence of events (29, 46).
The immunization regimen utilized has previously been shown to result in activation of the acquired immune response (7). That the acquired immune response led to greater tissue loss induced by P. gingivalis is supported by an increased area of soft tissue destruction, greater osteoclastogenesis, and enhanced bone loss. There are several avenues through which these may occur. Cytokine expression was markedly increased by adding the acquired immunity to the host response to P. gingivalis. In immunized mice, the expression of IL-6 and formation of a PMN infiltrate, both of which are associated with the innate response, were enhanced and more persistent than in sham-immunized control animals. Thus, one mechanism by which the acquired immune response may aggravate the destruction of tissue is through further up-regulation of innate immunity. This may occur through IFN-, which is one of the principal cytokines through which acquired immunity enhances the innate response. IFN- stimulates the induction of chemokines that recruit PMNs and monocytes and enhances activity in these cells (1, 36, 41).
The activity of mononuclear cells appears important in explaining the enhanced host responses observed. Mononuclear cells produce many of the factors that lead to connective tissue breakdown and bone resorption (23, 35), and these products include IL-6, IL-1, TNF, RANKL (receptor activator of NF-B ligand), and others that activate matrix metalloproteinases and stimulate osteoclastogenesis (6, 15, 44). The greater infiltration of mononuclear cells in immunized mice in response to P. gingivalis could be explained by the increased levels of MCP-1, a potent chemoattractant for monocytes and some lymphocytes (14). Immunization also led to greater expression of the chemokine MIP-2, which is a chemoattractant for PMNs. Thus, enhanced chemokine expression in immunized mice may represent a mechanism through which the acquired immune response may lead to a more pronounced and prolonged inflammatory infiltrate (1, 36, 41).
The loss of cells through infection-induced apoptosis is widely recognized as contributing to gastritis (21, 32). A similar process may be important in periodontal disease, where it has been shown that apoptosis of fibroblasts is enhanced (12, 24, 26, 34, 40). It has also recently been reported that apoptosis of fibroblastic cells is associated with loss of attachment (11). In the present study, immunized mice had severalfold-higher numbers of apoptotic fibroblasts in comparison to the sham-immunized controls. This may occur since mediators induced by the acquired response may act synergistically with those induced by the innate response to maximally induce apoptosis (33, 47). In support of this, immunized mice had significantly higher levels of the proapoptotic factors TNF-, FasL, and caspase 3. Thus, activation of the acquired immune response may tip the intracellular balance toward apoptosis by induction of proapoptotic genes.
The critical role of plaque bacteria in periodontal pathogenesis is well accepted, and the interactions between bacteria and host are known to be myriad and complex. Bacteria that are shed from the biofilm on the tooth surface invade the periodontium and initiate a cascade that results in cell and tissue loss. Periodontal pathogens, such as P. gingivalis, also express potent proteases that enhance their virulence (3, 22). Given that humans have antibodies against the major periodontal pathogens, invasion by these bacteria or their products is likely to elicit both an innate and acquired immune response (10). We specifically examined proinflammatory gene expression at the mRNA level to measure the in vivo response to a bacterial stimulus with and without prior immunization. Because the levels of expression of the proinflammatory genes examined were significantly higher in the immunized group than in the nonimmunized group, we believe that the response to P. gingivalis is heightened by preimmunization and that this has clinical relevance in the context of chronic periodontitis. However, this study does not address whether the acquired immune response is ultimately protective by limiting invasion, since our goal was to investigate the consequence of the acquired immune response once bacteria were inoculated into a connective tissue environment in close proximity to bone.
To conclude, findings reported here indicate that the acquired immune response enhances the expression of cytokines and chemokines and promotes a more pronounced and prolonged recruitment of inflammatory cells. This in turn is associated with greater area of tissue destruction and enhanced fibroblast apoptosis. These results support earlier studies which indicate that the acquired immune response may play a significant role in the loss of tissue that occurs in response to periodontal pathogens (5, 13, 39). The addition of the acquired immune response may contribute to enhanced loss of tissue through several mechanisms, including up-regulation of the innate response, enhanced osteoclastogenesis, and greater loss of matrix-producing cells through apoptosis.
ACKNOWLEDGMENTS
We thank Renee Cabral for assistance in preparing the histology specimens and Alicia Ruff for help in preparing the manuscript.
This work was supported by NIH grants R01DE07559, R01DE15989, and HL76801.
These authors contributed equally to this project.
REFERENCES
1. Ahlin, A., G. Elinder, and J. Palmblad. 1997. Dose-dependent enhancements by interferon-gamma on functional responses of neutrophils from chronic granulomatous disease patients. Blood 89:3396-3401.
2. Alikhani, M., Z. Alikhani, and D. Graves. 2005. FOXO1 functions as a master switch that regulates gene expression necessary for TNF-induced fibroblast apoptosis. J. Biol. Chem. 280:12096-12102.
3. Andrian, E., D. Grenier, and M. Rouabhia. 2004. In vitro models of tissue penetration and destruction by Porphyromonas gingivalis. Infect. Immun. 72:4689-4698.
4. Assuma, R., T. Oates, D. Cochran, S. Amar, and D. Graves. 1998. IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis. J. Immun. 160:403-409.
5. Baker, P., M. Dixon, R. Evans, L. Dufour, E. Johnson, and D. Roopenian. 1999. CD4+ T cells and the proinflammatory cytokines gamma interferon and interleukin-6 contribute to alveolar bone loss in mice. Infect. Immun. 67:2804-2809.
6. Birkedal-Hansen, H. 1993. Role of cytokines and inflammatory mediators in tissue destruction. J. Periodontal Res. 28:500-510.
7. Chae, P., M. Im, F. Gibson, Y. Jiang, and D. Graves. 2002. Mice lacking monocyte chemoattractant protein 1 have enhanced susceptibility to an interstitial polymicrobial infection due to impaired monocyte recruitment. Infect. Immun. 70:3164-3169.
8. Delima, A., T. Oates, R. Assuma, Z. Schwartz, D. Cochran, S. Amar, and D. Graves. 2001. Soluble antagonists to interleukin-1 (IL-1) and tumor necrosis factor (TNF) inhibits loss of tissue attachment in experimental periodontitis. J. Clin. Periodontol. 28:233-240.
9. Delima, A., K. Spyros, S. Amar, and D. T. Graves. 2002. Inflammation and tissue loss caused by periodontal pathogens is reduced by IL-1 antagonists. J. Infect. Dis. 186:511-516.
10. Ebersole, J. L., M. A. Taubman, D. J. Smith, and D. E. Frey. 1986. Human immune responses to oral microorganisms: patterns of systemic antibody levels to Bacteroides species. Infect. Immun. 51:507-513.
11. Ekuni, D., T. Tomofuji, R. Yamanaka, K. Tachibana, T. Yamamoto, and T. Watanabe. 2005. Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases. J. Periodontol. 76:43-48.
12. Gamonal, J., A. Bascones, A. Acevedo, E. Blanco, and A. Silva. 2001. Apoptosis in chronic adult periodontitis analyzed by in situ DNA breaks, electron microscopy, and immunohistochemistry. J. Periodontol. 72:517-525.
13. Gemmell, E., C. Carter, D. Grieco, P. Sugerman, and G. Seymour. 2002. P. gingivalis-specific T-cell lines produce Th1 and Th2 cytokines. J. Dent. Res. 81:303-307.
14. Graves, D. 1999. The potential role of chemokines and inflammatory cytokines in periodontal disease progression. Clin. Infect. Dis. 28:482-490.
15. Graves, D., and D. Cochran. 2003. The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction. J. Periodontol. 74:391-401.
16. Graves, D., A. Delima, R. Assuma, S. Amar, T. Oates, and D. Cochran. 1998. Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis. J. Periodontol. 69:1419-1425.
17. Graves, D., Y. Jiang, and C. Genco. 2000. Periodontal disease: bacterial virulence factors, host response and impact on systemic health. Curr. Opin. Infect. Dis. 13:227-232.
18. Graves, D., M. Oskoui, S. Volejnikova, G. Naguib, S. Cai, T. Desta, A. Kakouras, and Y. Jiang. 2001. Tumor necrosis factor modulates fibroblast apoptosis, PMN recruitment, and osteoclast formation in response to P. gingivalis infection. J. Dent. Res. 80:1875-1879.
19. He, H., R. Liu, T. Desta, C. Leone, L. Gerstenfeld, and D. Graves. 2004. Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss. Endocrinology 145:447-452.
20. Holt, S., J. Ebersole, J. Felton, M. Brunsvold, and K. Kornman. 1988. Implantation of Bacteroides gingivalis in non-human primates initiates progression of periodontitis. Science 239:55-57.
21. Ierardi, E., A. Di Leo, M. Barone, S. Marangi, O. Burattini, A. Panares, M. Margiotta, R. Francavilla, C. Panella, A. Francavilla, and R. Cuomo. 2003. Tumor necrosis factor alpha and apoptosis in Helicobacter pylori related progressive gastric damage: a possible mechanism of immune system involvement in epithelial turnover regulation. Immunopharmacol. Immunotoxicol. 25:203-211.
22. Imamura, T., J. Travis, and J. Potempa. 2003. The biphasic virulence activities of gingipains: activation and inactivation of host proteins. Curr. Protein Pept. Sci. 4:443-450.
23. Ingham, E., and J. Fisher. 2005. The role of macrophages in osteolysis of total joint replacement. Biomaterials 26:1271-1286.
24. Isogai, E., H. Isogai, K. Kimura, N. Fujii, S. Takagi, K. Hirose, and M. Hayashi. 1996. In vivo induction of apoptosis and immune responses in mice by administration of lipopolysaccharide from Porphyromonas gingivalis. Infect. Immun. 64:1461-1466.
25. Kinane, D., and D. Lappin. 2001. Clinical, pathological and immunological aspects of periodontal disease. Acta Odontol. Scand. 59:154-160.
26. Koulouri, O., D. Lapping, M. Radvar, and D. Kinane. 1999. Cell division, synthetic capacity and apoptosis in periodontal lesions analyzed by in situ hybridization and immunohistochemistry. J. Clin. Periodontol. 26:552-559.
27. Li, H., J. Suzuki, E. Bayna, F. Zhang, M. Dalle, A. Clark, R. Engler, and W. Lew. 2002. Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT1 receptors. Am. J. Physiol. Heart Circ. Physiol. 282:H461-H467.
28. Li, K. L., R. Vogel, M. K. Jeffcoat, M. C. Alfano, M. A. Smith, J. G. Collins, and S. Offenbacher. 1996. The effect of ketoprofen creams on periodontal disease in rhesus monkeys. J. Periodontal Res. 31:525-532.
29. Liu, R., T. Desta, H. He, and D. Graves. 2004. Diabetes alters the response to bacteria by enhancing fibroblast apoptosis. Endocrinology 145:2997-3003.
30. Menaker, R. J., and N. L. Jones. 2003. Fascination with bacteria-triggered cell death: the significance of Fas-mediated apoptosis during bacterial infection in vivo. Microbes Infect. 5:1149-1158.
31. Nyman, S., H. Schroeder, and J. Lindhe. 1979. Suppression of inflammation and bone resorption by indomethacin during experimental periodontitis in dogs. J. Periodontol. 50:450-461.
32. Perfetto, B., E. Buommino, N. Canozo, I. Paoletti, F. Corrado, R. Greco, and G. Donnarumma. 2004. Interferon-gamma cooperates with Helicobacter pylori to induce iNOS-related apoptosis in AGS gastric adenocarcinoma cells. Res. Microbiol. 155:259-266.
33. Pommier, Y., O. Sordet, S. Antony, R. L. Hayward, and K. W. Kohn. 2004. Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 23:2934-2949.
34. Preshaw, P., R. Schifferle, and J. Walters. 1999. Porphyromonas gingivalis lipopolysaccharide delays human polymorphonuclear leukocyte apoptosis in vitro. J. Periodontal Res. 34:197-202.
35. Reddy, S. V. 2004. Regulatory mechanisms operative in osteoclasts. Crit. Rev. Eukaryot. Gene Expr. 14:255-270.
36. Schroder, K., P. Hertzog, T. Ravasi, and D. Hume. 2004. Interferon-gamma: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75:163-189.
37. Socransky, S., A. Haffajee, L. Ximenez-Fyvie, M. Feres, and D. Mager. 1999. Ecological considerations in the treatment of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis periodontal infections. Periodontol. 2000 20:341-362.
38. Teng, Y. 2003. The role of acquired immunity and periodontal disease progression. Crit. Rev. Oral Biol. Med. 14:237-252.
39. Teng, Y., H. Nguyen, X. Gao, Y. Kong, R. Gorczynski, B. Singh, R. Ellen, and J. Penninger. 2000. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J. Clin. Investig. 106:R59-R67.
40. Tonetti, M., D. Cortellini, and N. Lang. 1998. In situ detection of apoptosis at sites of chronic bacterially induced inflammation in human gingiva. Infect. Immun. 66:5190-5195.
41. Valente, A., J. Xie, M. Abramova, U. Wenzel, H. Abboud, and D. Graves. 1998. Transcriptional regulation of monocyte chemotactic protein-1 in human osteoblastic cells in response to interferon-. J. Immunol. 161:3719-3728.
42. Williams, R. 1990. Periodontal disease. N. Engl. J. Med. 322:373-382.
43. Williams, R., M. Jeffcoat, M. Kaplan, P. Goldhaber, H. Johnson, and W. Wechter. 1985. Flurbiprofen: a potent inhibitor of alveolar bone resorption in beagles. Science 227:640-642.
44. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio, N. Udagawa, N. Takahashi, and T. Suda. 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95:3597-3602.
45. Zappa, U., M. Reinking-Zappa, H. Graf, and D. Chase. 1992. Cell populations associated with active probing attachment loss. J. Periodontol. 63:748-752.
46. Zubery, Y., C. Dunstan, B. Story, L. Kesavalu, J. Ebersole, S. Holt, and B. Boyce. 1998. Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin. Infect. Immun. 66:4158-4162.
47. Zusman, I., P. Gurevich, E. Gurevich, and H. Ben-Hur. 2001. The immune system, apoptosis and apoptosis-related proteins in human ovarian tumors (a review). Int. J. Oncol. 18:965-972.(Cataldo W. Leone, Haneen )
ABSTRACT
It is well established that host-bacterium interactions play a critical role in the initiation and progression of periodontal diseases. By the use of inhibitors, it has been shown that mediators associated with the innate immune response significantly contribute to the disease process. Less is known regarding the role of the acquired immune response. To investigate mechanisms by which the acquired immune response to Porphyromonas gingivalis could affect connective tissue, we used a well-documented calvarial model to study host-bacterium interactions. Injection of P. gingivalis stimulated gamma interferon, interleukin 6, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 expression as determined by real-time PCR. Prior immunization against P. gingivalis significantly enhanced the mRNA levels of these cytokines and chemokines. Similarly, immunization significantly increased and prolonged the formation of a polymorphonuclear leukocyte and mononuclear cell infiltrate (P < 0.05). In addition, the area of connective tissue destruction, osteoclastogenesis, bone loss, mRNA expression of proapoptotic genes, and degree of fibroblast apoptosis were increased in immunized mice (P < 0.05). These results indicate that activation of the acquired immunity by P. gingivalis increases the inflammatory and destructive responses which occur in part through up-regulating the innate immune response and enhancing osteoclastogenesis and fibroblast apoptosis.
INTRODUCTION
Periodontal diseases are highly prevalent chronic inflammatory disorders induced by a bacterial biofilm that forms on teeth (25, 42). The destructive form of periodontal disease, periodontitis, affects approximately 15 to 20% of the U.S. population and is one of the most significant causes of tooth loss in adults. Periodontitis is characterized by irreversible destruction of both soft tissue and bone and is one of the most prevalent forms of bone pathology in humans. Periodontal tissue destruction appears to result from a complex interplay between the host response and specific plaque microorganisms, such as Porphyromonas gingivalis (15, 37). P. gingivalis is a gram-negative anaerobic bacterium that induces a host response that leads to soft and hard tissue loss in periodontitis (17, 20).
Both the innate and acquired arms of the immune response are believed to have roles in periodontal disease progression (15, 38). We have previously demonstrated that interleukin 1 (IL-1) and tumor necrosis factor (TNF) cytokines produced by the innate immune response are significant contributors to bone destruction in a nonhuman primate model (4, 8). Specific inhibitors of IL-1/TNF activity reduced recruitment of inflammatory cells in close proximity to bone, the number of osteoclasts formed, bone loss, and loss of attachment. Inhibition of IL-1 alone significantly reduced inflammation, connective tissue attachment loss, and bone resorption induced by periodontal pathogens (9). Similarly, inhibition of prostaglandins has been shown to significantly reduce inflammation and bone loss in animal models of periodontal disease (28, 43). Conversely, the pivotal role in periodontal bone loss of CD4+ T cells of the acquired immune system was demonstrated in studies of lymphocyte-immunodeficient mice challenged by oral infection with Porphyromonas gingivalis (5) or Actinobacillus actinomycetemcomitans (39). Furthermore, osteoprotegerin ligand, a bone-resorbing factor produced by activated lymphocytes, was a key modulator in a murine model of periodontal bone loss (39).
The inflammatory response in periodontal disease is thought to contribute to the loss of bone, connective tissue, and critical matrix-producing cells (15, 42). It is noteworthy that loss of fibroblasts is one of the most distinctive cellular changes that occur in progressing periodontal disease (45). Fibroblastic cells in patients with periodontitis have the highest rate of apoptosis among the various cells in the gingiva and are observed predominantly in areas where inflammatory cells have been recruited (26, 40). This association between leukocytes and fibroblast apoptosis was subsequently confirmed by the observation that P. gingivalis stimulates fibroblast apoptosis in vivo by induction of TNF activity of the innate immune response (18). The clinical significance of fibroblast apoptosis has recently been suggested by linking it to loss of attachment, an early feature of periodontitis that precedes loss of bone (11).
The purpose of experiments described here was to investigate the degree to which the addition of the acquired immune response augments cytokine expression, the destruction of soft tissue and bone, and apoptosis of fibroblastic cells induced by inoculation of P. gingivalis. These experiments provide insight into mechanisms by which host-bacterium interactions result in tissue loss in conditions such as periodontal disease.
MATERIALS AND METHODS
Animals. Eight-week-old CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were immunized and subsequently challenged with P. gingivalis as described below. Anesthesia was achieved with ketamine (80 mg/kg of body weight) and xylazine (10 mg/kg) in sterile phosphate-buffered saline (PBS) delivered intraperitoneally (i.p.). All animal protocols were approved by the Institutional Animal Care and Use Committee of Boston University Medical Center.
Bacterial inoculation and experimental design. We have previously demonstrated that P. gingivalis strain 381 reproducibly induces formation of an inflammatory infiltrate, proapoptotic gene expression, and fibroblast apoptosis when inoculated into connective tissue (19, 29). Broth-grown P. gingivalis strain 381 in logarithmic growth phase was collected and suspended in sterile PBS. For immunization, bacteria were fixed with 1% paraformaldehyde for 4 hours just prior to injection. An inoculum of 2.5 x 108 bacteria in 50 μl of sterile PBS was injected subcutaneously into the dorsal dermis of animals once weekly for three consecutive weeks. Animals undergoing this protocol represent the immunized group in this study, and we have previously shown that this protocol results in activation of the acquired immune response 1 week after the third inoculation (7). Control animals were sham immunized with an equal volume of sterile PBS according to the same scheme; these animals are the sham-immunized (i.e., nonimmunized) group in this study. Four weeks after the first inoculation, both the immunized and the sham-immunized animals were inoculated with live P. gingivalis in the scalp as described previously (18, 27). Briefly, mice were inoculated by supraperiosteal injection at the midline of the scalp between the ears with P. gingivalis (2 x 108 bacteria), and animals were subsequently euthanized 0, 5, or 8 days later for histologic analysis and at 0, 3, or 5 days for mRNA analysis. In some cases, vehicle alone, sterile PBS, was inoculated into the scalp.
Preparation of histologic specimens and RNA isolation. Calvaria with intact soft tissue were prepared for fixation, embedding in paraffin, and sectioning as previously described (19). For mRNA analysis, the soft tissue was immediately frozen in liquid nitrogen and pulverized, and total RNA was extracted twice with Trizol reagent (Life Technologies Inc., Rockville, MD) according to the manufacturer's instructions.
mRNA expression. The mRNA levels of cytokines, chemokines, and proapoptotic factors were assessed by real-time PCR using primers and probe sets purchased from Applied Biosystems (Foster City, CA) as previously described (2). Briefly, 2 μg of total RNA was used from each sample and Taqman reagents were used for first-strand cDNA synthesis and amplification. Results were normalized with an 18S ribosomal primer and probe set. Each experiment was performed three times, and the results from the three separate experiments were combined in order to derive mean values that could be tested for statistical significance. The mRNA levels of the experimental groups were compared to baseline levels which were established with RNA isolated from the scalp of animals that received no injections of P. gingivalis or vehicle. The mRNA values of proinflammatory and proapoptotic factors were normalized to the baseline value, which was set at 1, and data were expressed as increases in stimulation.
Histomorphometric analysis. To assess the inflammatory response, 5-μm hematoxylin-and-eosin-stained sections were examined at x1,000 magnification at the center of the inflammatory lesion and three flanking fields on each side so that a total of seven fields were examined per specimen. The number of polymorphonuclear leukocytes (PMNs) and mononuclear cells was determined in each field, and the results were expressed as mean values per field. The degree of tissue destruction 5 days after inoculation was determined by measuring the area that contained cells which were clearly necrotic. We have previously shown that this area is well defined at the 5-day time point (29).
Osteoclast counts and bone destruction. Osteoclasts were identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells lining the bone between coronal and occipital sutures. The area of bone loss was determined with van Geisson-stained slides using Image ProPlus software (Media Cybernetics, Silver Spring, MD), as previously described (19). Bone loss was assessed by subtracting each value from the value for the baseline group which did not receive injection of bacteria.
Apoptosis assay. Apoptotic cells were detected by an in situ terminal deoxynucleotidyltransferase-mediated dUTP (TdT)-biotin nick end labeling (TUNEL) assay using a TACS 2 TdT kit with nuclear fast red counterstain (Trevigen, Gaithersburg, MD). The number of TUNEL-positive fibroblastic cells between the occipital and coronal sutures was counted at x1,000 magnification. Apoptotic fibroblasts were distinguished by their characteristic appearance. In selected experiments, the number of apoptotic fibroblasts was confirmed by identifying cells that were both TUNEL positive and strongly vimentin positive as we have previously described (29).
Data analysis. Histologic sections were examined under blind conditions by one examiner, and measurements were independently confirmed by a second examiner. The mouse was the unit of measurement, and for each data point, there were at least six mice. Data are presented as means ± standard errors of the means (SEMs). Statistical analysis between immunized and sham-immunized groups for a given parameter was established by Student's t test, with P values of <0.05 considered statistically significant. The real-time PCR data from three separate experiments were combined, and results were expressed as means ± SEMs. Statistical analysis between immunized and sham-immunized groups for a given molecular mediator was established by Student's t test, with P values of <0.05 considered statistically significant.
RESULTS
Immunization enhances and prolongs the inflammatory response to bacterial infection. The effect of P. gingivalis on cytokine mRNA levels was established at 3 and 5 days after inoculation (Fig. 1). In the sham-immunized animals, P. gingivalis did not increase cytokine mRNA levels. In contrast, the mRNA levels of IL-6 and gamma interferon (IFN-) were significantly higher in immunized animals challenged with P. gingivalis at both time points (P < 0.05) than in sham-immunized animals (Fig. 1A and B). The levels of chemokines that recruit PMNs and mononuclear cells, monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2), were also examined (Fig. 1C and D). The mRNA levels of both chemokines were significantly higher (P < 0.05) at both time points in immunized mice than in sham-immunized mice.
The impact of immunization on the recruitment of PMNs and mononuclear cells was assessed. The numbers of PMNs were severalfold higher in immunized mice than in sham-immunized mice at both day 5 (Fig. 2A) and day 8 (Fig. 2B). Similarly, the numbers of mononuclear cells were severalfold higher in immunized mice than in sham-immunized mice at day 5 (Fig. 2C) and day 8 (Fig. 2D), and the values were statistically significant at both time points (P < 0.05). Immunized animals that were inoculated with PBS (vehicle alone) served as a negative control; these animals showed no measurable inflammatory cell recruitment.
Immunization enhances destructive events following bacterial infection. Since infection with P. gingivalis leads to bone resorption, the TRAP stain was used to quantify the number of osteoclasts in the histologic sections (Fig. 3). The immunized group had nearly twice the number of osteoclasts compared to sham-immunized mice, and the values were statistically significant (P < 0.05). The amount of bone loss followed a similar pattern (Fig. 3C). Immunized animals had more than twice the area of bone destruction that the sham-immunized animals had, and these values were also significant (P < 0.05). Immunized animals inoculated with PBS vehicle alone showed no measurable bone destruction (data not shown).
To study the impact of the acquired immune response on loss of connective tissue, the area of tissue destruction was measured. Connective tissue destruction was markedly enhanced by immunization (Fig. 4A). There was a 20-fold increased area of soft tissue destruction in the immunized animals 5 days following challenge with P. gingivalis compared to the sham-immunized animals. Immunized animals inoculated with PBS vehicle alone showed no measurable soft tissue destruction (data not shown).
A significant sequela of infection is the loss of cells through apoptosis (30). To determine whether the acquired immune response would significantly enhance apoptosis triggered by P. gingivalis inoculation, the TUNEL assay was carried out. When the mice were immunized, there was a 5.6-fold increase in fibroblast apoptosis on day 5 (Fig. 4B) and a 3.3-fold increase on day 8 (Fig. 4C), and both of these values were significant (P < 0.05). We also examined the mRNA levels of proapoptotic genes to establish how immunization might augment cell apoptosis. TNF-, caspase 3, and Fas ligand (FasL) mRNA levels were measured by real-time PCR (Fig. 5). The immunized group had significantly higher levels of expression of these three genes on both days 3 and 5 compared to the sham-immunized group (P < 0.05).
DISCUSSION
Periodontal pathogens initiate a cascade of events, which include destruction of connective tissue and loss of fibroblasts. Investigation of periodontal disease mechanisms have traditionally focused on the progressive loss of connective tissue integrity which results from the host inflammatory response to infection (16, 31, 43, 46). To date, most studies investigating destructive mechanisms of periodontal pathogens or their products, such as lipopolysaccharide, have focused on the innate immune response. However, there is increasing evidence that the acquired immune response may play an active role in this process (5, 38). The purpose of experiments described here was to investigate further the impact of adding the acquired immune response to P. gingivalis-host interactions in a model where many of the variables could be controlled. The murine calvarial model used in this study is well suited for examining host-bacterium interactions in vivo because it facilitates examination of host-bacterium interactions that affect both soft and hard tissue and allows for delivery of a bacterial inoculum of known size that induces a well-defined spatiotemporal sequence of events (29, 46).
The immunization regimen utilized has previously been shown to result in activation of the acquired immune response (7). That the acquired immune response led to greater tissue loss induced by P. gingivalis is supported by an increased area of soft tissue destruction, greater osteoclastogenesis, and enhanced bone loss. There are several avenues through which these may occur. Cytokine expression was markedly increased by adding the acquired immunity to the host response to P. gingivalis. In immunized mice, the expression of IL-6 and formation of a PMN infiltrate, both of which are associated with the innate response, were enhanced and more persistent than in sham-immunized control animals. Thus, one mechanism by which the acquired immune response may aggravate the destruction of tissue is through further up-regulation of innate immunity. This may occur through IFN-, which is one of the principal cytokines through which acquired immunity enhances the innate response. IFN- stimulates the induction of chemokines that recruit PMNs and monocytes and enhances activity in these cells (1, 36, 41).
The activity of mononuclear cells appears important in explaining the enhanced host responses observed. Mononuclear cells produce many of the factors that lead to connective tissue breakdown and bone resorption (23, 35), and these products include IL-6, IL-1, TNF, RANKL (receptor activator of NF-B ligand), and others that activate matrix metalloproteinases and stimulate osteoclastogenesis (6, 15, 44). The greater infiltration of mononuclear cells in immunized mice in response to P. gingivalis could be explained by the increased levels of MCP-1, a potent chemoattractant for monocytes and some lymphocytes (14). Immunization also led to greater expression of the chemokine MIP-2, which is a chemoattractant for PMNs. Thus, enhanced chemokine expression in immunized mice may represent a mechanism through which the acquired immune response may lead to a more pronounced and prolonged inflammatory infiltrate (1, 36, 41).
The loss of cells through infection-induced apoptosis is widely recognized as contributing to gastritis (21, 32). A similar process may be important in periodontal disease, where it has been shown that apoptosis of fibroblasts is enhanced (12, 24, 26, 34, 40). It has also recently been reported that apoptosis of fibroblastic cells is associated with loss of attachment (11). In the present study, immunized mice had severalfold-higher numbers of apoptotic fibroblasts in comparison to the sham-immunized controls. This may occur since mediators induced by the acquired response may act synergistically with those induced by the innate response to maximally induce apoptosis (33, 47). In support of this, immunized mice had significantly higher levels of the proapoptotic factors TNF-, FasL, and caspase 3. Thus, activation of the acquired immune response may tip the intracellular balance toward apoptosis by induction of proapoptotic genes.
The critical role of plaque bacteria in periodontal pathogenesis is well accepted, and the interactions between bacteria and host are known to be myriad and complex. Bacteria that are shed from the biofilm on the tooth surface invade the periodontium and initiate a cascade that results in cell and tissue loss. Periodontal pathogens, such as P. gingivalis, also express potent proteases that enhance their virulence (3, 22). Given that humans have antibodies against the major periodontal pathogens, invasion by these bacteria or their products is likely to elicit both an innate and acquired immune response (10). We specifically examined proinflammatory gene expression at the mRNA level to measure the in vivo response to a bacterial stimulus with and without prior immunization. Because the levels of expression of the proinflammatory genes examined were significantly higher in the immunized group than in the nonimmunized group, we believe that the response to P. gingivalis is heightened by preimmunization and that this has clinical relevance in the context of chronic periodontitis. However, this study does not address whether the acquired immune response is ultimately protective by limiting invasion, since our goal was to investigate the consequence of the acquired immune response once bacteria were inoculated into a connective tissue environment in close proximity to bone.
To conclude, findings reported here indicate that the acquired immune response enhances the expression of cytokines and chemokines and promotes a more pronounced and prolonged recruitment of inflammatory cells. This in turn is associated with greater area of tissue destruction and enhanced fibroblast apoptosis. These results support earlier studies which indicate that the acquired immune response may play a significant role in the loss of tissue that occurs in response to periodontal pathogens (5, 13, 39). The addition of the acquired immune response may contribute to enhanced loss of tissue through several mechanisms, including up-regulation of the innate response, enhanced osteoclastogenesis, and greater loss of matrix-producing cells through apoptosis.
ACKNOWLEDGMENTS
We thank Renee Cabral for assistance in preparing the histology specimens and Alicia Ruff for help in preparing the manuscript.
This work was supported by NIH grants R01DE07559, R01DE15989, and HL76801.
These authors contributed equally to this project.
REFERENCES
1. Ahlin, A., G. Elinder, and J. Palmblad. 1997. Dose-dependent enhancements by interferon-gamma on functional responses of neutrophils from chronic granulomatous disease patients. Blood 89:3396-3401.
2. Alikhani, M., Z. Alikhani, and D. Graves. 2005. FOXO1 functions as a master switch that regulates gene expression necessary for TNF-induced fibroblast apoptosis. J. Biol. Chem. 280:12096-12102.
3. Andrian, E., D. Grenier, and M. Rouabhia. 2004. In vitro models of tissue penetration and destruction by Porphyromonas gingivalis. Infect. Immun. 72:4689-4698.
4. Assuma, R., T. Oates, D. Cochran, S. Amar, and D. Graves. 1998. IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis. J. Immun. 160:403-409.
5. Baker, P., M. Dixon, R. Evans, L. Dufour, E. Johnson, and D. Roopenian. 1999. CD4+ T cells and the proinflammatory cytokines gamma interferon and interleukin-6 contribute to alveolar bone loss in mice. Infect. Immun. 67:2804-2809.
6. Birkedal-Hansen, H. 1993. Role of cytokines and inflammatory mediators in tissue destruction. J. Periodontal Res. 28:500-510.
7. Chae, P., M. Im, F. Gibson, Y. Jiang, and D. Graves. 2002. Mice lacking monocyte chemoattractant protein 1 have enhanced susceptibility to an interstitial polymicrobial infection due to impaired monocyte recruitment. Infect. Immun. 70:3164-3169.
8. Delima, A., T. Oates, R. Assuma, Z. Schwartz, D. Cochran, S. Amar, and D. Graves. 2001. Soluble antagonists to interleukin-1 (IL-1) and tumor necrosis factor (TNF) inhibits loss of tissue attachment in experimental periodontitis. J. Clin. Periodontol. 28:233-240.
9. Delima, A., K. Spyros, S. Amar, and D. T. Graves. 2002. Inflammation and tissue loss caused by periodontal pathogens is reduced by IL-1 antagonists. J. Infect. Dis. 186:511-516.
10. Ebersole, J. L., M. A. Taubman, D. J. Smith, and D. E. Frey. 1986. Human immune responses to oral microorganisms: patterns of systemic antibody levels to Bacteroides species. Infect. Immun. 51:507-513.
11. Ekuni, D., T. Tomofuji, R. Yamanaka, K. Tachibana, T. Yamamoto, and T. Watanabe. 2005. Initial apical migration of junctional epithelium in rats following application of lipopolysaccharide and proteases. J. Periodontol. 76:43-48.
12. Gamonal, J., A. Bascones, A. Acevedo, E. Blanco, and A. Silva. 2001. Apoptosis in chronic adult periodontitis analyzed by in situ DNA breaks, electron microscopy, and immunohistochemistry. J. Periodontol. 72:517-525.
13. Gemmell, E., C. Carter, D. Grieco, P. Sugerman, and G. Seymour. 2002. P. gingivalis-specific T-cell lines produce Th1 and Th2 cytokines. J. Dent. Res. 81:303-307.
14. Graves, D. 1999. The potential role of chemokines and inflammatory cytokines in periodontal disease progression. Clin. Infect. Dis. 28:482-490.
15. Graves, D., and D. Cochran. 2003. The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction. J. Periodontol. 74:391-401.
16. Graves, D., A. Delima, R. Assuma, S. Amar, T. Oates, and D. Cochran. 1998. Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis. J. Periodontol. 69:1419-1425.
17. Graves, D., Y. Jiang, and C. Genco. 2000. Periodontal disease: bacterial virulence factors, host response and impact on systemic health. Curr. Opin. Infect. Dis. 13:227-232.
18. Graves, D., M. Oskoui, S. Volejnikova, G. Naguib, S. Cai, T. Desta, A. Kakouras, and Y. Jiang. 2001. Tumor necrosis factor modulates fibroblast apoptosis, PMN recruitment, and osteoclast formation in response to P. gingivalis infection. J. Dent. Res. 80:1875-1879.
19. He, H., R. Liu, T. Desta, C. Leone, L. Gerstenfeld, and D. Graves. 2004. Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss. Endocrinology 145:447-452.
20. Holt, S., J. Ebersole, J. Felton, M. Brunsvold, and K. Kornman. 1988. Implantation of Bacteroides gingivalis in non-human primates initiates progression of periodontitis. Science 239:55-57.
21. Ierardi, E., A. Di Leo, M. Barone, S. Marangi, O. Burattini, A. Panares, M. Margiotta, R. Francavilla, C. Panella, A. Francavilla, and R. Cuomo. 2003. Tumor necrosis factor alpha and apoptosis in Helicobacter pylori related progressive gastric damage: a possible mechanism of immune system involvement in epithelial turnover regulation. Immunopharmacol. Immunotoxicol. 25:203-211.
22. Imamura, T., J. Travis, and J. Potempa. 2003. The biphasic virulence activities of gingipains: activation and inactivation of host proteins. Curr. Protein Pept. Sci. 4:443-450.
23. Ingham, E., and J. Fisher. 2005. The role of macrophages in osteolysis of total joint replacement. Biomaterials 26:1271-1286.
24. Isogai, E., H. Isogai, K. Kimura, N. Fujii, S. Takagi, K. Hirose, and M. Hayashi. 1996. In vivo induction of apoptosis and immune responses in mice by administration of lipopolysaccharide from Porphyromonas gingivalis. Infect. Immun. 64:1461-1466.
25. Kinane, D., and D. Lappin. 2001. Clinical, pathological and immunological aspects of periodontal disease. Acta Odontol. Scand. 59:154-160.
26. Koulouri, O., D. Lapping, M. Radvar, and D. Kinane. 1999. Cell division, synthetic capacity and apoptosis in periodontal lesions analyzed by in situ hybridization and immunohistochemistry. J. Clin. Periodontol. 26:552-559.
27. Li, H., J. Suzuki, E. Bayna, F. Zhang, M. Dalle, A. Clark, R. Engler, and W. Lew. 2002. Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT1 receptors. Am. J. Physiol. Heart Circ. Physiol. 282:H461-H467.
28. Li, K. L., R. Vogel, M. K. Jeffcoat, M. C. Alfano, M. A. Smith, J. G. Collins, and S. Offenbacher. 1996. The effect of ketoprofen creams on periodontal disease in rhesus monkeys. J. Periodontal Res. 31:525-532.
29. Liu, R., T. Desta, H. He, and D. Graves. 2004. Diabetes alters the response to bacteria by enhancing fibroblast apoptosis. Endocrinology 145:2997-3003.
30. Menaker, R. J., and N. L. Jones. 2003. Fascination with bacteria-triggered cell death: the significance of Fas-mediated apoptosis during bacterial infection in vivo. Microbes Infect. 5:1149-1158.
31. Nyman, S., H. Schroeder, and J. Lindhe. 1979. Suppression of inflammation and bone resorption by indomethacin during experimental periodontitis in dogs. J. Periodontol. 50:450-461.
32. Perfetto, B., E. Buommino, N. Canozo, I. Paoletti, F. Corrado, R. Greco, and G. Donnarumma. 2004. Interferon-gamma cooperates with Helicobacter pylori to induce iNOS-related apoptosis in AGS gastric adenocarcinoma cells. Res. Microbiol. 155:259-266.
33. Pommier, Y., O. Sordet, S. Antony, R. L. Hayward, and K. W. Kohn. 2004. Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 23:2934-2949.
34. Preshaw, P., R. Schifferle, and J. Walters. 1999. Porphyromonas gingivalis lipopolysaccharide delays human polymorphonuclear leukocyte apoptosis in vitro. J. Periodontal Res. 34:197-202.
35. Reddy, S. V. 2004. Regulatory mechanisms operative in osteoclasts. Crit. Rev. Eukaryot. Gene Expr. 14:255-270.
36. Schroder, K., P. Hertzog, T. Ravasi, and D. Hume. 2004. Interferon-gamma: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75:163-189.
37. Socransky, S., A. Haffajee, L. Ximenez-Fyvie, M. Feres, and D. Mager. 1999. Ecological considerations in the treatment of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis periodontal infections. Periodontol. 2000 20:341-362.
38. Teng, Y. 2003. The role of acquired immunity and periodontal disease progression. Crit. Rev. Oral Biol. Med. 14:237-252.
39. Teng, Y., H. Nguyen, X. Gao, Y. Kong, R. Gorczynski, B. Singh, R. Ellen, and J. Penninger. 2000. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J. Clin. Investig. 106:R59-R67.
40. Tonetti, M., D. Cortellini, and N. Lang. 1998. In situ detection of apoptosis at sites of chronic bacterially induced inflammation in human gingiva. Infect. Immun. 66:5190-5195.
41. Valente, A., J. Xie, M. Abramova, U. Wenzel, H. Abboud, and D. Graves. 1998. Transcriptional regulation of monocyte chemotactic protein-1 in human osteoblastic cells in response to interferon-. J. Immunol. 161:3719-3728.
42. Williams, R. 1990. Periodontal disease. N. Engl. J. Med. 322:373-382.
43. Williams, R., M. Jeffcoat, M. Kaplan, P. Goldhaber, H. Johnson, and W. Wechter. 1985. Flurbiprofen: a potent inhibitor of alveolar bone resorption in beagles. Science 227:640-642.
44. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio, N. Udagawa, N. Takahashi, and T. Suda. 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95:3597-3602.
45. Zappa, U., M. Reinking-Zappa, H. Graf, and D. Chase. 1992. Cell populations associated with active probing attachment loss. J. Periodontol. 63:748-752.
46. Zubery, Y., C. Dunstan, B. Story, L. Kesavalu, J. Ebersole, S. Holt, and B. Boyce. 1998. Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin. Infect. Immun. 66:4158-4162.
47. Zusman, I., P. Gurevich, E. Gurevich, and H. Ben-Hur. 2001. The immune system, apoptosis and apoptosis-related proteins in human ovarian tumors (a review). Int. J. Oncol. 18:965-972.(Cataldo W. Leone, Haneen )