Elsevier

Archives of Oral Biology

Volume 48, Issue 12, December 2003, Pages 825-834
Archives of Oral Biology

Tumour necrosis factor-α and apoptosis in the rat temporomandibular joint

https://doi.org/10.1016/S0003-9969(03)00175-4Get rights and content

Abstract

The purpose of this investigation was to investigate the roles that tumour necrosis factor-α (TNF-α) and apoptosis play during acute inflammation of the temporomandibular joint (TMJ). Adult male Sprague–Dawley rats were injected with complete Freund’s adjuvant (CFA) into the TMJ or kept as uninjected controls. The TMJ tissues were removed 2 days post-injection to mimic conditions of acute inflammation and analysed for changes in expression of TNF-α, the receptor TNF-R1, caspase-3 and -8, and apoptosis. Concentrations of TNF-α, TNF-R1, caspase-3 and -8, and apoptosis were significantly elevated in CFA-injected animals compared to uninjected controls. Tissue incubation with TNF-α caused a significant increase in caspase-3 and -8. Also, levels of apoptosis were significantly increased during inflammation, which could be inhibited by the addition of either anti-TNF-α neutralising antibody or caspase inhibitors. TNF-α may play a significant role in the onset of acute CFA-induced TMJ inflammation, and activation of apoptosis signalling pathways may be involved.

Introduction

Joint inflammation, often associated with rheumatoid arthritis, is usually described as consisting of persistent synovial joint inflammation and invasive pannus that often leads to joint destruction.1., 2. Inflammation is a highly complex process occurring in a milieu of cell types and molecules that serve to maintain or alter the process.3 While acute inflammation acts in a protective manner within damaged tissues, it may lead to conditions where the inflammatory response contributes to undesirable effects that are characterised by the production of inflammatory mediators. Cytokines have been shown to play an important role in the onset of the inflammatory cascade and as signalling molecules that promote the release of other inflammatory mediators.4., 5. While the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) have been implicated in the onset and exacerbation of many inflammatory diseases, only TNF-α is linked to apoptosis via direct activation of death domains on its receptors.6., 7., 8., 9., 10.

Two different receptors exist for TNF-α, the p55 or TNF-R1 receptor and the p75 or TNF-R2 receptor. Studies indicate that the TNF-R1 receptor activates intracellular signals leading to apoptosis through the recruitment of intracellular proteins that activate the caspase family of intracellular cysteine proteases.11., 12., 13. Caspases, synthesised as inactive pro-enzymes that must be activated by proteolytic cleavage, are involved in the initiation and propagation of apoptosis.14., 15., 16. One manner of activation of the caspase cascade is an extrinsic pathway that involves ligand binding to “death receptors”, such as TNF-R1, and transmission of signals to the interior of the cell where initiator caspases, such as caspase-8, are recruited to stimulate activation of executioner caspases, typically caspase-3.17., 18. Furthermore, caspase-3 and -8 have both been shown to be in the signal transduction cascade downstream from TNF-R1 activation and play an important role in TNF-induced apoptosis.19., 20., 21. Thus, demonstration of caspase activation downstream from death receptors has become one recognised method in the detection of apoptosis.22., 23.

During the past few years, our understanding of apoptotic pathways and control mechanisms in development and disease processes has exploded. Apoptosis has typically been associated with cell death in the absence of inflammation.24., 25., 26. However, the current understanding of apoptotic events has been re-evaluated based on conserved molecular events, including caspase activation. Recent studies indicate that apoptosis may participate in rheumatoid arthritis.27., 28., 29., 30., 31., 32. Furthermore, TNF-α may be involved in this process.33., 34., 35. However, our understanding of the role that TNF-α plays in inflammatory disorders of the temporomandibular joint (TMJ) is limited.36 Thus, the goals of this study were to investigate the involvement of TNF-α during TMJ inflammation using a CFA-induced model and determine if apoptosis was involved in the process.

Section snippets

Surgical procedures and adjuvant injection

Adult male Sprague–Dawley rats (200–250 g) were anaesthetised and received bilateral injection into the TMJ of a mixture containing 50 μg complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MO) dissolved in 50 μl of paraffin oil. Additional animals were used as uninjected controls. All animals were sacrificed via carbon dioxide inhalation and decapitation 2 days after CFA using protocols approved by the Baylor College of Dentistry Institutional Animal Care and Use Committee. The TMJ tissues

TNF-α and TNF-R1 concentrations after CFA treatment

To determine the magnitude of change following a CFA-induced acute TMJ inflammation, TNF-α and TNF-R1 concentrations were examined 48 h after CFA treatment. A significant increase in the protein expression of TNF-α and TNF-R1 48 h after CFA treatment was observed (Fig. 1).

Caspase activity after CFA treatment

TNF-R1 has been linked to activation of specific caspase pathways. Therefore, we examined the concentration of two specific caspases downstream from TNF-R1, caspase-3 and -8, after CFA administration. Caspase-3 and -8 were both

Changes in response to CFA treatment

While a few studies have used the CFA model to examine effects at the TMJ,37., 38., 39., 40., 41., 42., 43. there are few reports in the literature investigating the role that the pro-inflammatory cytokine TNF-α, caspases, or apoptosis may play in this process. The use of CFA is a documented model for the induction of inflammation within joint tissues similar to rheumatoid arthritis.44., 45., 46., 47., 48., 49., 50., 51., 52. We have previously used a CFA model for the induction of inflammation

Acknowledgements

This study was supported in part by NIH grant T35 DE07188-06, the Center for Craniofacial Research and Diagnosis at Baylor College of Dentistry—The Texas A&M University System Health Science Center, Baylor College of Dentistry—The Texas A&M University System Health Science Center Research Funds, and the Baylor Oral Health Foundation.

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