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Increased Gene Expression of Toll-like Receptors and Antigen-Presenting Cell–related Molecules in the Onset of Experimentally Induced Furcation Lesions of Endodontic Origin in Rat Molars
Basic Research—Biology
Increased Gene Expression of Toll-like Receptors and Antigen-Presenting Cell–related Molecules in the Onset of Experimentally Induced Furcation Lesions of Endodontic Origin in Rat Molars Uraiwan Chokechanachaisakul, DDS,* Tomoatsu Kaneko, DDS, PhD,* Takashi Okiji, DDS, PhD,† Reika Kaneko, DDS, PhD,‡ Mitsuhiro Kaneko, DDS, PhD,* Jun Kawamura, DDS,* Mitsuhiro Sunakawa, DDS, PhD,*§ and Hideaki Suda, DDS, PhD* Abstract Introduction: Early immunopathogenic mechanisms behind pulp infection–induced furcal inflammation have not been well understood. To address the immunopathology of the pulp infection–induced furcal region of the periodontal ligament (PDL), we performed immunohistochemical and quantitative gene expression analyses for toll-like receptors (TLRs) in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. Methods: Furcal inflammation in rat molars was generated by making unsealed pulpotomies that were exposed to the oral environment for 24 hours. Pulpotomized teeth sealed with a temporary filling material and untreated normal teeth served as controls. Gene expression was analyzed with laser capture real-time polymerase chain reaction for TLR-2, TLR-4, and antigen presenting cell (APC)-related molecules (class II MHC, CD83, and CD86). Immunohistochemistry for TLR-2 and TLR-4 was also performed. Results: Messenger RNA expression levels of TLRs and the APC-related molecules in the furcal periodontal ligament were significantly up-regulated in teeth with unsealed pulpotomy. Immunohistochemistry for unsealed pulpotomized teeth revealed that TLRs-expressing cells were predominantly distributed within the PDL beneath the furcal dentin. Conclusions: These results suggested the involvement of innate immune mechanisms involving TLRs and resulting activation of APCs in the early pathogenesis of pulp infection-induced furcal inflammation. (J Endod 2010;36:251–255)
Key Words Class II MHC, gene expression analysis, periodontal ligament, pulpotomy, toll-like receptor
T
he periodontal ligament (PDL) is the soft specialized connective tissue that forms the structural attachment between the tooth and the surrounding alveolar bone (1). Moreover, defense cells such as macrophages and dendritic cells (DCs) reside in the PDL, most likely to maintain local homeostasis and cope with infectious challenges (2, 3). We have recently shown that resident type of DCs in the rat PDL show heterogeneity in terms of morphology and cell surface antigen expression in different regions of the PDL, probably reflecting the differences in the microenvironment, such as the amount of external antigens, in different regions (4). In particular, DCs in the furcal region frequently show the morphologic and phenotypic features of activated antigen-presenting cells (APCs), characterized by their CD86 expression, electrondense lysosomes, and cell-to-cell contacts with lymphocytes. This may be associated with the anatomic proximity of the furcal area to the oral environment. It has been well documented that pulp infection may cause furcal inflammation, most probably via furcal and lateral canals (5–9). However, there is limited information regarding cellular and molecular mechanisms behind the immunopathogenesis of such furcal lesions of endodontic origin. In a previous study, we have shown that messenger RNA expression levels of APC-related molecules are upregulated in the furcal PDL of rat molars after 24 hours of pulp exposure, although the number of class II major histocompatibility complex (MHC) molecule-expressing DCs does not show any increase (4). This suggests the activation/maturation of resident DCs in response to bacterial challenge. Toll-like receptors (TLRs) are known as a family of transmembrane receptors that play a pivotal role in the modulation of immune response by recognizing pathogenassociated molecular patterns (10, 11). The recognition subsequently stimulates a sequence of signaling mechanisms resulting in the production of various cytokines that serve as a link between innate and specific immune mechanisms. TLR-2 and TLR-4 are the most well-defined members of the TLR superfamily: TLR-4 recognizes lipopolysaccharides in gram-negative bacteria, whereas TLR-2 plays a major role in the recognition of various bacterial components such as lipoteichoic acid in gram-positive bacteria, lipoproteins, and peptide glycans (12, 13). The up-regulation of TLR-2 and/or TLR-4 has been shown in bacterially challenged dental pulp, which suggests innate immune responses involving the TLRs as signaling receptors contribute to the
From *Pulp Biology and Endodontics, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; †Division of Cariology, Operative Dentistry and Endodontics, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan; ‡Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan; and §Infection Control Team, University Hospital, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan. Supported by Grants-in-Aid for Scientific Research (No. 19209059 to H.S., No. 18791395 to M.K., No. 18390504 to M.S., No. 20390483 to T. O., and No. 21592411 to T. K.) from the Japan Society for the Promotion of Sciences. Address requests for reprints to Dr Tomoatsu Kaneko, Pulp Biology and Endodontics, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo 113-8549, Japan. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2009.10.005
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Basic Research—Biology pathogenesis of pulp inflammation (14, 15). However, the roles of TLRs in pulp infection-induced furcal inflammation are poorly understood. To further analyze the immunopathology of the pulp infection– induced furcal region of the PDL, we performed immunohistochemical and quantitative gene expression analyses for TLRs in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. Quantitative gene expression analysis for APC-related molecules was also performed to address the link between expression of TLRs and APC activation.
Materials and Methods All experiments were conducted under the approval of the Animal Care Committee at Tokyo Medical and Dental University.
Induction of Furcal Inflammation Seven-week-old adult male Wistar rats (N = 21), each weighting around 200 g, were divided into the following three groups (n = 7 for each group): group 1 (untreated control), normal rats without pulpotomy; group 2 (experimental group), rats with unsealed pulpotomy; and group 3 (sealed control), rats with sealed pulpotomy. In the experimental group (group 2), unsealed pulpotomy was performed in mandibular first molars of the left side. Under pentobarbital sodium anesthesia (30 mg/kg, intraperitoneally), the pulp was exposed from the occlusal surface and most of the coronal pulp tissue was removed with no. 1/2 round burs with the aid of an operating microscope. The pulp chamber was irrigated with 6% sodium hypochlorite solution and 15% EDTA. The teeth were then left open to the oral environment for 24 hours. In sealed controls (group 3), pulpotomy was made in the same manner, and then the cavity was sealed with a temporary restorative material (Caviton; GC Corporation, Tokyo, Japan). Normal rats without pulpotomy (group 1) served as untreated controls. Tissue Preparation Twenty-four hours after the pulpotomy, the animals for light microscopic analysis were anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg) and killed by transcardiac perfusion of 4% paraformaldehyde in phosphate-buffered solution. Both sides of the lower mandibles were retrieved and kept in the same fixative overnight and then demineralized in 14% EDTA on the rotator at 4 C. Immunohistochemistry For immunohistochemistry, the samples (n = 3 for each group) were cut mesiodistally in a linear slicer (LEICA CM 1900; Leica Microsystems, Wetzlar, Germany) at 40 mm in thickness. Immunostaining by the avidin-biotin-peroxidase complex method was performed on 24well plate as previously described (4). After incubation in 3% H2O2 for 5 minutes, the sections were incubated with one of the primary antibodies diluted with phosphate-buffered saline for 24 hours at 4 C. The first polyclonal antibodies used in this study were TLR-2 and TLR-4 (Santa Cruz Biotechnology, CA, USA; dilution: 1:100 and 1:500) with normal horse serum (R.T.U. VECTASTAIN Universal Quick Kit; Vector Laboratories, Inc, Burlingame, CA). The sections were then sequentially incubated with a biotinylated pan-specific secondary antibody (rat adsorbed, R.T.U. VECTASTAIN Universal Quick Kit) for 1 hour and avidinbiotin-peroxidase complex (VECTASTAIN Universal Quick Kit) for 1 hour. The sections were then developed with a 3,3’-diaminobenzidine–H2O2 solution (DAB Substrate Kit, Vector Laboratories) for 3 minutes, counterstained with hematoxylin, and examined under a light microscope. Negative control staining was always performed in parallel by incubating sections with nonimmune serum or phosphate-buffered saline instead of the primary antibodies. 252
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Figure 1. The overall strategy used here for imuunohistochemical and quantitative gene expression analysis of furcal region of the rat molar.
Quantitative Gene Expression Analysis For laser capture real-time polymerase chain reaction analysis, the samples (n = 4 for each group) were prepared in a similar manner as described earlier with the same fixation, and prepared tissues were cut mesiodistally at 80 mm in a linear slicer (Pro 7; Dosaka EM, Kyoto, Japan). The tissue sections were mounted on glass foiled pen slides (Leica). The furcal regions of PDL were dissected and collected by using a laser capture microdissection microscope Leica AS LMD (Leica) (16). In these samples, the total RNA from each sample was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with RNeasy Mini kits (Qiagen, Valencia, CA) in accordance with manufacturers’ protocols as we described before (17). The optical density (OD)260/280 ratio was used to evaluate the purity of the nucleic acid samples, and the quality of the extracted total RNA was determined with the BioPhotometer (Eppendorf, Hamburg, Germany). Then, first-strand complementary DNA synthesis was performed with TaqMan reverse transcription reagents (Applied Biosystems). Probe and primer sets of TaqMan Gene Expression Assays (NM_019178.1;TLR-4, NM_198769.2; TLR-2, Rn01768599_g1; Class II MHC DM alpha, Rn01434593_m1; CD83, Rn00571654_m1; CD86, Rn99999916_s1; and glyceraldehyde 3-phosphate dehydrogenase) were obtained from Applied Biosystems. Total RNA at 0.02 mg/30 mL of reaction mixture was prepared by TaqMan Universal PCR Master Mix (Applied Biosystems). For standard RNA, we used TaqMan One-step RT-PCR Master Mix Reagents (Applied Biosystems). The reactions were performed in a 96-well clear optical reaction plate using ABI7700 Sequence Detection System (Applied Biosystems), and the data were normalized by the data of glyceraldehyde 3-phosphate dehydrogenase controls. All reactions were performed in triplicate, with each run containing at least one negative and one positive control, and three independent experiments were performed to verify reproducibility of results. JOE — Volume 36, Number 2, February 2010
Basic Research—Biology
Figure 2. Immunohistochemistry and gene expression analysis of TLR-2 and TLR-4 at the furcal region of the PDL. (A-D) Representative images of (A) TLR-2 in group 1 (untreated control; original magnification, 20), (B) TLR-2 in group 2 (unsealed pulpotomy; original magnification, 20), (C) TLR-4 in group 1 (original magnification, 40), and (D) TLR-4 in group 2 (D: dentin; original magnification, 40). (E) Real-time polymerase chain reaction analysis of TLR-2 and TLR-4 relative messenger RNA expression in the furcal PDL tissue in each group. Data presented from real-time polymerase chain reaction experiments reflect the expression level of TLR-2 or TLR-4 normalized by glyceraldehyde 3-phosphate dehydrogenase (mean and standard error of the mean, n = 4 in each group). The single asterisks stand for p < 0.05 (Tukey test).
The overall strategy used here for immunohistochemical and quantitative gene expression analysis of the furcal region of the rat molar is presented in Figure 1. After the mandibles retrieval, samples were processed for immunohistochemistry using the avidin-biotinperoxidase method (n = 3 for each group) or polymerase chain reaction analysis after the laser capture microdissection (n = 4 for each group) was performed (Fig. 1).
Statistical Analysis For statistical comparison, a one-way analysis of variance (Tukey multiple comparison test) using Prism 5.0 (Graphpad, San Diego, CA) was used. JOE — Volume 36, Number 2, February 2010
Results Immunohistochemical and Quantitative Gene Expression Analysis for TLR-2 and TLR-4 Immunohistochemical analysis showed that in group 2 (pulpotomy without restoration), a number of TLR-2– and TLR-4–expressing cells were observed in the furcal region of the PDL (Fig. 2). The expressing cells were predominantly distributed just beneath the furcal dentin. In groups 1 and 3, however, only a small number of TLR-2– and TLR-4– expressing cells were distributed in the furcal region of the PDL (Fig. 2). Real-time polymerase chain reaction analysis showed that in group 2 the expression levels of TLR-2 messenger RNAs showed significant increases in the inflamed furcal region compared with both group 1 (untreated control) and group 3 (sealed control) (Fig. 2E). TLR-4
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Basic Research—Biology
Figure 3. Real-time polymerase chain reaction analysis of class II MHC DM alpha, CD83, and CD86 relative messenger RNA expression in the furcal PDL tissue in each group. Data presented from real-time polymerase chain reaction experiments reflect the expression level of class II MHC DM alpha, CD83, or CD86 normalized by glyceraldehyde 3-phosphate dehydrogenase (mean and standard error of the mean, n = 4 in each group). The single asterisks stand for p < 0.05 (Tukey test).
messenger RNA expression in group 2 was also significantly higher than that in groups 1 and 3 (Fig. 2E).
Quantitative Gene Expression Analysis for APC-related Molecules In group 2, the expression levels of class II MHC, CD83, and CD86 messenger RNAs showed significant increases compared with groups 1 and 3 (Fig. 3).
Discussion Ulmansky et al (18) reported that furcation canals on the chamber floor were found in rat molars. In this analysis, obvious pathosis in furcal region was not observed if the cavity restoration was performed after pulpotomy. This may indicate that endodontic pathogens and/or pathogenic substances that originate from the oral environment are able to spread most likely through the accessory canals and/or dentinal tubules from the pulp chamber to the PDL in the furcal region. TLRs play a pivotal role in innate immune response by activating immune cells. TLR-4 detects lipopolysaccharide (LPS) in gram-negative bacteria. LPS interacts with immune cells by binding to surface CD14 molecules or by forming a complex with soluble CD14 and then binding to TLR-4 on monocytes, macrophages, dendritic cells, and endothelial cells (11, 19). TLR-4 messenger RNA expression is up-regulated by LPS (20). There are reports suggesting that these signaling processes by TLR-4 and CD14 occur in human dental pulp (21, 22). Notably, in the real-time polymerase chain reaction analysis, the messenger RNA expression level of TLR-4 was upregulated in the teeth in which unsealed pulpotomy was performed as compared with the teeth with sealed pulpotomy. We also tested the gene expression analysis for TLR-2. TLR-2 recognizes unknown cell-wall components of Porphyromonas gingivalis rather than LPS itself (23). In the real-time polymerase chain reaction analysis, the extent of TLR-2 messenger RNA up-regulation in group 2 was higher than that of TLR-4 in the same group. This result suggested that TLR-2 is mainly upregulated in the early stage of PDL inflammation. This finding is in accordance with the recent data that TLR-2 is mainly upregulated during the early stage of pulp inflammation triggered by bacterial infection in the dental pulp as compared with TLR-4 (14). Recently, we have shown the heterogeneity of resident type of DCs in the rat PDL (4, 24). A considerable proportion of DCs in the furcal region are in a more mature/activated state compared with those in the other regions, which may reflect the environmental differences such as 254
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the amount of external antigens (4). Our previous semiquantitative data showed that the up-regulation of APC related molecules such as class II MHC, CD83, and CD86 was recognized at 24 hours after pulp exposure as compared with normal furcal region of the PDL (4). The up-regulation of class II MHC correlates with APC activity (25, 26). In humans, CD83 expression is a hallmark of mature DCs (27) and activated DCs (28). CD86 (B7-2) plays a major role in T-cell activation (29). It is also known that immature DCs start to mature and up-regulate CD86 after exposure to microbial pathogens and receiving TLR signaling (30). In this real-time polymerase chain reaction analysis, we also showed that the gene expression levels of class II MHC, CD83, and CD86 were increased at 24 hours after unsealed pulpotomy, whereas significant up-regulation of these molecules was not observed when the cavity was sealed. These results strongly support the notion that oral pathogens that invade into the pulp chamber are able to cause activation of APCs in the furcal PDL, most probably because of their spread via furcal canals and/or open dentinal tubules. In conclusion, the results of the present study suggest the involvement of innate immune mechanisms involving TLR-2 and TLR-4 and resulting activation of antigen-presenting cells in the early pathogenesis of pulp infection-induced furcal inflammation.
References 1. Lekic P, McCulloch CA. Periodontal ligament cell population: the central role of fibroblasts in creating a unique tissue. Anat Rec 1996;245:327–41. 2. Kawahara I, Takano Y, Sato O, et al. Histochemical and immunohistochemical demonstration of macrophages and dendritic cells in the lingual periodontal ligament of rat incisors. Arch Histol Cytol 1992;55:211–7. 3. Kan L, Okiji T, Kaneko T, et al. Localization and density of myeloid leucocytes in the periodontal ligament of normal rat molars. Arch Oral Biol 2001;46:509–20. 4. Kaneko T, Okiji T, Kaneko R, et al. Characteristics of resident dendritic cells in various regions of rat periodontal ligament. Cell Tissue Res 2008;331:413–21. 5. Vertucci FJ, Williams RG. Furcation canals in the human mandibular first molar. Oral Surg Oral Med Oral Pathol 1974;38:308–14. 6. Gutmann JL. Prevalence, location, and patency of accessory canals in the furcation region of permanent molars. J Periodontol 1978;49:21–6. 7. Goldberg F, Massone EJ, Soares I, et al. Accessory orifices: anatomical relationship between the pulp chamber floor and the furcation. J Endod 1987;13:176–81. 8. Gentner MR, Meyers IA, Symons AL. The floor of the pulp chamber following pulpotomy. J Clin Pediatr Dent 1991;16:20–4. 9. Aqrabawi J, Jarbawi MM. The healing potential of periodontal-endodontic lesions. Int Dent J 2004;54:166–70. 10. Fearon DT. Seeking wisdom in innate immunity. Nature 1997;388:323–4. 11. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388: 394–7.
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Basic Research—Biology 12. Zhang FX, Kirschning CJ, Mancinelli R, et al. Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J Biol Chem 1999;274: 7611–4. 13. Yoshimura A, Lien E, Ingalls RR, et al. Cutting edge: recognition of gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J Immunol 1999;163:1–5. 14. Mutoh N, Tani-Ishii N, Tsukinoki K, et al. Expression of toll-like receptor 2 and 4 in dental pulp. J Endod 2007;33:1183–6. 15. Mutoh N, Watabe H, Chieda K, et al. Expression of Toll-like receptor 2 and 4 in inflamed pulp in severe combined immunodeficiency mice. J Endod 2009;35:975–80. 16. Kaneko T, Okiji T, Kaneko R, et al. Antigen-presenting cells in human radicular granulomas. J Dent Res 2008;87:553–7. 17. Kaneko T, Okiji T, Kaneko R, et al. Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection—a technical report. Microsc Res Tech May 7, 2009 (epub ahead of print). 18. Ulmansky M, Sela J, Hirschfeld Z. Accessory canals in rat molars. J Dent Res 1972; 51:879. 19. Awawdeh L, Lundy FT, Shaw C, et al. Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue from painful and healthy human teeth. Int Endod J 2002;35:30–6. 20. Lin Y, Lee H, Berg AH, et al. The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes. J Biol Chem 2000;275:24255–63.
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21. Wadachi R, Hargreaves KM. Trigeminal nociceptors express TLR-4 and CD14: a mechanism for pain due to infection. J Dent Res 2006;85:49–53. 22. Botero TM, Shelburne CE, Holland GR, et al. TLR-4 mediates LPS-induced VEGF expression in odontoblasts. J Endod 2006;32:951–5. 23. Dillon S, Agrawal S, Banerjee K, et al. Yeast zymosan, a stimulus for TLR-2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. J Clin Invest 2006;116:916–28. 24. Zhao L, Kaneko T, Okiji T, et al. Immunoelectron microscopic analysis of CD11cpositive dendritic cells in the periapical region of the periodontal ligament of rat molars. J Endod 2006;32:1164–7. 25. Cella M, Engering A, Pinet V, et al. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 1997;388:782–7. 26. Wilson NS, El-Sukkari D, Villadangos JA. Dendritic cells constitutively present self antigens in their immature state in vivo and regulate antigen presentation by controlling the rates of MHC class II synthesis and endocytosis. Blood 2004;103:2187–95. 27. Zhou LJ, Tedder TF. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J Immunol 1995;154:3821–35. 28. Gao Z, Mackenzie IC, Rittman BR, et al. Immunocytochemical examination of immune cells in periapical granulomata and odontogenic cysts. J Oral Pathol 1988;17:84–90. 29. Takahashi K, Asagoe K, Zaishun J, et al. Heterogeneity of dendritic cells in human superficial lymph node. In vitro maturation of immature dendritic cells into mature or activated interdigitating reticulum cells. Am J Pathol 1998;153:745–55. 30. Randolph GJ, Angeli V, Swartz MA. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat Rev Immunol 2005;5:617–28.
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Increased Gene Expression of Toll-like Receptors and Antigen-Presenting Cell–related Molecules in the Onset of Experimentally Induced Furcation Lesions of Endodontic Origin in Rat Molars Uraiwan Chokechanachaisakul, DDS,* Tomoatsu Kaneko, DDS, PhD,* Takashi Okiji, DDS, PhD,† Reika Kaneko, DDS, PhD,‡ Mitsuhiro Kaneko, DDS, PhD,* Jun Kawamura, DDS,* Mitsuhiro Sunakawa, DDS, PhD,*§ and Hideaki Suda, DDS, PhD* Abstract Introduction: Early immunopathogenic mechanisms behind pulp infection–induced furcal inflammation have not been well understood. To address the immunopathology of the pulp infection–induced furcal region of the periodontal ligament (PDL), we performed immunohistochemical and quantitative gene expression analyses for toll-like receptors (TLRs) in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. Methods: Furcal inflammation in rat molars was generated by making unsealed pulpotomies that were exposed to the oral environment for 24 hours. Pulpotomized teeth sealed with a temporary filling material and untreated normal teeth served as controls. Gene expression was analyzed with laser capture real-time polymerase chain reaction for TLR-2, TLR-4, and antigen presenting cell (APC)-related molecules (class II MHC, CD83, and CD86). Immunohistochemistry for TLR-2 and TLR-4 was also performed. Results: Messenger RNA expression levels of TLRs and the APC-related molecules in the furcal periodontal ligament were significantly up-regulated in teeth with unsealed pulpotomy. Immunohistochemistry for unsealed pulpotomized teeth revealed that TLRs-expressing cells were predominantly distributed within the PDL beneath the furcal dentin. Conclusions: These results suggested the involvement of innate immune mechanisms involving TLRs and resulting activation of APCs in the early pathogenesis of pulp infection-induced furcal inflammation. (J Endod 2010;36:251–255)
Key Words Class II MHC, gene expression analysis, periodontal ligament, pulpotomy, toll-like receptor
T
he periodontal ligament (PDL) is the soft specialized connective tissue that forms the structural attachment between the tooth and the surrounding alveolar bone (1). Moreover, defense cells such as macrophages and dendritic cells (DCs) reside in the PDL, most likely to maintain local homeostasis and cope with infectious challenges (2, 3). We have recently shown that resident type of DCs in the rat PDL show heterogeneity in terms of morphology and cell surface antigen expression in different regions of the PDL, probably reflecting the differences in the microenvironment, such as the amount of external antigens, in different regions (4). In particular, DCs in the furcal region frequently show the morphologic and phenotypic features of activated antigen-presenting cells (APCs), characterized by their CD86 expression, electrondense lysosomes, and cell-to-cell contacts with lymphocytes. This may be associated with the anatomic proximity of the furcal area to the oral environment. It has been well documented that pulp infection may cause furcal inflammation, most probably via furcal and lateral canals (5–9). However, there is limited information regarding cellular and molecular mechanisms behind the immunopathogenesis of such furcal lesions of endodontic origin. In a previous study, we have shown that messenger RNA expression levels of APC-related molecules are upregulated in the furcal PDL of rat molars after 24 hours of pulp exposure, although the number of class II major histocompatibility complex (MHC) molecule-expressing DCs does not show any increase (4). This suggests the activation/maturation of resident DCs in response to bacterial challenge. Toll-like receptors (TLRs) are known as a family of transmembrane receptors that play a pivotal role in the modulation of immune response by recognizing pathogenassociated molecular patterns (10, 11). The recognition subsequently stimulates a sequence of signaling mechanisms resulting in the production of various cytokines that serve as a link between innate and specific immune mechanisms. TLR-2 and TLR-4 are the most well-defined members of the TLR superfamily: TLR-4 recognizes lipopolysaccharides in gram-negative bacteria, whereas TLR-2 plays a major role in the recognition of various bacterial components such as lipoteichoic acid in gram-positive bacteria, lipoproteins, and peptide glycans (12, 13). The up-regulation of TLR-2 and/or TLR-4 has been shown in bacterially challenged dental pulp, which suggests innate immune responses involving the TLRs as signaling receptors contribute to the
From *Pulp Biology and Endodontics, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; †Division of Cariology, Operative Dentistry and Endodontics, Niigata University, Graduate School of Medical and Dental Sciences, Niigata, Japan; ‡Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan; and §Infection Control Team, University Hospital, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan. Supported by Grants-in-Aid for Scientific Research (No. 19209059 to H.S., No. 18791395 to M.K., No. 18390504 to M.S., No. 20390483 to T. O., and No. 21592411 to T. K.) from the Japan Society for the Promotion of Sciences. Address requests for reprints to Dr Tomoatsu Kaneko, Pulp Biology and Endodontics, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo 113-8549, Japan. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2009.10.005
JOE — Volume 36, Number 2, February 2010
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Basic Research—Biology pathogenesis of pulp inflammation (14, 15). However, the roles of TLRs in pulp infection-induced furcal inflammation are poorly understood. To further analyze the immunopathology of the pulp infection– induced furcal region of the PDL, we performed immunohistochemical and quantitative gene expression analyses for TLRs in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. Quantitative gene expression analysis for APC-related molecules was also performed to address the link between expression of TLRs and APC activation.
Materials and Methods All experiments were conducted under the approval of the Animal Care Committee at Tokyo Medical and Dental University.
Induction of Furcal Inflammation Seven-week-old adult male Wistar rats (N = 21), each weighting around 200 g, were divided into the following three groups (n = 7 for each group): group 1 (untreated control), normal rats without pulpotomy; group 2 (experimental group), rats with unsealed pulpotomy; and group 3 (sealed control), rats with sealed pulpotomy. In the experimental group (group 2), unsealed pulpotomy was performed in mandibular first molars of the left side. Under pentobarbital sodium anesthesia (30 mg/kg, intraperitoneally), the pulp was exposed from the occlusal surface and most of the coronal pulp tissue was removed with no. 1/2 round burs with the aid of an operating microscope. The pulp chamber was irrigated with 6% sodium hypochlorite solution and 15% EDTA. The teeth were then left open to the oral environment for 24 hours. In sealed controls (group 3), pulpotomy was made in the same manner, and then the cavity was sealed with a temporary restorative material (Caviton; GC Corporation, Tokyo, Japan). Normal rats without pulpotomy (group 1) served as untreated controls. Tissue Preparation Twenty-four hours after the pulpotomy, the animals for light microscopic analysis were anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg) and killed by transcardiac perfusion of 4% paraformaldehyde in phosphate-buffered solution. Both sides of the lower mandibles were retrieved and kept in the same fixative overnight and then demineralized in 14% EDTA on the rotator at 4 C. Immunohistochemistry For immunohistochemistry, the samples (n = 3 for each group) were cut mesiodistally in a linear slicer (LEICA CM 1900; Leica Microsystems, Wetzlar, Germany) at 40 mm in thickness. Immunostaining by the avidin-biotin-peroxidase complex method was performed on 24well plate as previously described (4). After incubation in 3% H2O2 for 5 minutes, the sections were incubated with one of the primary antibodies diluted with phosphate-buffered saline for 24 hours at 4 C. The first polyclonal antibodies used in this study were TLR-2 and TLR-4 (Santa Cruz Biotechnology, CA, USA; dilution: 1:100 and 1:500) with normal horse serum (R.T.U. VECTASTAIN Universal Quick Kit; Vector Laboratories, Inc, Burlingame, CA). The sections were then sequentially incubated with a biotinylated pan-specific secondary antibody (rat adsorbed, R.T.U. VECTASTAIN Universal Quick Kit) for 1 hour and avidinbiotin-peroxidase complex (VECTASTAIN Universal Quick Kit) for 1 hour. The sections were then developed with a 3,3’-diaminobenzidine–H2O2 solution (DAB Substrate Kit, Vector Laboratories) for 3 minutes, counterstained with hematoxylin, and examined under a light microscope. Negative control staining was always performed in parallel by incubating sections with nonimmune serum or phosphate-buffered saline instead of the primary antibodies. 252
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Figure 1. The overall strategy used here for imuunohistochemical and quantitative gene expression analysis of furcal region of the rat molar.
Quantitative Gene Expression Analysis For laser capture real-time polymerase chain reaction analysis, the samples (n = 4 for each group) were prepared in a similar manner as described earlier with the same fixation, and prepared tissues were cut mesiodistally at 80 mm in a linear slicer (Pro 7; Dosaka EM, Kyoto, Japan). The tissue sections were mounted on glass foiled pen slides (Leica). The furcal regions of PDL were dissected and collected by using a laser capture microdissection microscope Leica AS LMD (Leica) (16). In these samples, the total RNA from each sample was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with RNeasy Mini kits (Qiagen, Valencia, CA) in accordance with manufacturers’ protocols as we described before (17). The optical density (OD)260/280 ratio was used to evaluate the purity of the nucleic acid samples, and the quality of the extracted total RNA was determined with the BioPhotometer (Eppendorf, Hamburg, Germany). Then, first-strand complementary DNA synthesis was performed with TaqMan reverse transcription reagents (Applied Biosystems). Probe and primer sets of TaqMan Gene Expression Assays (NM_019178.1;TLR-4, NM_198769.2; TLR-2, Rn01768599_g1; Class II MHC DM alpha, Rn01434593_m1; CD83, Rn00571654_m1; CD86, Rn99999916_s1; and glyceraldehyde 3-phosphate dehydrogenase) were obtained from Applied Biosystems. Total RNA at 0.02 mg/30 mL of reaction mixture was prepared by TaqMan Universal PCR Master Mix (Applied Biosystems). For standard RNA, we used TaqMan One-step RT-PCR Master Mix Reagents (Applied Biosystems). The reactions were performed in a 96-well clear optical reaction plate using ABI7700 Sequence Detection System (Applied Biosystems), and the data were normalized by the data of glyceraldehyde 3-phosphate dehydrogenase controls. All reactions were performed in triplicate, with each run containing at least one negative and one positive control, and three independent experiments were performed to verify reproducibility of results. JOE — Volume 36, Number 2, February 2010
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Figure 2. Immunohistochemistry and gene expression analysis of TLR-2 and TLR-4 at the furcal region of the PDL. (A-D) Representative images of (A) TLR-2 in group 1 (untreated control; original magnification, 20), (B) TLR-2 in group 2 (unsealed pulpotomy; original magnification, 20), (C) TLR-4 in group 1 (original magnification, 40), and (D) TLR-4 in group 2 (D: dentin; original magnification, 40). (E) Real-time polymerase chain reaction analysis of TLR-2 and TLR-4 relative messenger RNA expression in the furcal PDL tissue in each group. Data presented from real-time polymerase chain reaction experiments reflect the expression level of TLR-2 or TLR-4 normalized by glyceraldehyde 3-phosphate dehydrogenase (mean and standard error of the mean, n = 4 in each group). The single asterisks stand for p < 0.05 (Tukey test).
The overall strategy used here for immunohistochemical and quantitative gene expression analysis of the furcal region of the rat molar is presented in Figure 1. After the mandibles retrieval, samples were processed for immunohistochemistry using the avidin-biotinperoxidase method (n = 3 for each group) or polymerase chain reaction analysis after the laser capture microdissection (n = 4 for each group) was performed (Fig. 1).
Statistical Analysis For statistical comparison, a one-way analysis of variance (Tukey multiple comparison test) using Prism 5.0 (Graphpad, San Diego, CA) was used. JOE — Volume 36, Number 2, February 2010
Results Immunohistochemical and Quantitative Gene Expression Analysis for TLR-2 and TLR-4 Immunohistochemical analysis showed that in group 2 (pulpotomy without restoration), a number of TLR-2– and TLR-4–expressing cells were observed in the furcal region of the PDL (Fig. 2). The expressing cells were predominantly distributed just beneath the furcal dentin. In groups 1 and 3, however, only a small number of TLR-2– and TLR-4– expressing cells were distributed in the furcal region of the PDL (Fig. 2). Real-time polymerase chain reaction analysis showed that in group 2 the expression levels of TLR-2 messenger RNAs showed significant increases in the inflamed furcal region compared with both group 1 (untreated control) and group 3 (sealed control) (Fig. 2E). TLR-4
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Figure 3. Real-time polymerase chain reaction analysis of class II MHC DM alpha, CD83, and CD86 relative messenger RNA expression in the furcal PDL tissue in each group. Data presented from real-time polymerase chain reaction experiments reflect the expression level of class II MHC DM alpha, CD83, or CD86 normalized by glyceraldehyde 3-phosphate dehydrogenase (mean and standard error of the mean, n = 4 in each group). The single asterisks stand for p < 0.05 (Tukey test).
messenger RNA expression in group 2 was also significantly higher than that in groups 1 and 3 (Fig. 2E).
Quantitative Gene Expression Analysis for APC-related Molecules In group 2, the expression levels of class II MHC, CD83, and CD86 messenger RNAs showed significant increases compared with groups 1 and 3 (Fig. 3).
Discussion Ulmansky et al (18) reported that furcation canals on the chamber floor were found in rat molars. In this analysis, obvious pathosis in furcal region was not observed if the cavity restoration was performed after pulpotomy. This may indicate that endodontic pathogens and/or pathogenic substances that originate from the oral environment are able to spread most likely through the accessory canals and/or dentinal tubules from the pulp chamber to the PDL in the furcal region. TLRs play a pivotal role in innate immune response by activating immune cells. TLR-4 detects lipopolysaccharide (LPS) in gram-negative bacteria. LPS interacts with immune cells by binding to surface CD14 molecules or by forming a complex with soluble CD14 and then binding to TLR-4 on monocytes, macrophages, dendritic cells, and endothelial cells (11, 19). TLR-4 messenger RNA expression is up-regulated by LPS (20). There are reports suggesting that these signaling processes by TLR-4 and CD14 occur in human dental pulp (21, 22). Notably, in the real-time polymerase chain reaction analysis, the messenger RNA expression level of TLR-4 was upregulated in the teeth in which unsealed pulpotomy was performed as compared with the teeth with sealed pulpotomy. We also tested the gene expression analysis for TLR-2. TLR-2 recognizes unknown cell-wall components of Porphyromonas gingivalis rather than LPS itself (23). In the real-time polymerase chain reaction analysis, the extent of TLR-2 messenger RNA up-regulation in group 2 was higher than that of TLR-4 in the same group. This result suggested that TLR-2 is mainly upregulated in the early stage of PDL inflammation. This finding is in accordance with the recent data that TLR-2 is mainly upregulated during the early stage of pulp inflammation triggered by bacterial infection in the dental pulp as compared with TLR-4 (14). Recently, we have shown the heterogeneity of resident type of DCs in the rat PDL (4, 24). A considerable proportion of DCs in the furcal region are in a more mature/activated state compared with those in the other regions, which may reflect the environmental differences such as 254
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the amount of external antigens (4). Our previous semiquantitative data showed that the up-regulation of APC related molecules such as class II MHC, CD83, and CD86 was recognized at 24 hours after pulp exposure as compared with normal furcal region of the PDL (4). The up-regulation of class II MHC correlates with APC activity (25, 26). In humans, CD83 expression is a hallmark of mature DCs (27) and activated DCs (28). CD86 (B7-2) plays a major role in T-cell activation (29). It is also known that immature DCs start to mature and up-regulate CD86 after exposure to microbial pathogens and receiving TLR signaling (30). In this real-time polymerase chain reaction analysis, we also showed that the gene expression levels of class II MHC, CD83, and CD86 were increased at 24 hours after unsealed pulpotomy, whereas significant up-regulation of these molecules was not observed when the cavity was sealed. These results strongly support the notion that oral pathogens that invade into the pulp chamber are able to cause activation of APCs in the furcal PDL, most probably because of their spread via furcal canals and/or open dentinal tubules. In conclusion, the results of the present study suggest the involvement of innate immune mechanisms involving TLR-2 and TLR-4 and resulting activation of antigen-presenting cells in the early pathogenesis of pulp infection-induced furcal inflammation.
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