Cyclooxygenase-2 in synovial tissue and fluid of dysfunctional temporomandibular joints with internal derangement

J Oral Maxillofac Surg 58:1229-1232, 2000

Cyclooxygenase-2 in Synovial Tissue and Fluid of Dysfunctional Temporomandibular Joints With Internal Derangement James H. Quinn, DDS,* John N. Kent, DDS,† Allison Moise,‡ and Walter J. Lukiw, PhD§ Purpose:

This study investigated cyclooxygenase-2 (COX-2) gene expression in temporomandibular joint (TMJ) synovial tissue and fluid from patients with internal derangement. Patients and Methods: Seventeen synovial tissue biopsy specimens and 16 synovial fluid samples were obtained from patients (1 male and 11 female) during arthroscopic TMJ surgery. The samples were frozen at ⫺70°C and, by using Northern and reverse transcription polymerase chain reaction (RT-PCR) analysis, the levels of COX-2 RNA in relation to ␤-actin RNA message levels were determined. Results: COX-2 RNA message was detected in 16 of 17 synovial tissue samples (94%) and 12 of 16 synovial fluid samples (75%) by using ␤-actin RNA levels in the same sample (either tissue or fluid) as an internal control. Samples were not quantified because of the same sample mass. Conclusion: COX-2, an important inflammatory mediator, is present in the TMJ synovial tissue and fluid from patients with internal derangement. Therefore, COX-2 antagonists may be indicated in the treatment of TMJ arthralgia. © 2000 American Association of Oral and Maxillofacial Surgeons The pathophysiology of temporomandibular joint (TMJ) pain caused by the microtrauma of stress-related bruxism is not well understood. In 1990, Quinn and Bazan1 first identified the presence of prostaglandin E2 (PGE2) and leukotriene B4 in the inflamed TMJ and showed a significant correlation with the level of acute synovitis seen arthroscopically. Numerous articles have been published since, identifying other pain mediators, including interleukin-1B (IL-1B)2-5; interleukin-6 (IL-6)3-5; tumor necrosis factor (TNF)3,5; neuropeptides: Y, substance P, neurokinin-A, calcitoningenerated peptide6,7; and serotonin (5HT).2 These findings have aided in our understanding of TMJ arthralgia. However, critical inflammatory mediators * Clinical Professor, LSU School of Dentistry, New Orleans, LA. † Boyd Professor, Chair, LSU School of Dentistry, New Orleans, LA. ‡ Senior Dental Student, LSU School of Dentistry, New Orleans, LA. § Instructor of Research, LSU Neuroscience Center of Excellence, New Orleans, LA. Address correspondence and reprint requests to Dr Quinn: 4224 Houma Blvd, Suite 670, Metairie, LA 70006 © 2000 American Association of Oral and Maxillofacial Surgeons

0278-2391/00/5811-0005$3.00/0 doi:10.1053/joms.2000.16619

may be present, such as cyclooxygenase-2 (COX-2), that require identification. Prostaglandins (PGs) are important lipid mediators that are produced at elevated levels in inflamed tissues, including the rheumatoid synovium.8,9 PGs likely contribute to synovial inflammation by increasing blood flow and potentiating the effects of mediators such as IL-1B, which induces vasopermeability. PGs are biologically active derivatives of arachidonic acid that modulate neuronal cell function. Although injury and inflammation greatly stimulate the generation of prostaglandins, there is no clear understanding of the significance of this event. Prostaglandin synthesis is initiated by the activation of cPLA2 that releases free AA. Cyclooxygenases, the next step in prostaglandin synthesis, catalyze the conversion of AA into PGH2, the precursor of the prostaglandins. Until 1991, it was thought that there was only 1 cyclooxygenase. However, since then, 2 genes located in different chromosomes were found to encode a COX-1 (constitutive) and COX-2, highly inducible by proinflammatory cytokines and other factors.10,11 These isoenzymes are the central targets for nonsteroidal anti-inflammatory drugs.12 Regulation of COX-2 gene expression has been documented in both human and rodent synovial tissues. Before the identification of selective probes for each

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1230 COX isoform, Sano et al13 observed intense intracellular COX immunostaining in synovial lining cells, fibroblasts, and macrophages of rheumatoid arthritis patients, whereas osteoarthritis synovia showed weak and diffuse COX staining, and staining was absent in normal synovial tissue. COX immunostaining was also observed in inflamed paw tissue of Lewis rats with streptococcal cell wall or adjuvant-induced arthritis; staining was absent in normal paw tissue. The level of COX expression was also found to parallel the development of clinical disease and correlate with synovial mononuclear cell infiltration in experimentally induced arthritis. In a comparison of COX-1 and COX-2 gene expression in synovial tissue samples of patients with inflammatory arthritis, the expression of COX-2, but not COX-1, was found to be elevated in a disease-related pattern.14 Furthermore, NF-KB, a transcription factor that binds to the COX-2 promoter region, has been identified in the early stages of human knee-joint synovial tissue.15 Taken together, these findings suggest that the enhanced production of PGs in both acutely and chronically inflamed tissues results from selective, local upregulation of COX-2 biosynthesis. In this study, using the reverse transcriptase-polymerase chain reaction (RTPCR), the presence of COX-2 mRNA is demonstrated for the first time in inflamed TMJ synovial tissue and fluid, suggesting the possible use of selective COX-2 inhibitors in the treatment of TMJ dysfunction.

Patients and Methods Seventeen synovial tissue biopsy specimens and 16 synovial fluid samples were obtained from 12 patients with chronically, anteriorly dislocated TMJ discs verified by clinical and magnetic resonance imaging examination. None of the 12 patients (1 male and 11 females) had responded to appropriate nonsurgical therapy. Synovial fluid samples were obtained preparatory to TMJ arthroscopic surgery by injecting 1.5 mL saline into the superior joint space and translating the condyle anteriorly and posteriorly to mix the saline with the synovial fluid, followed by aspirating the fluid and placing it in a collection vial. During TMJ arthroscopy, a tissue specimen approximately 2 mm wide was obtained from the inflammed retrodiscal synovial tissue with biopsy forceps. All specimens were then frozen at ⫺70°C until assayed. ISOLATION OF TOTAL RNA FROM TMJ SYNOVIAL FLUID AND TISSUE

Total RNA was isolated from TMJ synovial fluid and tissue by using TRIzol reagent (BRL).16 Phenylmethylsulfonyl fluoride (1 mmol/L), leupeptin (0.05 ␮g/␮L), and 1 U/␮L RNasin (human placenta ribonuclease inhibitor; Promega, Madison, WI) were added to the

COX-2 IN SYNOVIAL TISSUE AND FLUID

extraction media to inhibit protease and ribonuclease A, B, C, and H activity. QUANTITATION OF COX-2 RNA MESSAGE LEVELS BY RT-PCR ANALYSIS

Total RNA samples of A260/A280 ⬇ 2.0 were used as templates for reverse transcription into cDNA. Mini cDNA libraries were constructed from total RNA by using RNase H Moloney murine leukemia virus–reverse transcriptase and hexamer primers (Superscript II/One-Step RT-PCR system; BRL, detectability: 10 molecules of RNA template). Human-specific ␤-actin using the sense primer 5⬘-TGACGGGGTCACCCACACTGTGCCCATCTA-3⬘ and antisense primer 5⬘-CTAGAAGCA-TTTGCGGTGGACGATGGAGGG-3⬘ yielded a 661-bp primary polymerase chain reaction product.17,18 Human-specific COX-2 signals were generated with a double hotstart technique that used HotStart 50 reaction tubes, Amplitaq Gold DNA polymerase (Perkin-Elmer, Norwich, CT), the COX-2 sense 5⬘TTCAAATGAGATTGTGGGAAA-ATTGCT-3⬘ and antisense primers 5⬘-AGATCATCTCTGCCTGAGTATCTT-3⬘, yielding 304- and 305-bp primary PCR products, respectively.16,19 For signal quantitation, primers were singly end-labeled by using [32P]dATP (3,000 Ci/mmol, Amersham redivue, Piscataway, NJ) and T4 PNK (Promega) before use in PCRs. As previously described, PCR was performed on the up-ramp of cycling so that at 25 cycles of amplification the log of primary COX-2 PCR products generated was a linear function of the log of cDNA template added.16,19 End-radiolabeled primary PCR products generated were analyzed on 5% acrylamide/1 ⫻ TBE (90 mmol/L Tris, pH 8.4/90 mmol/L boric acid/1 mmol/L ethylenediaminetetra-acetic acid) gels and were dried under vacuum on 2-mm Whatman filter paper at 80°C for 2 hours and then autoradiographed on phosphorImager screens (BioRad GS-250 Imaging screen B-1). DNA SEQUENCE ANALYSIS, DATA ANALYSIS, AND QUANTITATION

The dried gels were exposed to PhosphorImaging storage screens, and the resulting signals were analyzed on a GS-250 Molecular Imager (Bio-Rad Molecular Analyst Software version 1.4.1, Chicago, IL) or a Fuji FLA2000 Bio-Imaging Analyzer (Fuji, Stamford, CT). Relative intensities of ␤-actin gelshifted species or COX-2 RNA message levels were quantitated by using the PhosphorImager analysis and the data acquisition/statistical analysis packages provided with each instrument. All P values were derived from protected t-tests or least-square means from a 2-way factorial analysis of variance (ANOVA).

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FIGURE 1. Strong expression of COX-2 in 5 of 7 TMJ synovial tissue samples (JB) and in 7 of 8 synovial fluid (SF) samples.

Results TMJ synovial fluid and joint biopsy samples showed a COX-2 signal that was strongly expressed. COX-2 RNA message was detected by using RT-PCR in 16 of 17 synovial tissue samples (94%) and in 12 of 16 synovial fluid samples (75%) by using B-actin RNA levels in the same sample (either tissue or fluid) as an internal control. Because of the small quantity of tissue and fluid extracted during the TMJ biopsies, the samples could not be quantified. COX-2 mRNA signals are shown in Figure 1.

Discussion The results of this study indicate that the development of synovial inflammation in dysfunctional TMJs is associated with the presence of COX-2 in the affected tissues. Although COX-2 is known to be constitutively expressed in some tissues (eg, brain), in most tissues it is expressed as the inducible form. The regulation of COX-2 gene expression involves both a transcriptional and a translational mechanism,20 but the details of the mechanism remain elusive. Ristimaki et al21 recently showed that IL-1␣ induces a rapid but transient activation of COX-2 transcription in inflammatory cells and stabilizes the COX-2 mRNA in the absence of transcription. It is likely that the adjuvant induces the production of IL-1 as well as TNF-␣ in the affected joints and that IL-1 plays an important role in the regulation of sustained COX-2 polypeptide synthesis in the inflammatory response. Nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin, are effective agents commonly used in the treatment of rheumatoid arthritis and osteoarthritis. NSAIDs prevent PG formation by inhibiting both the COX-1 and COX-2 enzymes.22,23 However, long-term NSAID treatment is often limited by the formation of gastrointestinal ulcers that result from the suppression of physiologic PG production in these tissues. Additional studies in this model will be needed to determine whether the expression of COX-2 is dependent on other inflammatory mediators, such as IL-1 production, and whether PGs regulate the expression of these mediators in inflamed synovial tissue. Overall,

this study shows that treatment of established TMJ arthralgia with a selective inhibitor of COX-2 may be indicated. These findings suggest that proinflammatory PG production in chronic synovial inflammation is mediated primarily by the activity of the induced COX-2 enzyme. Current studies are aimed at determining whether selective COX-2 inhibitors are as effective as NSAIDs in the treatment of TMJ dysfunction. Acknowledgment The authors acknowledge the important contribution made to the study by Nicolas G. Bazan; Head, LSU Neuroscience Center of Excellence, LSU Medical Center.

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