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RESEARCH REPORT |
1 Pulp Biology and Endodontics, Department of Restorative Sciences,
2 Molecular Pathology, Department of Oral Restitution, Division of Oral Health Sciences, Graduate School, Tokyo Medical & Dental University, and
3 Center of Excellence (COE) Program for Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical & Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan;
* corresponding author, kawashima.n.endo{at}tmd.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, tumor necrosis factor alpha.
KEY WORDS: nitric oxide synthase • pulpitis • cytokines • L-NAME • immunocompetent cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Nitric oxide (NO) was first reported to induce dilation of the blood vessels (Palmer et al., 1987), but it is now known to be a biological effector molecule in various systems, and is essential for these systems to function in the body (Moilanen et al., 1999). NO is synthesized by a group of enzymes called nitric oxide synthases (NOS). Three isoforms of this enzyme have been characterized: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are produced constitutively, while iNOS is induced in response to inflammatory stimuli, such as LPS and cytokines. Once iNOS is induced, it can produce copious quantities of NO for prolonged periods (Moilanen et al., 1999), which further affects the synthesis of the chemical mediators: IL1 (Hill et al., 1996), IL6 (Mossalayi et al., 1994), IL12 (Rothe et al., 1996), TNF (Mossalayi et al., 1994), and PGs (Posadas et al., 2000).
These properties of NO would make it an essential and important mediator in pulpal pathosis, if it were produced at the onset of pulpal inflammation. In this study, we evaluated whether iNOS expression was related to the progress of pulpitis, and whether an NOS inhibitor would effectively reduce pulpal inflammation.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR
The pulp tissues extracted from the left and right incisors of a rat were combined, and total RNA was isolated with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 2 µg of total RNA by MMLV RNaseH-reverse-transcriptase (Superscript II; Invitrogen). PCR amplification was performed with Taq polymerase (platinum PCR supermix; Invitrogen) and specific primers designed for rat iNOS, 5'-ATGGCTTGCCCC TGGAAGTTTCTC-3' and 5'-CCTCTGATGGTGCCATCGGG CATCTG-3' (Nunokawa et al., 1993); IL1, 5'-CACCTTCTGC TTCTAAAGTGCC-3' and 5'-AATTCTGCTGCTGAGGATGC-3'; IL1ß, 5'-ACCCAAGCACCTTCTTTTCC-3' and 5'-GTTT GGGATCCACACTCTCC-3'; IL6, 5'-ATGTTGTTGACAG CCACTGC-3' and 5'-AAACGGAACTCCAGAAGACC-3'; IL10, 5'-TAAGGGTTACTTGGGTTGCC-3' and 5'-TTCATGGCCTT GTAGACACC-3'; IL12, 5'-GGGTCCGGTTTGATGAT GTCCCTG-3' and 5'-GGAGAAACGGTGACCCTCACCT-3'; TNF, 5'-AGATGTGGAACTGGCAGAGG-3' and 5'-GGTTGTCTTTGAGATCCATGC-3'; COX2, 5'-AGTATCAG AACCGCATTGCC-3' and 5'-TAAGGTTTCAGGGAGAAGCG-3'; and ß-actin, 5'-AAGTACCCCATTGAACACGG-3' and 5'-ATCACAATGCCAGTGGTACG-3'. The density of each band in the agarose gels was quantified with the use of Scion Image software (http://www.scioncorp.com) and standardized against the amount of ß-actin. The band density in each group (n = 3) was evaluated statistically with one-way ANOVA and Tukey-Kramer tests. The amount of iNOS expression in the inflamed pulp was quantitatively compared with that in LPS-treated liver, by means of a competitive-PCR test (Takara, Kyoto, Japan).
Immunohistochemistry
Incisors were fixed with a periodate-lysine paraformaldehyde fixative at 4°C overnight, and then decalcified in 14% EDTA at 4°C for 3 wks. The frozen samples were serially sectioned at a thickness of 7 µm, with the use of a cryostat (Leica Microsystems AG, Wetzlar, Germany). Immunohistochemical staining (avidin-biotin peroxidase complex system, Elite ABC; Vector, Burlingame, CA, USA) was performed with an anti-rat iNOS antibody (polyclonal, x1250; Upstate Inc., Waltham, MA, USA), W3/13 (monoclonal, anti-rat CD43, x 2500; Serotec, Oxford, UK), ED1 (monoclonal, anti-rat CD68, x10,000; Serotec), and OX6 (monoclonal, anti-rat MHC class II, x10,000; Serotec). Diaminobenzidine-HCl (DAB; Vector) was used for visualization. Double-staining was performed with anti-rabbit IgG labeled with Alexa Fluor 594 (x200; Molecular Probes, Eugene, OR, USA) and anti-mouse IgG labeled with Alexa Fluor 488 (x200, Molecular Probes). Double-stained samples were evaluated under a confocal laser microscope (LSM5 Pascal; Carl Zeiss, Oberkochen, Germany). On 3 typical sections from each sample, we counted positively stained cells in whole pulp tissues, except for the inside of abscesses and blood vessels, under 400x magnification, using a 10mm x 10mm graticule, which was moved to cover the pulp tissues completely. Cell fragments without nuclei were excluded from the cell count. We calculated cell density by dividing the total cell number by the pulpal area (mm2), which we measured using bioimaging software (WinROOF, Mitani Co., Fukui, Japan). We confirmed the reliability of this method by preliminary experiments in which cell counts on the same sections were assessed to be reproducible. Average cell density of the 3 counts was considered to be representative of the sample, and was evaluated statistically with one-way ANOVA and Tukey-Kramer tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, 1B). The pathological findings were characterized by infiltration of many neutrophils and the dilation of blood vessels beneath the LPS-treated portion. In contrast, saline did not induce a severe inflammatory reaction, and only slight infiltration of neutrophils was observed in the coronal pulp at 6 hrs (Figs. 1C, 1D).
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). However, application of LPS to the pulp induced iNOS mRNA expression even at 3 hrs, and peak expression was noted at 6 hrs (Fig. 1E). The iNOS expression decreased thereafter, and only slight expression was observed at 12 hrs. No expression was observed at 24 hrs. Competitive PCR showed that the iNOS mRNA expression level in the LPS-treated pulp at 6 hrs was between 104 and 105 copies compared with that of the iNOS competitor, and that, in the LPS-treated liver at 6 hrs, it was about 105 copies of the iNOS competitor (Fig. 1F).
Evaluation of iNOS+ Cells
No iNOS+ cells were observed in either the saline-treated (3, 6, 9 hrs; Fig. 2A) or the non-treated (0 hr) pulp tissue. The cells started to infiltrate the pulp tissue 3 hrs after the application of LPS, and were observed around blood vessels beneath the exposed surface. Six hrs after the application of LPS, iNOS+ cells were scattered abundantly over the coronal pulp (Fig. 2A). ED1+ and Ia+ cells were observed in the coronal portion of LPS-treated pulp (Fig. 2A), but there were fewer Ia+ cells than ED1+ cells. Double-staining with iNOS and ED1 or OX6 revealed that iNOS+ cells were divided into ED1+ and ED1– cells, and that the population of iNOS+ cells was completely different from that of OX6+ cells (Fig. 2B).
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. Infiltration of iNOS+ cells into pulp was observed at 3 hrs and peaked at 9 hrs. At 3 hrs, many of the iNOS+ cells were granulocytes, but the population of ED1+ macrophages increased thereafter, and most of the iNOS+ cells at 9 hrs were ED1+ macrophages. The peaks of infiltration of W3/13+, ED1+, and OX6+ cells were observed at 3, 9, and 9 hrs, respectively.
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, IL1ß, and COX2 was observed, but other cytokines were not expressed in either the saline-treated pulp (data not shown) or the non-treated pulp tissue (0 hr, Fig. 3A). LPS application rapidly induced the expression of IL6 and TNF and also enhanced the expression of IL1, IL1ß, and COX2 at 3 hrs after the pulpal exposure. The expression of all the pro-inflammatory cytokines (IL1, IL1ß, IL6, and TNF) and COX2 peaked at 6 hrs, but a high expression level of IL1ß continued until 24 hrs. IL12 expression was observed only at 6 hrs (Fig. 3B). The expression of IL10 mRNA, an anti-inflammatory cytokine, was first observed at 6 hrs after the LPS application, and its expression level decreased thereafter.
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, IL1ß, and TNF) and COX2 at 6 hrs after the LPS application (Fig. 3B). In particular, expressions of IL1, IL1ß, and TNF were severely depressed in L-NAME-treated rat pulp, and COX2 expression in L-NAME-treated rats was about 60% of that in saline-treated ones. In contrast, expression of IL6 and IL10 was not affected by L-NAME administration. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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To characterize pulpal inflammation, we evaluated the kinetics of cytokine and COX2 expression. The expression of pro-inflammatory cytokines, such as IL1, IL1ß, and TNF, began or increased at 3 hrs after the application of LPS, corresponding to the onset of pulpitis. These pro-inflammatory cytokines are largely produced by inflammatory cells such as macrophages (Tani-Ishii et al., 1995), but the pulpal cells might produce chemical mediators in response to LPS (Hosoya and Matsushima, 1997), even though the amounts would be limited. IL12 expression was detected in rat pulpitis (Fig. 3B), but its expression was temporal and was observed only at 6 hrs; however, it has been reported to be a key cytokine in the course of inflammation (Trifilieff et al., 2000). Expressions of anti-inflammatory cytokines, such as IL10, were first observed at 6 hrs after the application of LPS. Production of anti-inflammatory cytokines should be induced as a result of the over-production of inflammatory cytokines, to control them (Kawashima and Stashenko, 1999). COX2 mRNA expression in pulp tissue was clearly observed from 3 to 9 hrs after application of LPS in this study, and the prostaglandin metabolites produced by COX2 might be involved in pulpal inflammatory reactions (Okiji et al., 1987, 1989).
To determine the effects of NOS inhibitors on cytokine and COX2 expression in pulp tissue, we applied L-NAME before applying LPS to the pulp. L-NAME, an arginine analogue, is widely used as a non-selective inhibitor of NOS enzymes (Moncada and Higgs, 1995). Our study revealed that L-NAME dramatically inhibited inflammatory cytokine and COX2 expression. Down-regulation of cytokine expression by L-NAME was also observed in arthritis and LPS-treated liver (Aono et al., 1997; de Mello et al., 1997). Considering the properties of NO that could induce chemical mediators (Mossalayi et al., 1994; Hill et al., 1996; Rothe et al., 1996; Posadas et al., 2000), the blockade of NO synthesis by an NOS inhibitor could be responsible for the down-regulation of cytokine and COX expression. However, we recently found that a selective iNOS inhibitor was effective for inhibiting the infiltration of inflammatory cells, such as neutrophils, macrophages, and Ia+ cells, into pulp (Kawanishi et al., 2004). Selective iNOS inhibitors have been reported to down-regulate chemokine expression during airway inflammation (Trifilieff et al., 2000), and limitations on chemokine production by NOS inhibitors could diminish the infiltration of inflammatory cells. The down-regulation of cytokine and COX2 synthesis in the pulp observed in this study may have been caused by this diminution of inflammatory cell infiltration into the pulp. The beneficial effects of NOS inhibitors on inflammation have been reported for other tissues (Colon et al., 2000; Trifilieff et al., 2000; Kankuri et al., 2001), although opposite effects have also been reported (Vos et al., 1997). Further study will be necessary to evaluate the effects of the mechanisms of NOS inhibitors on the progress of inflammation.
In conclusion, NO synthesis may be related to the initiation of mediator production, since NOS inhibitors down-regulated the synthesis of inflammatory cytokines and prostaglandins. NO would also cause severe relaxation of the blood vessel smooth muscle and enhancement of permeability in inflamed pulp. These phenomena might be harmful to ‘low-compliant’ pulp tissue (Kim and Dörscher-Kim, 1990). Therefore, over-production of NO would be harmful to pulp tissue, and so administration of an NOS- or iNOS-specific inhibitor may have a beneficial action on pulpal inflammation.
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ACKNOWLEDGMENTS |
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Received October 16, 2003; Last revision May 5, 2005; Accepted May 6, 2005
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REFERENCES |
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