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Immunohistochemical localization of cyclooxygenase-1 and -2 in synovial tissues from patients with internal derangement or osteoarthritis of the temporomandibular joint
Int. J. Oral Maxillofac. Surg. 2004; 33: 687–692 doi:10.1016/j.ijom.2004.01.026, available online at http://www.sciencedirect.com
Research and Emerging Technologies TMJ Disorders
Immunohistochemical localization of cyclooxygenase-1 and -2 in synovial tissues from patients with internal derangement or osteoarthritis of the temporomandibular joint
H. Seki1, M. Fukuda1, M. Iino1, T. Takahashi2, N. Yoshioka3 1 Division of Dentistry and Oral Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan; 2Second Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1, Manazuru, Kokurakitaku, Kitakyushu City 803-8580, Japan; 3Department of Forensic Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan
H. Seki, M. Fukuda, M. Iino, T. Takahashi, N. Yoshioka:Immunohistochemical localization of cyclooxygenase-1 and -2 in synovial tissues from patients with internal derangement or osteoarthritis of the temporomandibular joint. Int. J. Oral Maxillofac. Surg. 2004; 33: 687–692. # 2004 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. This study examined the immunohistochemical expression and localization of cyclooxygenase-1 and -2 (COX-1 and COX-2) in synovial tissues from patients with internal derangement (ID) or osteoarthritis (OA) of the temporomandibular joint (TMJ). Synovial tissues from patients with condylar fractures of the mandible were studied as control. Synovial tissues from 13 TMJs of 10 patients with ID or OA and from 5 TMJs of 4 patients with fractures were examined for COX-1 and COX-2 expression by immunohistochemical staining using two monoclonal antibodies. In addition, whether the COX-2 expression grade correlated with the synovitis score and clinical findings was assessed. COX-2 was expressed in the synovial lining, infiltrating mononuclear cells, fibroblast-like cells, and blood vessels, including CD31-positive endothelial cells, in the synovium of patients with ID or OA. Expression levels of COX-1 in synovial lining cells and endothelial cells were similar in the specimens obtained from the patients with ID or OA and those obtained from the controls. The expression of COX-2 positively correlated with arthroscopic findings of synovitis (r ¼ 0:55, P ¼ 0:023) and with joint pain (r ¼ 0:56, P ¼ 0:021). These results suggest that up-regulation of COX-2 in synovium may play a part in the pathogenesis of synovitis in patients with ID or OA of the TMJ.
Prostaglandins (PGs), synthesized by cyclooxygenase (COX), are important regulators of inflammation7. PGE2 has been detected in inflamed joints22 and in synovial fluid from patients with dysfunction 0901-5027/070687 + 06 $30.00/0
of the temporomandibular joint (TMJ), correlating with joint pain4,21. PGE2 inhibits chondrocyte growth, triggering osteoclastic bone resorption, up-regulates interleukin-1b (IL-1b) production, and
Key words: cyclooxygenase; immunohistochemistry; temporomandibular joint; internal derangement; synovium; synovitis; joint pain. Accepted for publication 13 January 2004 Available online 28 March 2004
mediates inflammation and bone destruction in rheumatoid arthritis (RA)7. COX exists in the isoforms of COX-1 and COX-228, both of which are inhibited by nonsteroidal anti-inflammatory
# 2004 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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drugs (NSAIDs). COX-1 is constitutively expressed in a number of cell types, whereas COX-2 is an inducible isoform that is up-regulated by variety of cytokines, growth factors, and free radicals at sites of inflammation28. Recent studies suggest that activation of COX-2 contributes to the pathogenesis of inflammatory arthritis, including RA6–15 and adjuvant-induced arthritis15. Immunohistochemical studies have shown that COX-2 is expressed in synovial lining, as well as in fibroblast-like cells, vascular endothelial cells, and mononuclear inflammatory cells in the synovium of patients with RA or osteoarthritis (OA)6,15,17. Moreover, COX-2 mRNA is detected in synovial tissue and fluid obtained from patients with internal derangement (ID) of the TMJ20. The production of COX-2 is increased by IL-1b or PMA6,8 and by nitric oxide (NO)13 in cultured RA synovial cells. We previously demonstrated elevated concentrations of IL-1b26 and nitric oxide (NO)27 in synovial fluid and expression of inducible nitric oxide synthase (iNOS) in synovial tissue of human TMJs with ID12,25. These findings suggest that increased production of PGE2 synthesized by COX-2 may play a part in the pathogenesis of synovitis and degenerative changes in articular cartilage and subchondral bone in the TMJ. However, the expression and localization of COX-1 and COX-2 in the synovium of patients with ID or OA of the TMJ is unclear. In this study, we examined the immunohistochemical expression and localization of COX-1 and COX-2 and the correlation of these enzymes with arthroscopic evidence of synovitis and clinical findings in patients with ID or OA of the TMJ. Patients with condylar fractures of the mandible were studied as control. Materials and methods Subjects
Tissue specimens were obtained from 13 TMJs with ID or OA in 10 patients (1 man and 9 women, mean age: 41 years, range: 25–68 years) during arthroscopic surgery. As control, tissue specimens were obtained from 5 TMJs of 4 patients (2 men and 2 women, mean age: 33.5 years, range: 22–49 years) with fractures of the mandible during arthrocentesis and arthroscopic examination immediately before fracture reduction. Informed consent was obtained from all subjects before obtaining the tissue samples. Magnetic resonance imaging (MRI) was
performed in the patients with ID or OA of the TMJ, and all patients were confirmed to have anterior disc displacement without reduction. Clinical examinations were performed, and any signs and symptoms were recorded. Each patient subjectively evaluated the intensity of joint pain according to a visual analog scale (VAS). The active range of vertical maximal mouth opening (MMO) was measured as the distance between the upper and lower incisors, including overbite. Symptoms in the patients with ID or OA had not responded to a minimum of 3-months’ nonsurgical treatment, including splint therapy, physical therapy, and arthrocentesis. None of the patients with condylar fractures had had any symptoms of the TMJ before injury. The patients were given no NSAIDs for at least 2 months, except for two patients in whom NSAIDs were stopped 1 week before arthroscopic surgery (joints 7 and 14). None of the patients had any relevant immunological conditions or a history of systemic inflammation. The clinical characteristics of the subjects are summarized in Table 1. Arthroscopy
Arthroscopy was performed in the superior joint compartment with a 2.3-mm rodlens arthroscope (Stryker, Santa Clara, CA, USA) under general anesthesia. Synovial biopsy specimens were taken by triangulation technique from the posterior synovial pouch of the superior compart-
ment, using a small basket forceps under direct visualization. Tissue preparation
The tissue samples were all 1–2 mm wide and were fixed immediately in 4% paraformaldehyde (pH 7.4) at 4 8C for 24 h. After washing in phosphate-buffered saline (PBS, pH 7.2), the samples were dehydrated through an ethanol series and embedded in paraffin according to routine methods. Then, 4-mm-thick serial sections of each sample were prepared with a microtome and mounted on silane coated slides. For histological evaluation, each slide was stained with hematoxylin and eosin. Immunohistochemical staining
Primary antibodies to ovine COX-1 (Takara, Tokyo, Japan), human COX-2 (Takara), human CD68 (DAKO, Carpinteria, CA, USA), and human CD31 (DAKO) mouse monoclonal were purchased commercially. Immunohistochemical staining was performed with the use of a Catalyzed Signal Amplification (CSA) System (DAKO) for COX-2 and Histofine Simple Stain (HSS) MAX-MO (Nichirei, Tokyo, Japan) for COX-1, CD31, and CD68 according to the manufacturers’ instructions. Briefly, paraffin sections were dewaxed and rehydrated through an ethanol series. The specimens for CD68 were incubated with 0.01% trypsin
Table 1. Clinical features and COX-2 expression in synovial tissue of patients and controls Number Diagnosis Gender 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
OA OA OA OA OA OA OA ID ID ID ID ID ID F F F F F
F F F F F F F F F M M F F M F F F M
Age (years) 58 46 68 37 25 25 29 38 38 25 25 48 37 22 46 17 17 49
MMO (mm) 32 37 33 39 37 37 38 27 27 25 25 33 32 24 35 30 30 43
(30 (37 (30 (39 (28 (28 (38 (22 (22 (25 (25 (28 (28 (24 (30 (30 (30 (40
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
2) 0) 3) 0) 9) 9) 0) 5) 5) 0) 0) 5) 4) 0) 5) 0) 0) 3)
VAS
Duration
50 72 85 68 50 50 50 55 55 44 44 34 60 0 0 0 0 0
6 months 9 months 9 months 9 months 87 months 87 months 51 months 27 months 27 months 4 months 4 months 5 months 63 months 7 days 7 days 9 days 9 days 7 days
Synovitis COX-2 score score 2 2 2 1 2 2 0 1 2 2 1 2 1 0 1 1 1 0
4 1 2 1 2 1 0 3 3 3 1 0 1 0 0 0 0 1
Number 1–13, patients; number 14–18, controls. Diagnosis: OA, osteoarthritis; ID, internal derangement; F, fracture; MMO, maximal mouth opening; VAS, visual analog scale of pain (1–100). Number 1–13, patients; number 14–18, control. Duration, duration from onset of symptoms or injury to surgery. Synovitis score: 0, normal; 1, slight; 2, pronounce. COX-2 score: 0, no staining; 1, 0–25%; 2, 25–50%; 3, 50–75%; 4, more than 75%.
Cox expression in synovial tissue (Sigma, St. Louis, USA) for 20 min in a humid chamber at 37 8C. To inactivate endogenous peroxidase, all sections were incubated for 20 min in methanol containing 0.03% hydrogen peroxide. In the CSA System, the specimens were then incubated for 5 min with a protein-blocking reagent to suppress nonspecific binding, followed by incubation with mouse monoclonal antibody against COX-2 for 15 min in a humid chamber at room temperature. After washing in PBS, the sections were incubated with biotinylated link antibody, streptavidin–biotin–peroxidase complex, amplification reagent, and streptavidin–peroxidase for 15 min each at room temperature. In the HSS, the specimens were then incubated for 5 min with a protein-blocking reagent (DAKO) to suppress nonspecific binding, followed by incubation with mouse monoclonal antibody against CD31, CD68, and COX-1 overnight at 4 8C. After washing in PBS, the sections were incubated with HSS MAX-PO for 20 min at room temperature. Finally, substrate reagent (3,3-diaminobenzidine tetrahydrochloride,
Dojindo, Mashiki-machi, Japan) was added to all specimens. Counterstaining was performed with hematoxylin, and the sections were mounted. For negative control specimens, the primary antibody was replaced with normal mouse IgG (DAKO). The intensity of immunohistochemical staining was scored as described previously5: Grade 0, no staining of cells in any microscopic field; Grade 1, less than 25% of tissue stained positive; Grade 2, between 25 and 50% stained positive; Grade 3, between 50 and 75% stained positive; and Grade 4, more than 75% stained positive. Each slide was evaluated by two independent observers blinded to the clinical findings. Evaluation of the arthroscopic criteria for synovitis
Arthroscopic findings of synovitis were evaluated and scored as reported previously11, with slight modification: Grade 0 (normal), no capillary hyperemia and/or vascularity; Grade 1 (slight), capillary
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hyperemia and/or increased vascularity in a localized area; and Grade 2 (pronounced), three or more of the following features: capillary hyperemia, increased vascularity, thickening of the lateral capsule, fibrosis, villi, or synovial hyperplasia. Statistical analysis
Statistical analyses to assess correlations of COX-2 expression with the synovitis score and clinical findings were performed with the use of Spearman’s rank correlation coefficient or the Mann– Whitney U test. Results
Specimens from joints with ID or OA showed proliferation of synovial lining cells, increased vascularity, and/or capillary hyperemia and slight infiltration of inflammatory cells. In contrast, in the specimens of joints with fractures, synovial lining cells were limited to one or two layers, vascularity was absent or minimal, and few infiltrating inflamma-
Fig. 1. Immunohistochemical staining for COX-2 in synovial tissues from patients with ID or OA and from controls. (A) Definite expression was observed in both CD68-positive and -negative layers of synovial lining cells (a). (B) Strong expression was observed in blood vessels including CD31-positive endothelial cells (b), round mononuclear cells (arrows), and fibroblast-like cells (arrowheads). (C, D) In contrast, staining was absent or weak in the synovial lining and blood vessels. Original magnification 400.
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Fig. 2. Immunohistochemical staining for COX-1 in synovial tissues from patients with ID or OA and from controls. COX-1 was well expressed in the lining cell layer (A), round mononuclear cells (arrows), fibroblast-like cells (arrowheads), and blood vessels (B) in the diseased tissues. In the control tissues, COX-1 was well expressed in the lining cell layer (C) and blood vessels (D). Original magnification 400.
tory cells were observed. The arthroscopic findings of synovitis are summarized in Table 1. In most joints with ID or OA, capillary hyperemia, increased vascularity, thickening of the lateral capsule, fibrosis, and synovial hyperplasia were observed. In contrast, the joints with fractures had no or slight vascularity in localized areas, with no other evidence of injury-related damage. On the basis of these results, we evaluated joints with ID or OA as diseased joints, and those with fractures as control. Eleven of 13 diseased tissue specimens showed strong or definite expression of COX-2 in infiltrating mononuclear cells, CD68-positive and -negative synovial lining cells, fibroblast-like cells beneath the synovial lining, and blood vessels, including CD31-positive endothelial cells (Fig. 1). Among these four cell types, mononuclear cells, fibroblast-like cells, and blood vessels were very intensely stained. In contrast, no or weak expression of COX-2 was seen in synovial lining and blood vessels in the five control specimens. The COX-2 expression score of the diseased specimens was higher
than that of the control specimens (P ¼ 0:014). Expression of COX-1 was observed in infiltrating mononuclear cells, synovial lining cells, fibroblast-like cells in the sublining layer, and blood vessels in the diseased specimens. In the control specimens, COX-1 was observed in synovial lining cells, fibroblast-like
cells in the sublining layer, and blood vessels. The expression of COX-1 in these cells was similar in the diseased and control specimens (Fig. 2). Eight of the 13 diseased joints had Grade 2 synovitis. In five specimens of these eight joints, COX-2 expression was stronger than that in the other joints
Fig. 3. Correlation between COX-2 expression and synovitis (r ¼ 0:55, P ¼ 0:023).
Cox expression in synovial tissue
Fig. 4. Correlation between COX-2 expression and joint pain (r ¼ 0:56, P ¼ 0:021).
(Table 1). COX-2 expression positively correlated with the arthroscopic findings of synovitis (r ¼ 0:55, P ¼ 0:023) (Fig. 3) and with joint pain (r ¼ 0:561, P ¼ 0:021) (Fig. 4). Discussion
The present study sheds new light on the participation of COX expression and localization in ID or OA of the TMJ. QUINN et al.20 showed that COX-2 mRNA is detected in the synovial tissue (94%) and synovial fluid (75%) of patients with ID of the TMJ. YOSHIDA et al.29 immunohistochemically demonstrated expression of COX-2 in fibroblast-like cells and chondrocyte-like cells within joint discs, as well as in fibroblast-like cells, endothelial cells, and synovial layer cells in the posterior and/or anterior loose connective tissues in human TMJ samples with ID, but not in control human TMJ samples. The pattern of COX-2 expression in the synovium was consistent with the results of our study. Previous studies of COX expression in the synovium of other joints with RA and OA have reported various results. CROFFORD et al.6 reported that COX-2 was expressed in infiltrating mononuclear cells, endothelial cells, and subsynovial fibroblast-like cells, but only weakly expressed in synovial lining cells. KANG et al.15 detected COX-2 expression in lining cells, lymphoid aggregates, and blood vessels in RA and OA tissues, but not in normal tissues. In OA tissues, the number of stained cells and the staining intensity were lower than those of RA tissues. LEE et al.17 detected COX-2 expression in synovial lining cells, inflammatory cells, stromal
fibroblast-like cells, and vascular endothelial cells of RA or OA synovium. There was no significant difference in the cell types expressing COX-2 in the synovium between RA and OA. In our study, similar types of cells expressed COX-2, suggesting that the inflammatory mechanisms modulated by COX-2 in ID or OA of the TMJ are similar to those in RA and OA of other joints. The production of COX-2 is enhanced by various cytokines, growth factors, and free radicals in different types of cells. COX-2 mRNA is markedly increased by treatment with IL-1b in cultured RA synovial cells6,8. Tumor necrosis factor a (TNF-a) also stimulates COX-2 production and up-regulates the expression of nuclear factor kappa B (NF-kB), a transcriptional factor of COX-2, in cultured OA synovial cells2. HONDA et al.13 reported that induction of COX-2 expression is enhanced, while COX-1 is unaffected in synovial cells treated with S-nitros-N-acetyl-D,L-penicillamine, an NO donor. Proinflammatory cytokines also stimulate NO production in RA joint cells18, and NOS inhibitors inhibit both NO and PGE2 production23. Recently, proinflammatory cytokines and free radicals, such as IL-1b, TNF-a and NO, have been detected in synovial fluid and synovial tissues from diseased joints1,3,9,10,14,16,24. We have previously demonstrated high levels of IL-1b, TNFa, and interferon-g in the synovial fluid from patients with TMD26. We have also detected high levels of NO in synovial fluid from patients with TMD27, and iNOS expression in synovial tissues of these patients12,25. Furthermore, we have
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confirmed the immunohistochemical expression of NF-kB in diseased tissues, especially the synovial lining and blood vessels (data not shown). These results suggest that two pathways may up-regulate COX-2 in the synovium of patients with ID or OA. First, increased production of COX-2 in synovial cells may be stimulated directly by proinflammatory cytokines. Second, increased production of COX-2 is perhaps stimulated by NO that has been up-regulated by cytokines. However, several other investigations assessing interactions between PGE2 and NO production in different types of cells have yielded inconsistent results28. Therefore, further in vitro studies, using cultured synovial cell systems and/or organ culture systems of synovium from the TMJ, are needed to confirm the pathways involved and to examine interactions between PGE and NO in synovium of the TMJ. Our study showed that COX-2 expression was directly related to synovitis and joint pain. PGs are known to sensitize pain receptors to other mediators7. PGE2 is detected in synovial fluid from painful joints3,21. These findings suggest that PGs synthesized by COX-2 play an important, albeit secondary role in joint pain and inflammation of the synovium. In fact, joint pain has been demonstrated to be related to the degree of synovitis19. NSAIDs are known to inhibit COX expression. Most of our patients received no NSAIDs for at least 2 months before arthroscopic surgery, and COX-1 expression was not inhibited in these patients. We therefore consider that NSAIDs did not affect our results. Two patients, however, discontinued NSAIDs only 1 week before surgery, and COX-2 expression was evaluated to be Grade 0. In these two patients, the COX-2 expression score might have been affected by prior treatment with NSAIDs. References 1. Aghabeigi B, Cintra N, Meghji S, Evans A, Hopper C. Measurement of nitric oxide in temporomandibular joint saline aspirates. Int J Oral Maxillofac Surg 2003: 32: 401–403. 2. Alaaeddine N, Di Battista JA, Pelletier J-P, Kiansa K, Cloutier J-M, Martel-Pelletier J. Inhibition of tumor necrosis factor a-induced prostaglandin E2 production by the antiinflammatory cytokines interleukin-4, interleukin10, and interleukin-13 in osteoarthritic synovial fibroblasts. Arthritis Rheum 1999: 42: 710–718.
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3. Alstergren P, Ernberg M, Kvarnstro¨m M, Kopp S. Interleukin-1b in synovial fluid from the arthritic temporomandibular joint and its relation to pain, mobility, and anterior open bite. J Oral Maxillofac Surg 1998: 56: 1059– 1065. 4. Alstergren P, Kopp S. Prostaglandin E2 in temporomandibular joint synovial fluid and its relation to pain and inflammatory disorders. J Oral Maxillofac Surg 2000: 58: 180–186. 5. Brennan PA, Palacios-Callender M, Umar T, Tant S, Langdon JD. Expression of type 2 nitric oxide synthase and p21 in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2002: 31: 200–205. 6. Crofford LJ, Wilder RL, Ristima¨ki AP, Sano H, Remmers EF, Epps HR, Hla T. Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissue. Effect of interleukin-1b, phorbol ester, and corticosteroids. J Clin Invest 1994: 93: 1095– 1101. 7. Davies P, Bailey PJ, Goldenberg MM. The role of arachidonic acid oxygenation products in pain and inflammation. Annu Rev Immunol 1984: 2: 335–357. 8. Fahmi H, He Y, Zhang M, MartelPelletier J, Pelletier JP, Di Battista JA. Nimesulide reduces interleukin-1binduced cyclooxygenase-2 gene expression in human synovial fibroblasts. Osteoarthritis Cartilage 2001: 9: 332– 340. 9. Fu K, Ma X, Zhenkang Z, Chen W. Tumor necrosis factor in synovial fluid of patients with temporomandibular disorders. J Oral Maxillofac Surg 1995: 53: 424–426. 10. Hirota W. Intra-articular injection of hyaluronic acid reduces total amounts of leukotriene C4, 6-keto-prostaglandin F1a, prostaglandin F2a, and interleukin1b in synovial fluid of patients with internal derangement in disorders of the temporomandibular joint. Br J Oral Maxillofac Surg 1998: 36: 35–38. 11. Holmlund AB, Gynther G, Reinholt FP. Rheumatoid arthritis and disk derangement of the temporomandibular joint. A comparative arthroscopic study. Oral Surg Oral Med Oral Pathol 1992: 73: 273–277. 12. Homma H, Takahashi T, Seki H, Ohtani M, Kondoh T, Fukuda M. Immunohistochemical localization of nitric oxide synthase in synovial tissue of human temporomandibular joints with
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internal derangement. Arch Oral Biol 2001: 46: 93–97. Honda S, Migiga K, Hirai Y, Ueki Y, Yamasaki S, Urayama S, Kawabe Y, Fukuda T, Kawakami A, Kamachi M, Kita M, Ida H, Aoyagi T, Eguchi K. Induction of COX-2 expression by nitric oxide in rheumatoid synovial cells. Biochem Biophys Res Commun 2000: 268: 928–931. Kardel R, Ulfgren A-K, Reinholt FP, Holmlund A. Inflammatory cell and cytokine patterns in patients with painful clicking and osteoarthritis in the temporomandibular joint. Int J Oral Maxillofac Surg 2003: 32: 390–396. Kang RY, Freire-Moar J, Sigal E, Chu C-Q. Expression of cyclooxygenase-2 in human and an animal model of rheumatoid arthritis. Br J Rheumatol 1996: 35: 711–718. Kubota E, Imamura H, Kubota T, Shibata T, Murakami K-I. Interleukin-1b and stromelysin (MMP3) activity of synovial fluid as possible markers of osteoarthritis in temporomandibular joint. J Oral Maxillofac Surg 1997: 55: 20–27. Lee YH, Choi SJ, Kim A, Kim CH, Ji JD, Song GG. Expression of cyclooxygenase-1 and -2 in rheumatoid arthritis synovium. J Korean Med Sci 2000: 15: 88–92. LeGrand A, Fermor B, Fink C, Pisetsky DS, Weinberg JB, Vail TP, Guilak F. Interleukin-1, tumor necrosis factor a, and interleukin-17 synergistically up-regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci. Arthritis Rheum 2001: 44: 2078–2083. Murakami K-I, Segami N, Fujimura K, Iizuka T. Correlation between pain and synovitis in patients with internal derangement of the temporomandibular joint. J Oral Maxillofac Surg 1991: 49: 1159–1161. Quinn JH, Kent JN, Noise A, Lukiw WJ. Cyclooxygenase-2 in synovial tissue and fluid of dysfunctional temporomandibular joints with internal derangement. J Oral Maxillofac Surg 2000: 58: 1229– 1232. Quinn JH, Bazan NG. Identification of prostaglandin E2 and leukotriene B4 in the synovial fluid of painful dysfunctional temporomandibular joints. J Oral Maxillofac Surg 1990: 48: 968–971. Robinson DR, McGuire MB. Prostaglandins in the rheumatic diseases. Ann NY Acad Sci 1975: 13: 318–329.
23. Salvemini D, Manning PT, Zweifel BS, Seibert K, Connor J, Currie MG, Needleman P, Masferrer JL. Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors. J Clin Invest 1995: 96: 301–308. 24. Shafer DM, Assael L, White LB, Rossomando EF. Tumor necrosis factor-a as a biochemical marker of pain and outcome in temporomandibular joints with internal derangement. J Oral Maxillofac Surg 1994: 52: 786–791. 25. Takahashi T, Homma H, Nagai H, Seki H, Kondoh T, Yamazaki Y, Fukuda M. Specific expression of inducible nitric oxide synthase in the synovium of the diseased temporomandibular joint. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003: 95: 174– 181. 26. Takahashi T, Kondoh T, Fukuda M, Yamazaki Y, Toyosaki T, Suzuki R. Proinflammatory cytokines detectable in synovial fluids from patients with temporomandibular disorders. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998: 85: 135–141. 27. Takahashi T, Kondoh T, Kamei K, Seki H, Fukuda M, Nagai H, Takano H, Yamazaki Y. Elevated levels of nitric oxide in synovial fluid from patients with temporomandibular disorders. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996: 82: 505–509. 28. Weinberg JB. Nitric oxide synthase 2 and cyclooxygenase 2 interactions in inflammation. Immunol Res 2000: 22: 319–341. 29. Yoshida H, Fukumura Y, Fujita S, Nishida M, Iizuka T. The expression of cyclooxygenase-2 in human temporomandibular joint samples: an immunohistochemical study. J Oral Rehabil 2002: 29: 1146–1152. Address: Hiroshi Seki Division of Dentistry and Oral Surgery Akita University School of Medicine 1-1-1 Hondo Akita City Akita 010-8543 Japan. Tel: þ81 18 84 6188 Fax: þ81 18 884 6451 E-mail: [email protected]
Research and Emerging Technologies TMJ Disorders
Immunohistochemical localization of cyclooxygenase-1 and -2 in synovial tissues from patients with internal derangement or osteoarthritis of the temporomandibular joint
H. Seki1, M. Fukuda1, M. Iino1, T. Takahashi2, N. Yoshioka3 1 Division of Dentistry and Oral Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan; 2Second Department of Oral and Maxillofacial Surgery, Kyushu Dental College, 2-6-1, Manazuru, Kokurakitaku, Kitakyushu City 803-8580, Japan; 3Department of Forensic Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan
H. Seki, M. Fukuda, M. Iino, T. Takahashi, N. Yoshioka:Immunohistochemical localization of cyclooxygenase-1 and -2 in synovial tissues from patients with internal derangement or osteoarthritis of the temporomandibular joint. Int. J. Oral Maxillofac. Surg. 2004; 33: 687–692. # 2004 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. This study examined the immunohistochemical expression and localization of cyclooxygenase-1 and -2 (COX-1 and COX-2) in synovial tissues from patients with internal derangement (ID) or osteoarthritis (OA) of the temporomandibular joint (TMJ). Synovial tissues from patients with condylar fractures of the mandible were studied as control. Synovial tissues from 13 TMJs of 10 patients with ID or OA and from 5 TMJs of 4 patients with fractures were examined for COX-1 and COX-2 expression by immunohistochemical staining using two monoclonal antibodies. In addition, whether the COX-2 expression grade correlated with the synovitis score and clinical findings was assessed. COX-2 was expressed in the synovial lining, infiltrating mononuclear cells, fibroblast-like cells, and blood vessels, including CD31-positive endothelial cells, in the synovium of patients with ID or OA. Expression levels of COX-1 in synovial lining cells and endothelial cells were similar in the specimens obtained from the patients with ID or OA and those obtained from the controls. The expression of COX-2 positively correlated with arthroscopic findings of synovitis (r ¼ 0:55, P ¼ 0:023) and with joint pain (r ¼ 0:56, P ¼ 0:021). These results suggest that up-regulation of COX-2 in synovium may play a part in the pathogenesis of synovitis in patients with ID or OA of the TMJ.
Prostaglandins (PGs), synthesized by cyclooxygenase (COX), are important regulators of inflammation7. PGE2 has been detected in inflamed joints22 and in synovial fluid from patients with dysfunction 0901-5027/070687 + 06 $30.00/0
of the temporomandibular joint (TMJ), correlating with joint pain4,21. PGE2 inhibits chondrocyte growth, triggering osteoclastic bone resorption, up-regulates interleukin-1b (IL-1b) production, and
Key words: cyclooxygenase; immunohistochemistry; temporomandibular joint; internal derangement; synovium; synovitis; joint pain. Accepted for publication 13 January 2004 Available online 28 March 2004
mediates inflammation and bone destruction in rheumatoid arthritis (RA)7. COX exists in the isoforms of COX-1 and COX-228, both of which are inhibited by nonsteroidal anti-inflammatory
# 2004 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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drugs (NSAIDs). COX-1 is constitutively expressed in a number of cell types, whereas COX-2 is an inducible isoform that is up-regulated by variety of cytokines, growth factors, and free radicals at sites of inflammation28. Recent studies suggest that activation of COX-2 contributes to the pathogenesis of inflammatory arthritis, including RA6–15 and adjuvant-induced arthritis15. Immunohistochemical studies have shown that COX-2 is expressed in synovial lining, as well as in fibroblast-like cells, vascular endothelial cells, and mononuclear inflammatory cells in the synovium of patients with RA or osteoarthritis (OA)6,15,17. Moreover, COX-2 mRNA is detected in synovial tissue and fluid obtained from patients with internal derangement (ID) of the TMJ20. The production of COX-2 is increased by IL-1b or PMA6,8 and by nitric oxide (NO)13 in cultured RA synovial cells. We previously demonstrated elevated concentrations of IL-1b26 and nitric oxide (NO)27 in synovial fluid and expression of inducible nitric oxide synthase (iNOS) in synovial tissue of human TMJs with ID12,25. These findings suggest that increased production of PGE2 synthesized by COX-2 may play a part in the pathogenesis of synovitis and degenerative changes in articular cartilage and subchondral bone in the TMJ. However, the expression and localization of COX-1 and COX-2 in the synovium of patients with ID or OA of the TMJ is unclear. In this study, we examined the immunohistochemical expression and localization of COX-1 and COX-2 and the correlation of these enzymes with arthroscopic evidence of synovitis and clinical findings in patients with ID or OA of the TMJ. Patients with condylar fractures of the mandible were studied as control. Materials and methods Subjects
Tissue specimens were obtained from 13 TMJs with ID or OA in 10 patients (1 man and 9 women, mean age: 41 years, range: 25–68 years) during arthroscopic surgery. As control, tissue specimens were obtained from 5 TMJs of 4 patients (2 men and 2 women, mean age: 33.5 years, range: 22–49 years) with fractures of the mandible during arthrocentesis and arthroscopic examination immediately before fracture reduction. Informed consent was obtained from all subjects before obtaining the tissue samples. Magnetic resonance imaging (MRI) was
performed in the patients with ID or OA of the TMJ, and all patients were confirmed to have anterior disc displacement without reduction. Clinical examinations were performed, and any signs and symptoms were recorded. Each patient subjectively evaluated the intensity of joint pain according to a visual analog scale (VAS). The active range of vertical maximal mouth opening (MMO) was measured as the distance between the upper and lower incisors, including overbite. Symptoms in the patients with ID or OA had not responded to a minimum of 3-months’ nonsurgical treatment, including splint therapy, physical therapy, and arthrocentesis. None of the patients with condylar fractures had had any symptoms of the TMJ before injury. The patients were given no NSAIDs for at least 2 months, except for two patients in whom NSAIDs were stopped 1 week before arthroscopic surgery (joints 7 and 14). None of the patients had any relevant immunological conditions or a history of systemic inflammation. The clinical characteristics of the subjects are summarized in Table 1. Arthroscopy
Arthroscopy was performed in the superior joint compartment with a 2.3-mm rodlens arthroscope (Stryker, Santa Clara, CA, USA) under general anesthesia. Synovial biopsy specimens were taken by triangulation technique from the posterior synovial pouch of the superior compart-
ment, using a small basket forceps under direct visualization. Tissue preparation
The tissue samples were all 1–2 mm wide and were fixed immediately in 4% paraformaldehyde (pH 7.4) at 4 8C for 24 h. After washing in phosphate-buffered saline (PBS, pH 7.2), the samples were dehydrated through an ethanol series and embedded in paraffin according to routine methods. Then, 4-mm-thick serial sections of each sample were prepared with a microtome and mounted on silane coated slides. For histological evaluation, each slide was stained with hematoxylin and eosin. Immunohistochemical staining
Primary antibodies to ovine COX-1 (Takara, Tokyo, Japan), human COX-2 (Takara), human CD68 (DAKO, Carpinteria, CA, USA), and human CD31 (DAKO) mouse monoclonal were purchased commercially. Immunohistochemical staining was performed with the use of a Catalyzed Signal Amplification (CSA) System (DAKO) for COX-2 and Histofine Simple Stain (HSS) MAX-MO (Nichirei, Tokyo, Japan) for COX-1, CD31, and CD68 according to the manufacturers’ instructions. Briefly, paraffin sections were dewaxed and rehydrated through an ethanol series. The specimens for CD68 were incubated with 0.01% trypsin
Table 1. Clinical features and COX-2 expression in synovial tissue of patients and controls Number Diagnosis Gender 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
OA OA OA OA OA OA OA ID ID ID ID ID ID F F F F F
F F F F F F F F F M M F F M F F F M
Age (years) 58 46 68 37 25 25 29 38 38 25 25 48 37 22 46 17 17 49
MMO (mm) 32 37 33 39 37 37 38 27 27 25 25 33 32 24 35 30 30 43
(30 (37 (30 (39 (28 (28 (38 (22 (22 (25 (25 (28 (28 (24 (30 (30 (30 (40
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
2) 0) 3) 0) 9) 9) 0) 5) 5) 0) 0) 5) 4) 0) 5) 0) 0) 3)
VAS
Duration
50 72 85 68 50 50 50 55 55 44 44 34 60 0 0 0 0 0
6 months 9 months 9 months 9 months 87 months 87 months 51 months 27 months 27 months 4 months 4 months 5 months 63 months 7 days 7 days 9 days 9 days 7 days
Synovitis COX-2 score score 2 2 2 1 2 2 0 1 2 2 1 2 1 0 1 1 1 0
4 1 2 1 2 1 0 3 3 3 1 0 1 0 0 0 0 1
Number 1–13, patients; number 14–18, controls. Diagnosis: OA, osteoarthritis; ID, internal derangement; F, fracture; MMO, maximal mouth opening; VAS, visual analog scale of pain (1–100). Number 1–13, patients; number 14–18, control. Duration, duration from onset of symptoms or injury to surgery. Synovitis score: 0, normal; 1, slight; 2, pronounce. COX-2 score: 0, no staining; 1, 0–25%; 2, 25–50%; 3, 50–75%; 4, more than 75%.
Cox expression in synovial tissue (Sigma, St. Louis, USA) for 20 min in a humid chamber at 37 8C. To inactivate endogenous peroxidase, all sections were incubated for 20 min in methanol containing 0.03% hydrogen peroxide. In the CSA System, the specimens were then incubated for 5 min with a protein-blocking reagent to suppress nonspecific binding, followed by incubation with mouse monoclonal antibody against COX-2 for 15 min in a humid chamber at room temperature. After washing in PBS, the sections were incubated with biotinylated link antibody, streptavidin–biotin–peroxidase complex, amplification reagent, and streptavidin–peroxidase for 15 min each at room temperature. In the HSS, the specimens were then incubated for 5 min with a protein-blocking reagent (DAKO) to suppress nonspecific binding, followed by incubation with mouse monoclonal antibody against CD31, CD68, and COX-1 overnight at 4 8C. After washing in PBS, the sections were incubated with HSS MAX-PO for 20 min at room temperature. Finally, substrate reagent (3,3-diaminobenzidine tetrahydrochloride,
Dojindo, Mashiki-machi, Japan) was added to all specimens. Counterstaining was performed with hematoxylin, and the sections were mounted. For negative control specimens, the primary antibody was replaced with normal mouse IgG (DAKO). The intensity of immunohistochemical staining was scored as described previously5: Grade 0, no staining of cells in any microscopic field; Grade 1, less than 25% of tissue stained positive; Grade 2, between 25 and 50% stained positive; Grade 3, between 50 and 75% stained positive; and Grade 4, more than 75% stained positive. Each slide was evaluated by two independent observers blinded to the clinical findings. Evaluation of the arthroscopic criteria for synovitis
Arthroscopic findings of synovitis were evaluated and scored as reported previously11, with slight modification: Grade 0 (normal), no capillary hyperemia and/or vascularity; Grade 1 (slight), capillary
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hyperemia and/or increased vascularity in a localized area; and Grade 2 (pronounced), three or more of the following features: capillary hyperemia, increased vascularity, thickening of the lateral capsule, fibrosis, villi, or synovial hyperplasia. Statistical analysis
Statistical analyses to assess correlations of COX-2 expression with the synovitis score and clinical findings were performed with the use of Spearman’s rank correlation coefficient or the Mann– Whitney U test. Results
Specimens from joints with ID or OA showed proliferation of synovial lining cells, increased vascularity, and/or capillary hyperemia and slight infiltration of inflammatory cells. In contrast, in the specimens of joints with fractures, synovial lining cells were limited to one or two layers, vascularity was absent or minimal, and few infiltrating inflamma-
Fig. 1. Immunohistochemical staining for COX-2 in synovial tissues from patients with ID or OA and from controls. (A) Definite expression was observed in both CD68-positive and -negative layers of synovial lining cells (a). (B) Strong expression was observed in blood vessels including CD31-positive endothelial cells (b), round mononuclear cells (arrows), and fibroblast-like cells (arrowheads). (C, D) In contrast, staining was absent or weak in the synovial lining and blood vessels. Original magnification 400.
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Fig. 2. Immunohistochemical staining for COX-1 in synovial tissues from patients with ID or OA and from controls. COX-1 was well expressed in the lining cell layer (A), round mononuclear cells (arrows), fibroblast-like cells (arrowheads), and blood vessels (B) in the diseased tissues. In the control tissues, COX-1 was well expressed in the lining cell layer (C) and blood vessels (D). Original magnification 400.
tory cells were observed. The arthroscopic findings of synovitis are summarized in Table 1. In most joints with ID or OA, capillary hyperemia, increased vascularity, thickening of the lateral capsule, fibrosis, and synovial hyperplasia were observed. In contrast, the joints with fractures had no or slight vascularity in localized areas, with no other evidence of injury-related damage. On the basis of these results, we evaluated joints with ID or OA as diseased joints, and those with fractures as control. Eleven of 13 diseased tissue specimens showed strong or definite expression of COX-2 in infiltrating mononuclear cells, CD68-positive and -negative synovial lining cells, fibroblast-like cells beneath the synovial lining, and blood vessels, including CD31-positive endothelial cells (Fig. 1). Among these four cell types, mononuclear cells, fibroblast-like cells, and blood vessels were very intensely stained. In contrast, no or weak expression of COX-2 was seen in synovial lining and blood vessels in the five control specimens. The COX-2 expression score of the diseased specimens was higher
than that of the control specimens (P ¼ 0:014). Expression of COX-1 was observed in infiltrating mononuclear cells, synovial lining cells, fibroblast-like cells in the sublining layer, and blood vessels in the diseased specimens. In the control specimens, COX-1 was observed in synovial lining cells, fibroblast-like
cells in the sublining layer, and blood vessels. The expression of COX-1 in these cells was similar in the diseased and control specimens (Fig. 2). Eight of the 13 diseased joints had Grade 2 synovitis. In five specimens of these eight joints, COX-2 expression was stronger than that in the other joints
Fig. 3. Correlation between COX-2 expression and synovitis (r ¼ 0:55, P ¼ 0:023).
Cox expression in synovial tissue
Fig. 4. Correlation between COX-2 expression and joint pain (r ¼ 0:56, P ¼ 0:021).
(Table 1). COX-2 expression positively correlated with the arthroscopic findings of synovitis (r ¼ 0:55, P ¼ 0:023) (Fig. 3) and with joint pain (r ¼ 0:561, P ¼ 0:021) (Fig. 4). Discussion
The present study sheds new light on the participation of COX expression and localization in ID or OA of the TMJ. QUINN et al.20 showed that COX-2 mRNA is detected in the synovial tissue (94%) and synovial fluid (75%) of patients with ID of the TMJ. YOSHIDA et al.29 immunohistochemically demonstrated expression of COX-2 in fibroblast-like cells and chondrocyte-like cells within joint discs, as well as in fibroblast-like cells, endothelial cells, and synovial layer cells in the posterior and/or anterior loose connective tissues in human TMJ samples with ID, but not in control human TMJ samples. The pattern of COX-2 expression in the synovium was consistent with the results of our study. Previous studies of COX expression in the synovium of other joints with RA and OA have reported various results. CROFFORD et al.6 reported that COX-2 was expressed in infiltrating mononuclear cells, endothelial cells, and subsynovial fibroblast-like cells, but only weakly expressed in synovial lining cells. KANG et al.15 detected COX-2 expression in lining cells, lymphoid aggregates, and blood vessels in RA and OA tissues, but not in normal tissues. In OA tissues, the number of stained cells and the staining intensity were lower than those of RA tissues. LEE et al.17 detected COX-2 expression in synovial lining cells, inflammatory cells, stromal
fibroblast-like cells, and vascular endothelial cells of RA or OA synovium. There was no significant difference in the cell types expressing COX-2 in the synovium between RA and OA. In our study, similar types of cells expressed COX-2, suggesting that the inflammatory mechanisms modulated by COX-2 in ID or OA of the TMJ are similar to those in RA and OA of other joints. The production of COX-2 is enhanced by various cytokines, growth factors, and free radicals in different types of cells. COX-2 mRNA is markedly increased by treatment with IL-1b in cultured RA synovial cells6,8. Tumor necrosis factor a (TNF-a) also stimulates COX-2 production and up-regulates the expression of nuclear factor kappa B (NF-kB), a transcriptional factor of COX-2, in cultured OA synovial cells2. HONDA et al.13 reported that induction of COX-2 expression is enhanced, while COX-1 is unaffected in synovial cells treated with S-nitros-N-acetyl-D,L-penicillamine, an NO donor. Proinflammatory cytokines also stimulate NO production in RA joint cells18, and NOS inhibitors inhibit both NO and PGE2 production23. Recently, proinflammatory cytokines and free radicals, such as IL-1b, TNF-a and NO, have been detected in synovial fluid and synovial tissues from diseased joints1,3,9,10,14,16,24. We have previously demonstrated high levels of IL-1b, TNFa, and interferon-g in the synovial fluid from patients with TMD26. We have also detected high levels of NO in synovial fluid from patients with TMD27, and iNOS expression in synovial tissues of these patients12,25. Furthermore, we have
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confirmed the immunohistochemical expression of NF-kB in diseased tissues, especially the synovial lining and blood vessels (data not shown). These results suggest that two pathways may up-regulate COX-2 in the synovium of patients with ID or OA. First, increased production of COX-2 in synovial cells may be stimulated directly by proinflammatory cytokines. Second, increased production of COX-2 is perhaps stimulated by NO that has been up-regulated by cytokines. However, several other investigations assessing interactions between PGE2 and NO production in different types of cells have yielded inconsistent results28. Therefore, further in vitro studies, using cultured synovial cell systems and/or organ culture systems of synovium from the TMJ, are needed to confirm the pathways involved and to examine interactions between PGE and NO in synovium of the TMJ. Our study showed that COX-2 expression was directly related to synovitis and joint pain. PGs are known to sensitize pain receptors to other mediators7. PGE2 is detected in synovial fluid from painful joints3,21. These findings suggest that PGs synthesized by COX-2 play an important, albeit secondary role in joint pain and inflammation of the synovium. In fact, joint pain has been demonstrated to be related to the degree of synovitis19. NSAIDs are known to inhibit COX expression. Most of our patients received no NSAIDs for at least 2 months before arthroscopic surgery, and COX-1 expression was not inhibited in these patients. We therefore consider that NSAIDs did not affect our results. Two patients, however, discontinued NSAIDs only 1 week before surgery, and COX-2 expression was evaluated to be Grade 0. In these two patients, the COX-2 expression score might have been affected by prior treatment with NSAIDs. References 1. Aghabeigi B, Cintra N, Meghji S, Evans A, Hopper C. Measurement of nitric oxide in temporomandibular joint saline aspirates. Int J Oral Maxillofac Surg 2003: 32: 401–403. 2. Alaaeddine N, Di Battista JA, Pelletier J-P, Kiansa K, Cloutier J-M, Martel-Pelletier J. Inhibition of tumor necrosis factor a-induced prostaglandin E2 production by the antiinflammatory cytokines interleukin-4, interleukin10, and interleukin-13 in osteoarthritic synovial fibroblasts. Arthritis Rheum 1999: 42: 710–718.
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