Expression of tumour necrosis factor-alpha in the rat dental follicle
Introduction
Tumour necrosis factor-α is a proinflammatory cytokine that often is overexpressed in a number of disease states such as sepsis syndrome, rheumatoid arthritis, inflammatory bowel disease (see review by1) and periodontitis.2., 3., 4., 5. Although this role in the mediation of various diseases is viewed as the primary function of TNF-α, recent studies with TNF-α null mice have shown that they do not form germinal centres after immunization, have a reduced Ig response to thymus-dependent antigen, and a disorganized inflammatory response followed by death to heat-killed Corynebacterium pervum.6 Thus, these studies and others, suggest that TNF-α may have a homeostatic effect in limiting the extent of an inflammatory response6 (review by Ma7), as well as acting as an antimalarial agent8 and functioning in intra-membranous bone repair.9
The possibility that TNF-α is involved in normal physiological processes is supported by its function in osteoclastogenesis. First, a member of the TNF ligand family, RANKL, present on osteoblasts and marrow stromal cells, promotes osteoclastogenesis by signalling osteoclast precursors to form osteoclasts, as well as activating osteoclasts.10., 11. Second, a TNF-receptor family member, osteoprotegerin, inhibits osteoclastogenesis by acting as a soluble decoy receptor for RANKL.12., 13., 14. Third, TNF-α itself promotes osteoclastogenesis, perhaps by a mechanism independent of the RANKL stimulation pathway.15., 16. Other studies suggest that TNF-α promotes osteoclastogenesis by osteoclast precursors provided that the precursors have been pretreated with RANKL.17., 18.
The potential role of TNF-α in osteoclastogenesis is of significance both for tooth eruption and periodontitis. Tooth eruption requires osteoclastogenesis and bone resorption.19., 20., 21., 22., 23. A loose connective tissue sac that surrounds each unerupted tooth, the dental follicle, is required for eruption to occur.24., 25. It also is the precursor of the periodontal ligament. The follicle has been implicated in the production of excess TNF-α seen in the gingival crevice of deciduous molars with ankylosis.26 Moreover, the dental follicle expresses osteoprotegerin,20 and on incubation with parathyroid hormone-related protein, expresses RANKL.27 The periodontal ligament becomes degraded in periodontitis along with excess resorption of the alveolar bone, and TNF-α has been implicated in this inflammatory condition.28., 29.
As mentioned previously, it was our aim now to determine whether TNF-α is normally expressed in the dental follicle, to examine its chronological expression in vivo in the follicle, and to investigate the effect of various potential eruption molecules on its expression and the effect of TNF-α itself on the expression of genes in the follicle cells that may relate to either tooth eruption or periodontitis.
Section snippets
Rat dental follicles and thymus
Harlan Sprague–Dawley rats were housed in AAALAC-approved facilities at Louisiana State University. The dental follicles from first mandibular molars and the thymus were removed on postnatal days 1, 3, 5, 7, 9 and 11 for RNA isolation; this procedure was repeated on three different litters. In addition, a thymus was removed from an adult rat (about 1.5 months old) for RNA isolation.
Culture of dental follicle cells
Dental follicles were surgically isolated from 4- to 6-day-old rats, then trypsinized and cultured as described by
Results
In vivo, a slight increase in TNF-α expression was seen, starting at day 3 postnatally, but maximal expression was seen at day 9 (Fig. 1), which was statistically significantly higher than all other days postnatally. This finding was confirmed by real-time RT–PCR, which showed that TNF-α expression at day 9 was 5.5 times higher than at day 1 (Table 2). In contrast, TNF-α was equally expressed in the thymus at all days examined, including the adult (not shown). In vitro, TNF-α was not expressed
Discussion
This study demonstrates that the rat dental follicle can express the gene for TNF-α both in vitro and in vivo. Follicle cells in culture did not express constitutively the TNF-α gene but did express it strongly after incubation with IL-1α. Moreover, the cells were moderately immunostained for TNF-α after treatment with IL-1α (not shown). IL-1α is present in the stellate reticulum adjacent to the follicle but the IL-1 type 1 receptor is present on the dental follicle cells.34 Although the teeth
Acknowledgements
We thank Mr. Keith Tribble for his technical assistance with the thymus, and Ms. Cindy Daigle for typing the manuscript. This work was supported by an R01 NIH Grant DE08911 to Gary E. Wise.
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