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RESEARCH REPORT |
1 Departments of Oral Anatomy and Cell Biology,
2 Removable Prosthodontics, and
3 Fixed Prosthodontics, Faculty of Oral Science, Kyushu University Graduate School of Dental Science, Fukuoka 812-8582, Japan;
* corresponding author, yamazata{at}dent.kyushu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: temporomandibular joint synovitis • nitric oxide • iNOS • peroxynitrite • DNA single-strand break
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B activation in TMJ synovitis after condylar hypermovement (Yamaza et al., 2003). The high levels of NO produced by iNOS in response to inflammatory stimuli (Nathan, 1995) significantly contribute to free-radical- and oxidant-mediated injuries in inflammatory diseases (Moncada et al., 1991). Peroxynitrite, which is formed during NO reaction with oxygen superoxide, is more cytotoxic than either NO or superoxide (Beckman et al., 1990). Peroxynitrite produces nitration of tyrosine residues, thereby leading to the formation of nitrotyrosine (Ischiropoulos et al., 1992). The levels of NO breakdown products, nitrite and nitrate (Farrell et al., 1992) and nitrotyrosine (Kaur and Halliwell, 1994), are increased in the plasma samples and/or synovial fluids of arthritis patients (Kaur and Halliwell, 1994).
DNA is damaged by environmental genotoxic agents and by endogenous cellular reactions. Peroxynitrite and hydroxy radicals are key triggers of DNA strand breakage, which is related to cell proliferation, cell differentiation, and cell death (Szabo, 1996). Genotoxic events have been implicated in synovial inflammation in rheumatoid arthritis (RA) patients.
To elucidate the involvement of oxidative stress in synovitis progression during degenerative TMJ, we investigated: (i) whether NO and peroxynitrite were expressed in the synovium of TMJ after hypermobility of the mandibular condyle, by analyzing the localization of iNOS and nitrotyrosine as markers of NO and peroxynitrite production, respectively; and (ii) whether DNA damage occurred in synovial cells following condylar hypermovement, by analyzing DNA strand breaks.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue Preparation
After the last bout of hypermobility, under pentobarbital anesthesia (50 mg/kg i.p.), the rats were perfused transcardially with 4% paraformaldehyde/0.01 M phosphate-buffered saline (PBS, pH 7.4), with or without 0.05% glutaraldehyde. The TMJs were decalcified in 5% EDTA in PBS. Serial 10-µm-thick sagittal and 60-µm-thick frontal cryosections were used for the histochemical analyses and immunoelectron microscopy, respectively.
Immunolight Microscopy
We performed immunohistochemistry using the avidin biotinylated peroxidase (ABC) kit (Vector Laboratories, Burlingame, CA, USA) as described previously (Yamaza et al., 2003). Briefly, 10-µm-thick cryosections were treated with 0.3% H2O2. After being blocked, the sections were stained overnight at 4°C with anti-iNOS (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or anti-nitrotyrosine (1:100; Upstate Biotechnology, Charlottesville, VA, USA) antibody, followed by biotinylated antibody (1:200) and ABC (1:100). The sections were then treated with 0.02% 3,3'-diaminobenzidine (DAB; Dojin Laboratories, Kumamoto, Japan) and 0.006% H2O2/0.05 M Tris (pH 7.6), and stained with hematoxylin. The immunohistochemical controls were treated similarly, except that non-immune IgG or PBS alone was added instead of primary antibodies.
Immunoelectron Microscopy
Immunocytochemistry by the pre-embedding method has been described previously (Masuda et al., 2002). Briefly, 60-µm-thick sections were pre-treated with 0.1 M L-lysine and 0.3% H2O2. After being blocked, the sections were stained overnight at 4°C with anti-nitrotyrosine antibody (1:10), followed by biotinylated antibody and ABC at 4°C overnight. The sections were reacted with 0.02% DAB and 0.006% H2O2/0.05 M Tris (pH 7.6). The immunocytochemical controls were treated in the same way as the immunohistochemical controls. The samples were post-fixed with 1% OsO4, dehydrated in a graded ethanol series, and embedded in Quetol 652 resin (Nissin EM, Tokyo, Japan). Ultrathin sections were examined, without being counterstained, in a JEM 1210 transmission electron microscope (JEOL, Tokyo, Japan) at 80 kV.
Staining of DNA Strand Breaks
Ten-µm-thick sections were pre-treated with or without 1 µg/mL proteinase K (PK)/PBS (Sigma, St. Louis, MO, USA) for 20 min at 37°C. For in situ nick translation (ISNT) (Hashimoto et al., 1995), sections were reacted for 3 hrs at 37°C in 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.1 mM dithiothreitol, 50 µg/mL bovine serum albumin (BSA), 200 U/mL DNA polymerase I (Takara Co., Tokyo, Japan), and 20 µM each of dATP, dGTP, dCTP, and biotin-11-dUTP (Takara). For terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining (Hashimoto et al., 1995), sections were immersed for 30 min in 200 mM potassium cacodylate, 25 mM Tris (pH 6.6), and 0.25 mg/mL BSA, followed by incubation with 1.5 mM CoCl2, 0.2 U/µL terminal deoxynucleotidyl transferase (TdT) (Roche Diagnostics, Mannheim, Germany), 20 µM dATP, and 10 µM biotin-16-dUTP (Takara). After being blocked, all of the sections were reacted for 3 hrs with horseradish-peroxidase-labeled anti-biotin antibody (Vector Laboratories), treated with 0.5 mg/mL DAB, 0.025% CoCl2, 0.02% NiSO4(NH4)SO4, and 0.01% H2O2, and stained with eosin. The ISNT and TUNEL controls were incubated without DNA polymerase I or TdT, and/or an equivalent volume of TTP was added instead of biotinylated dUTP.
Quantitative Scoring of Synovitis
The experimental (N = 20) and control (N = 6) sagittal sections were assessed according to a quantitative scoring system for synovitis, which is based on the degree of thickness of the synovial cell layer and is graded as follows: grade 0, 1–3 layers; grade 1, 4–6 layers; and grade 2, 7 or more layers (Yamaza et al., 2003).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), the findings for the posterior portion facing the upper joint compartment, particularly in the synovial fold, are shown as typical for all the regions of the synovitis-induced and non-induced TMJs.
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, 1c). There were no differences in the nitrotyrosine and iNOS distribution patterns. Nitrotyrosine- or iNOS-positive cells were observed in the subsynovial layer. In non-forced animals, positive reactions were found in all of the synovia (Figs. 1d, 1e). The iNOS- or nitrotyrosine-positive superficial cell layers in the synovitis-induced TMJs were thicker than those in the non-forced TMJs (Figs. 1b–1e). Multi-layered changes (synovitis grades 1 and 2) were identified in > 80% of the synovial membranes in each portion of the experimental TMJ (Fig. 1f). However, hyperplasia was not found in the control synovium. In immunohistochemical controls, nitrotyrosine- and iNOS-reactivities were not found in either forced or non-forced TMJs (data not shown).
Immunoelectron Microscopy of Nitrotyrosine in the Synovial Cells of Synovitis-induced TMJs
Based on the ultrastructural classification (Masuda et al., 2002), the synovial lining cells were divided into two types: type A cells, which had abundant large vacuoles and vesicles, and cytoplasmic processes that extended into the joint compartment; and type B cells, which contained the rough endoplasmic reticulum, as well as a few vacuoles and vesicles. In synovitis-induced TMJs, nitrotyrosine-positive products were deposited throughout the cytosol and on the plasma membranes of type A cells (Figs. 2a, 2b), as well as in the vesicular and vacuolar structures and near the mitochondria. Nitrotyrosine-positive reactivity was also present in the intercellular spaces of the suprasynovial layer. On the other hand, nitrotyrosine-positive products were found in the cytoplasm or in the euchromatin of the nuclei of type B cells (Figs. 2c, 2d). These products were also located in the cytoplasm, vesicles, and nuclei of the fibroblasts in the subsynovial layer (Fig. 2e). In non-forced TMJs, nitrotyrosine immunoreactivity was found in the cytoplasm, but not in the nuclei of the synovial cells (data not shown). In immunocytochemical controls, immunoreactivity was not detected in the TMJ cells (data not shown).
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). In contrast, strong ISNT-positive reactivity was found in the nuclei of most of the synovial-lining cells in the PK-treated sections (Fig. 3b). ISNT-positive reactivity was also detected in some of the fibroblast-like cells of the subsynovial layer. On the other hand, the cell nuclei of the synovial membrane were TUNEL-negative in sections prepared either without or with PK treatment (Figs. 3c, 3d). In non-forced TMJs, the synovia in the PK-treated and untreated sections were negative for ISNT (Figs. 4a, 4b) and TUNEL (Figs. 4c, 4d). In ISNT and TUNEL controls, there was no reactivity in the sections from non-forced or forced TMJs with or without PK treatment (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Koji (1996) proposes that DNA single-strand breaks (DNA-SSBs) in the nuclei of cells at various stages of the cell cycle be divided into two types: (1) the protease-dependent type, which requires protease treatment for the detection of DNA-SSBs; and (2) the protease-independent type, which does not require protease treatment for detection by the ISNT method. The protease-dependent type is detected in the nuclei during replicative DNA synthesis, and the protease-independent type is found in terminally differentiated cells. In apoptotic cells, DNA-SSB cells are categorized as protease-dependent types, and those in necrotic cells are of the protease-independent type. In this study, the detectable DNA strand breaks in the synovia of mechanically loaded TMJs were only of the protease-dependent type. Furthermore, TUNEL-positive cells did not appear in the sections with and without PK treatment. Although extensive DNA damage has been implicated in the synovial hyperplasia of RA (Firestein et al., 1997), the synovial cells of RA patients do not show apoptotic changes (Sugiyama et al., 1996). These findings are in agreement with those of the present experimental and control TMJs, which indicate that neither necrosis nor apoptosis occurs in the synovium of forced and non-forced TMJs. Thus, mechanical loading of the TMJ appears to cause synovial cell proliferation, which leads to synovial hyperplasia. The results of this study are consistent with those of the previous study, in that significant synovial cell proliferation is found in the synovia of patients with RA (Yamasaki et al., 2001).
Peroxynitrite, rather than NO, is a key trigger of DNA-SSBs (Szabo, 1996). In this study, a marker of peroxynitrite, nitrotyrosine, was detected in the nuclei and cytoplasm of the synovial lining cells and in the fibroblasts of the inflamed TMJ. These findings support the notion of DNA-SSB generation in the synovia of mechanically loaded TMJs. DNA-SSBs promote the activation of a DNA nick sensor enzyme, poly(ADP-ribose) polymerase (PARP) (Ucta and Hayashi, 1985). PARP controls cell replication and proliferation (Simbulan-Rosenthal et al., 1996) and regulates the transcription of various proteins. PARP inhibition down-regulates cytokine-induced iNOS mRNA expression (Hauschildt et al., 1992). NF-B-dependent transcriptional activation is defective in PARP-deficient mice (Oliver et al., 1999). Furthermore, PARP appears during synovial hyperplasia in arthritis (Szabo et al., 1998). Recently, we investigated NF-B activation in relation to the progression of synovial hyperplasia in synovitis-induced TMJs (Yamaza et al., 2003). PARP and proliferative cell nuclear antigen were localized to the synovial cell nuclei, especially those of the synovial lining, in synovitis-induced TMJs (our unpublished data). Therefore, DNA-SSBs caused by NO-generated peroxynitrite appear to trigger the development and progression of TMJ synovitis. In contrast, interleukin-1 (IL-1) is a key regulator of arthritis in animal models (Horai et al., 2000), and accelerates superoxide anion (O2•–) release from cultured synovial cells, which is followed by DNA damage (Ahmadzadeh et al., 1990). Both IL-1 and the IL-1 receptor have been demonstrated in the synovial fluids of patients with TMJ disorders (Kubota et al., 1998), and in the synovial lining cells of TMJ (Masuda et al., 2002). The production of hydroxy radicals increases in the presence of the O2•– scavenger, superoxide dismutase, in the synovial fluids of IL-1-induced TMJ arthritis (Kawai et al., 2000). Therefore, it appears that genotoxic oxygen causes DNA damage, which induces synovitis, including synovial hyperplasia, in diseased TMJs.
In conclusion, we are the first to demonstrate that peroxynitrite production and DNA-SSBs occur in the synovial lining cells of synovitis-induced rat TMJs. We believe that oxidative stress, which involves the production of NO and peroxynitrite, causes DNA damage in response to excessive mechanical loading, which in turn promotes synovial hyperplasia in the TMJ.
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ACKNOWLEDGMENTS |
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Received September 22, 2003; Last revision April 7, 2004; Accepted May 28, 2004
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REFERENCES |
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7 layers thick) synovitis of the synovitis-induced TMJ (synov-TMJ) (b) than in the grade 0 (G0; synovial lining is 1–3 layers thick) synovitis of the non-forced TMJ (non-TMJ) (d). F: synovial fold. (c,e) Increased iNOS immunoreactivity is seen in the G2 synovitis of the synovitis-induced TMJ (c), as compared with the grade 0 synovitis of the non-induced TMJ (e). Hematoxylin staining. Original magnification: x400 (b-e). Bar = 25 µm (b-e). (f) Scoring the thickness of the synovial cell layers of the synovitis-induced and non-induced rat TMJs. The thickness of the synovial cell layer (synovitis) is graded as follows: grade 0, 1–3 layers; grade 2, 4–6 layers; grade 2, 
