The synergistic effect of peptidoglycan and lipopolysaccaride on osteoclast formation

The synergistic effect of peptidoglycan and lipopolysaccaride on osteoclast formation Jin Jiang, DDS, MS, PhD,a Jian Zuo, MD,b I. Rita Hurst, DMD,c and L. Shannon Holliday, PhD,d Gainesville, Fla UNIVERSITY OF FLORIDA

Purpose. The purpose of this study was to investigate the mechanisms leading to periapical tissue destruction by the Gram-positive bacterial cell component, peptidoglycan (PGN) and Gram-negative bacterial cell component lipopolysaccaride (LPS). Study design. Osteoclast precursor RAW 264.7 cells were cultured with 50 ng/ml receptor activator of NF-␬B ligand (RANKL) for 72 hours. RANKL was then removed and the cells were treated with various concentrations of PGN in the presence or absence of various concentration of LPS for an additional 48 hours. RT-PCR analysis was performed to examine the presence of receptors on osteoclasts known to be involved in mediating cellular activation in response to PGN (TLR2) and LPS (TLR 4). Results. PGN dose dependently and reproducibly stimulated TRAP-positive multinucleated osteoclast-like (OCL) cell formation. PGN and LPS had synergistic effects on the induction of OCL cell formation. Both unstimulated and RANKL-stimulated RAW 264.7 cells expressed TLR2 and TLR4 mRNA. Conclusion. The results underscore the importance of considering both Gram-positive and Gram-negative bacteria when interpreting findings associated with primary and secondary periapical lesions. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:738-43)

Microorganisms and their products play a primary role in the etiology of periradicular disease.1-3 Primary endodontic infections are mixed Gram-negative and Grampositive, predominately anaerobic bacteria.3,4 However, the microbiota associated with persistent secondary infections is usually composed of a single species or by a lower number of species when compared with primary infections. Gram-positive bacteria are predominant.5-7 With the increased identification of Gram-positive species in secondary endodontic infection, studies of the mechanisms leading to periapical tissue destruction by components of these organisms have appeared.8 The most commonly studied component of Gram-positive bacterial cell walls is peptidoglycan (PGN). PGN is composed of long sugar chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues, which are highly cross-linked via peptide side chains. The peptide side chain consists of alternating L- and D-amino acids, up to 4 or 5 in length, and is a

Assistant Professor, Department of Endodontics, College of Dentistry, University of Florida. b Research Instructor, Department of Orthodontics, College of Dentistry, University of Florida. c Orthodontic Resident, Department of Orthodontics, College of Dentistry, University of Florida. d Assistant Professor, Department of Orthodontics, College of Dentistry, and Department of Anatomy & Cell Biology, College of Medicine, University of Florida. © 2003, Mosby, Inc. All rights reserved. 1079-2104/2003/$30.00 ⫹ 0 doi:10.1016/j.tripleo.2003.08.006

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connected to the COOH group of N-acetylmuramic acid. Among different bacterial species, the structure of the sugar chains is highly conserved, while the composition of the peptide subunits varies.9 Toll-like receptors (TLRs) are a family of mammalian proteins homologous to Drosophila Toll..10 Toll was first identified as a key protein controlling dorsoventral pattern formation during the early development of Drosophila. Recent findings show that Toll plays important roles in the host defense against pathogens.11 TLRs in mammals are believed to be pattern-recognition receptors, which recognize common bacterial structures. Recent evidence has established a model for lipopolysaccaride (LPS) recognition that involves both CD14, a glycosylphosphatidylinositol-linked protein, and an associated signal transducer, Toll-like receptor 4 (TLR4). Therefore, TLR4 is a critical receptor involved in the LPS detection system.12 By contrast, PGN, does not appear to activate TLR4, but rather functions through a second TLR, namely TLR2, to activate an immune response.13 Increased osteoclastic bone resorption plays an important pathogenic role in periradicular bone destruction. Osteoclasts are derived from hematopoietic precursor cells of the monocyte-macrophage lineage. The precursors differentiate into multinucleated mature forms and activate to resorb bone in the specialized microenviroment of bone.14 Stromal cells and osteoblast precursors express a member of the tumor necrosis factor ligand family called receptor activator of NF-␬B ligand (RANKL), also known as osteoprote-

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gerin-ligand (OPGL), osteoclast differentiation factor, and TRANCE. This cell surface ligand stimulates osteoclastogenesis and osteoclast activity by binding to its cognate receptor, RANK, on the surface of osteoclast precursors.15,16 Macrophage-colony stimulating factor (M-CSF) is also required for osteoclast differentiation and survival.17 A soluble receptor, osteoprotegerin (OPG), is also expressed by osteoblast progeny and works to finely tune osteoclast activity.18 In the last step of osteoclast formation, mononuclear osteoclasts fuse with each other to form multinucleated cells (MNCs). We utilized a mouse monocyte cell line (RAW 264.7 cells) as an osteoclast model when stimulated with recombinant RANKL.19,20 RAW 264.7 cells express genes typical of mammalian osteoclasts, including tartrate-resistant acid phosphatase (TRAP), Cathepsin K, integrin ␣V␤3, c-src, and the calcitonin receptor (CTR). Morphologically, they are TRAP-positive multinucleated osteoclast-like cells with the capability of resorbing bone.21 We reported previously that LPS from Gram-negative cell wall components directly stimulated osteoclast formation.19 Several lines of evidence indicate that Gram-positive cell wall components have effects on bone resorption. Raisz et al reported that Gram-positive cell wall component can stimulate bone resorption in vitro as measured by the release of previously incorporated 45Ca.22 Stabholz and Sela showed Gram-positive organism Streptococcus mutans induced large periapical lesions with an intense inflammatory infiltrate in a feline model.23 Safavi and Nichols demonstrated that Gram-positive cell wall components induced bone resorbing cytokines from human monocytes.8 In this study, we examined the role of PGN in osteoclast formation and investigated the synergistic effect of PGN and LPS on osteoclast formation. Furthermore, we investigated the presence of receptors on osteoclasts known to be involved in mediating cellular activation in response to bacterial cell wall components. MATERIALS AND METHODS Cell culture and reagents A recombinant protein composed of glutathione-Stransferase (GST) attached to amino acids 158-316 of mouse RANKL was constructed, expressed in bacteria, and isolated as described previously.19 RAW 264.7 cells (American Type Culture Collection, Rockville, Md) were plated at a density of 20,000 cells/cm2 in 24 well plates with cover slips or 6-well plates in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were cultured in the presence of 50 ng/ml RANKL for 72 hours. After 72 hours, the medium was changed with

various agents and further incubated for another 48 hours for osteoclast differentiation. Each group was run in triplicate and repeated twice. All culture reagents were purchased from Gibco/BRL (Gaithersburg, Md). LPS from E. coli and PGN from S. aureus were purchased from Sigma (St Louis, Mo). TRAP assay Cytochemical staining of TRAP-positive cells was performed as described.24 TRAP-positive cells appeared dark red. TRAP-positive cells with more than 3 nuclei were counted. Values are expressed as mean ⫾ SD of triplicate cultures. Reverse transcription polymerase chain reaction (RT-PCR) RNA was extracted from unstimulated and RANKLstimulated RAW 264.7 cells using TRIZOL agent according to the manufacturer’s instructions (Invitrogen, Carlsbad, Calif). RNA (3 ␮g) was reverse transcripted into cDNA in the presence of oligo (dT) primers, RNase inhibitor, and SuperScript™II at 42°C (Invitrogen, Carlsbad, Calif). After heating at 80°C for 10 min, PCR was performed in a 50 ␮l reaction solution, containing 50 ␮mol/L of each primer, 40 nmol/L of each deoxynucleotide triphosphate, and 1.25 U Taq (Roche, Germany). A thermocycler (Stratagene, La Jolla, Calif) was used with an initial denaturation step of 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and a final elongation step of 72°C for 10 minutes. Amplified products were fractionated by electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. The PCR primers used in this study were TLR2: Forward primer: 5⬘-TCTGGGCAGTCTTGAACATTTG-3⬘ Reverse primer: 5⬘-AGAGTCAGGTGATGGATGTCG-3⬘ TLR4: Forward primer: GCAATGTCTCTGGCAGGTGTA-3⬘ Reverse primer: 5⬘-CAAGGGATGAGAACGCTGAGA-3⬘ GAPDH: Forward primer: 5⬘-TGAAGGTCGGTGTGAACGGATTTGGC-3⬘ Reverse primer: 5⬘CATGTAGGCCATGAGGTCCACCAC-3⬘ RESULTS PGN induces osteoclast formation in a dosedependent manner To test whether PGN directly stimulates osteoclast formation, RAW 264.7 cells were cultured in the presence and absence of various concentrations of PGN.

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Fig 1. PGN and LPS dose– dependently induced osteoclastlike cell formation from RANKL-pretreated RAW 264.7 cells. The number of TRAP-positive multinucleated cells in cultures was counted. Negative control group was culture treated with vehicle (0 ng/ml LPS or 0 ␮g/ml PGN). Values are expressed as mean ⫾ SD of the triplicate samples. *P ⬍ .01 vs. vehicle group.

We found that RAW 264.7 cells did not form osteoclast-like cells (OCLs) in the presence of PGN alone. While LPS does not directly stimulate osteoclast precursors to differentiate into osteoclasts, it does stimulate the later stages of differentiation if the early stages are induced by RANKL.19,25 To test if PGN has a similar action as LPS, osteoclast precursors were cultured with RANKL for 72 hours. At the end of this period of time there were 0-10 OCL cells per well. Cultures were then stimulated for an additional 48 hours with vehicle (as a negative control), LPS (as a positive control) or PGN. Vehicle group as a negative control only generated 12-14 OCL cells per well (Fig 1). In the presence of PGN, these cultures generated up to 253 ⫾ 43 TRAP-positive multinucleated OCL cells. PGN dose dependently and reproducibly stimulated TRAP-positive OCL formation. There was some variation between experiments in the maximal response seen, but the dose response was reproducible (Fig 1). OCL formation in response to stimulation with LPS, in contrast, was greater than PGN stimulation (Fig 1). Synergistic effects of PGN and LPS on induction of osteoclast formation To investigate whether PGN and LPS synergize for induction of osteoclast formation, RAW 264.7 cells were stimulated with RANKL for 72 hours, then stimulated with various concentrations of PGN in the presence or absence of various concentrations of LPS for 48 hours. TRAP-positive OCL cells were counted. As demonstrated in Fig 2, A, a suboptimal dose of LPS (10

Fig 2. Synergistic effect of PGN and LPS on osteoclast-like cell formation in RAW 264.7 cells. A, RAW 264.7 cells were stimulated with various concentration of PGN in the presence or absence of 10 ng/ml of LPS following a 72-hour pretreatment with RANKL. B, RAW 264.7 cells were stimulated with various concentration of LPS in the presence or absence of 10 ␮g/ml of PGN following a 72-hour pretreatment with RANKL. Values are expressed as mean ⫾ SD of the triplicate samples. *P ⬍ .01; significantly different from respective control (A: PGN alone; B: LPS alone).

ng/ml) synergistically enhanced PGN-mediated osteoclast formation. Moreover, a suboptimal dose of PGN (10 ␮g/ml) synergistically enhanced LPS-stimulated osteoclast formation (Fig 2, B). This synergistic effect of PGN and LPS for osteoclast formation was observed not only at low concentrations of PGN and LPS, but also with higher concentrations, which induced maximal amount of osteoclast formation. Osteoclasts express receptors for PGN and LPS To explore potential roles of Toll-like receptors (TLRs) during osteoclast formation, we tested whether

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Fig 3. Expression of LPS and PGN receptors in osteoclastlike cells. Total RNA was prepared from unstimulated and RANKL-stimulated RAW 264.7 cells. Templates for PCR were synthesized with (⫹RT) or without (⫺RT) reverse transcriptase. ⫺RT negative samples were included as controls for genomic DNA contamination. GAPDH mRNA expression was analyzed to verify similar cDNA loading.

mRNA for TLR2 and TLR4 were present in osteoclasts. Both unstimulated (osteoclast precursors) and RANKL-stimulated RAW 264.7 cells (mature osteoclasts) expressed TLR2 and TLR4 mRNA (Fig 3). We did not detect dramatic differences in the expression levels of TLR2 and TLR4 between unstimulated and RANKL-stimulated RAW 264.7 cells. DISCUSSION We show in this study that peptidoglycan from Gram-positive bacterial cell walls can support the late stages of osteoclast formation if the early stages are induced by RANKL. Coincubation of Gram-positive and -negative bacterial components, yielded synergistic effects on osteoclast formation. Moreover, we demonstrated the presence of receptors for PGN and LPS in osteoclasts. Bacteria and their products are the primary cause of pulpal necrosis and periapical lesions. Primary endodontic infection is mixed, comprising Gram-negative and positive and mostly anaerobic microorganisms,1-4 Root canal microbiota of teeth with failed endodontic treatment differs from that normally found in untreated teeth and appears to be a very limited number of Grampositive microbial species.5-7 Persisting bacteria in root

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canals may be those originally present in the primary infection that survive the biomechanical procedures or survive in the missed canals or uninstrumented areas of the canals.26,27 Alternatively, bacteria may originate from contamination during28 or coronal leakage after root canal treatment.29,30 In most of the cases, bacteria do not invade the periapical tissue, although the occurrence of extraradicular infection has been reported in treated and untreated endodontic cases.31-33 How bacteria stimulate bone pathology is still unclear. The accepted paradigm is that the release of soluble bacterial virulence factors invade into periapical tissue and induce local pathology.34,35 They can promote cellular processes that stimulate the degradation of bone and/or inhibit the synthesis of bone matrix.36 We previously reported that LPS was able to induce osteoclast formation directly.19 Because of the enormous clinical significance of Gram-positive infections in the root canal system, we investigated the role of the Gram-positive bacterial cell wall component PGN in osteoclast formation. We found that PGN induced the late stages of osteoclast formation, although less potently than LPS. However, Takami et al recently found that stimulation with LPS, PGN, and other TLR ligands inhibited osteoclast differentiation.37 The discrepancy might be related to different culture conditions of osteoclasts. In the study by Takami et al, either PGN or LPS was added to the cultures with RANKL at the early stage of osteoclast differentiation. They found LPS or PGN inhibited osteoclast differentiation into mature osteoclasts.37 In our study, we showed that LPS or PGN could substitute to RANKL with respect to osteoclast formation during the late stages of differentiation. It is well accepted that osteoclasts are derived from hematopoietic precursor cells of the monocyte-macrophage lineage. Osteoclast precursors produce proinflammatory cytokines such as TNF-␣, IL-1␤, and IL-6 in response to LPS or PGN.36-38 However, LPS does not stimulate mature osteoclasts to produce cytokines.38 In addition, osteoclast precursors possess phagocytic function. When osteoclasts mature, they lose their phagocytic function but develop the ability to resorb bone. It is speculated that at early stages of osteoclast differentiation, LPS or PGN inhibits the differntiation of monocytes/macrophages into nonphagocytic, nonimmune cells such as mature osteoclasts. Meanwhile, they promote cytokine production by the undifferentiated precursors. This process pushes multipotential precursors away from the osteoclast pathway and towards a role in the proinflammatory system. But once the differentiation pathway of osteoclasts is determined, their inflammatory response to LPS or PGN is lost and LPS or PGN promotes their differentiation

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into mature osteoclasts. Further studies will be necessary to elucidate the mechanisms of regulation of cytokine production and differentiation into osteoclasts. Our results show that PGN and LPS have a synergistic effect on the induction of osteoclast formation. Others have reported synergistic effects of PGN with LPS. Flak et al reported synergistic interactions between PGN and LPS in the induction of inflammatory process within hamster trachea epithelial cells.39 Wang et al reported that coadministration of PGN with LPS caused significantly increased concentrations of TNF-␣ and IL-6 in cultures of whole human blood.40 The results of these studies underscore the importance of considering both Gram-positive and Gram-negative bacteria when interpreting findings associated with primary and secondary periapical lesions. The molecular mechanisms involved in bacteria-induced bone pathology still remain obscure. This study represents a step towards unraveling the recognition mechanism for these potentially pathogenic components in the root canal system. Evidence from studies with C3H/HeJ LPS hyporesponsive mice and in vitro experiments led to the discovery that Toll-like receptors act as the signal-transducing coreceptors for the recognition of pathogenic microorganism. The characterization of TLR2- and TLR4-deficient mice demonstrated that TLR is involved in the recognition of LPS, and that TLR2 is involved in the response to PGN.41 Both LPS and PGN act by binding a TLR, activating various tyrosine kinases, and translocating the transcription factor NF-␬B, resulting in biological response.10 TLR4deficient mice have reduced periapical bone destruction following pulpal exposure and infection with controlled Gram-negative and Gram-positive endodontopathic bacteria,35 but have no difference in progression of periapical lesions compared to control mice when infected with uncontrolled bacterial contamination.42 The presence of TLR2 and TLR4 expression on unstimulated and stimulated RAW 264.7 cells is compatible with recent reports showing that osteoclast precursors and purified mature osteoclasts prepared from mouse bone marrow cells expressed TLR2 and TLR4 mRNA.37,38 By gaining a better understanding of the multiple receptors involved in bacterial virulence factor, clinically useful therapeutic approaches might be developed for the treatment of excessive bone resorption. Work was supported by NIH AR47959 to LSH. REFERENCES 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free conventional laboratory rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1965; 20:340-9.

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 2003 2. Moller AJR. Microbial examination of root canals and periapical tissues of human teeth. Odontol tidskrift 1966;74:1-380. 3. Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1994;78:522-30. 4. Sundqvist G. Associations between microbial species in dental root canal infections. Oral Microbiol Immunol 1992;7:257-62. 5. Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998; 31:1-7. 6. Sundqvist G, Figdor D, Persson S, Sjogren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93. 7. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M. Isolation of yeasts and enteric bacteria in root-filled teeth with chronic apical periodontitis. Int Endod J 2001;34:429-34. 8. Safavi KE, Nichols FC. Effects of a bacterial cell wall fragment on monocyte inflammatory function. J Endod 2000;26:153-5. 9. Schleifer KH, Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 1972;36: 407-77. 10. Lien E, Ingalls RR. Toll-like receptors. Crit Care Med 2002;30: S1-S11. 11. Kopp EB, Medzhitov R. The Toll-receptor family and control of innate immunity. Curr Opin Immunol 1999;11:13-8. 12. Takeuchi O, Akira S. Toll-like receptors; their physiological role and signal transduction system. Int Immunopharmacol 2001;1: 625-35. 13. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ. Peptidoglycan- and lipoteichoic acid–induced cell activation is mediated by toll-like receptor 2. J Biol Chem 1999;274:17406-9. 14. Teitelbaum SL. Bone resorption by osteoclasts. Science 2000; 289:1504-8. 15. Kong YY, Boyle WJ, Penninger JM. Osteoprotegerin ligand: a common link between osteoclastogenesis, lymph node formation and lymphocyte development. Immunol Cell Biol 1999;77:18893. 16. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315-323. 17. Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165-76. 18. Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998;139:1329-37. 19. Jiang J, Zuo J, Chen SH, Holliday LS. Calcium hydroxide reduces lipopolysaccharide-stimulated osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:348-54. 20. Krits I, Wysolmerski RB, Holliday LS, Lee BS. Differential Localization of Myosin II Isoforms in Resting and Activated Osteoclasts. Calcif Tissue Int 2002;71:530-8. 21. Hsu H, Lacey DL, Dunstan CR, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci USA 1999;96:3540-5. 22. Raisz LG, Alander C, Eilon G, Whitehead SP, Nuki K. Effects of two bacterial products, muramyl dipeptide and endotoxin, on bone resorption in organ culture. Calcif Tissue Int 1982;34: 365-9. 23. Stabholz A, Sela MN. The role of oral microorganisms in the pathogenesis of periapical pathosis. I. Effect of Streptococcus mutans and its cellular constituents on the dental pulp and periapical tissue of cats. J Endod 1983;9:171-5. 24. Holliday LS, Welgus HG, Fliszar CJ, Veith GM, Jeffrey JJ, Gluck SL. Initiation of osteoclast bone resorption by interstitial collagenase. J Biol Chem 1997;272:22053-8. 25. Zou W, Bar-Shavit Z. Dual modulation of osteoclast differentiation by lipopolysaccharide. J Bone Miner Res 2002;17:1211-8. 26. Fukushima H, Yamamoto K, Hirohata K, Sagawa H, Leung KP,

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27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

Walker CB. Localization and identification of root canal bacteria in clinically asymptomatic periapical pathosis. J Endod 1990;16: 534-8. Sjogren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997; 30:297-306. Siren EK, Haapasalo MP, Ranta K, Salmi P, Kerosuo EN. Microbiological findings and clinical treatment procedures in endodontic cases selected for microbiological investigation. Int Endod J 1997;30:91-5. Torabinejad M, Ung B, Kettering JD. In vitro bacterial penetration of coronally unsealed endodontically treated teeth. J Endod 1990;16:566-9. Ray HA, Trope M. Periapical status of endodontically treated teeth in relation to the technical quality of the root filling and the coronal restoration. Int Endod J 1995;28:12-8. Nair PN, Sjogren U, Krey G, Kahnberg KE, Sundqvist G. Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: a long-term light and electron microscopic follow-up study. J Endod 1990; 16:580-8. Sunde PT, Tronstad L, Eribe ER, Lind PO, Olsen I. Assessment of periradicular microbiota by DNA-DNA hybridization. Endod Dent Traumatol 2000;16:191-6. Siqueira JF Jr. Endodontic infections: concepts, paradigms, and perspectives. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:281-93. Nair PN. Apical periodontitis: a dynamic encounter between root canal infection and host response. Periodontol 2000 1997;13: 121-48. Hou L, Sasaki H, Stashenko P. Toll-like receptor 4 – deficient mice have reduced bone destruction following mixed anaerobic infection. Infect Immun 2000;68:4681-7. Nair SP, Meghji S, Wilson M, Reddi K, White P, Henderson B.

37.

38.

39.

40.

41.

42.

Bacterially induced bone destruction: mechanisms and misconceptions. Infect Immun 1996;64:2371-80. Takami M, Kim N, Rho J, Choi Y. Stimulation by toll-like receptors inhibits osteoclast differentiation. J Immunol 2002; 169:1516-23. Itoh K, Udagawa N, Kobayashi K, et al. Lipopolysaccharide promotes the survival of osteoclasts via toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages. J Immunol 2003;170: 3688-95. Flak TA, Heiss LN, Engle JT, Goldman WE. Synergistic epithelial responses to endotoxin and a naturally occurring muramyl peptide. Infect Immun 2000;68:1235-42. Wang ZM, Liu C, Dziarski R. Chemokines are the main proinflammatory mediators in human monocytes activated by Staphylococcus aureus, peptidoglycan, and endotoxin. J Biol Chem 2000;275:20260-7. Takeuchi O, Hoshino K, Kawai T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and grampositive bacterial cell wall components. Immunity 1999;11:44351. Fouad AF, Acosta AW. Periapical lesion progression and cytokine expression in an LPS hyporesponsive model. Int Endod J 2001;34:506-13.

Reprint requests: Dr. Jin Jiang, DDS, MS, Ph.D. Assistant Professor Department of Endodontics PO Box 100436 College of Dentistry University of Florida Gainesville, FL 32610-0436 [email protected]

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