Calcium Phosphate Particles Induce Interleukin-8 Expression in a Human Gingival Epithelial Cell Line via the Nuclear Factor-?B Signaling Pathway

• Autores: Yu Sakai
• Localización: Journal of periodontology, ISSN 0022-3492, Nº. 10, 2014, págs. 1464-1473
• Idioma: inglés
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• Resumen

  • Background: Dental calculus is calcified plaque composed primarily of calcium phosphate mineral salts, and there is a clear association between the presence of calculus and the initiation/progression of periodontitis. However, it is still inconclusive whether dental calculus can be a direct causative factor. The authors examined the effect of nano/microsized calcium phosphate particles, which may be generated in the process of early precipitation and/or dissolution of calcium phosphate mineral, on the expression of interleukin (IL)-8 in human gingival epithelial cells.

    Methods: Primary human gingival epithelial cells and/or a human gingival carcinoma cell line (Ca9-22) were stimulated with calcium phosphate particles. Gene and protein levels were assessed by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, respectively. The activity of nuclear factor (NF)-?B signaling was measured by an immunofluorescence assay to evaluate NF-?B p65 nuclear translocation.

    Results: The results show that nano/microsized particles stimulate IL-8 expression in human gingival epithelial cells at gene and protein levels. The activity to induce IL-8 expression depends on the particle size: particles with a diameter of 200 nm are more effective than those of 40-nm and 5-?m diameters. Calcium phosphate particles (diameter 200 nm) stimulated NF-?B activity. Pretreatment with BMS-345541, an NF-?B signaling inhibitor, inhibited the particle-mediated IL-8 gene induction, suggesting a requirement for the NF-?B signaling pathway.

    Conclusion: These findings suggest that calcium phosphate particles, which may be related to calculus development, may act as a direct causative factor in the pathogenesis of gingival epithelium.

    Dental calculus is mineralized plaque composed primarily of calcium phosphate mineral salts and is classified by its location on a tooth surface in relation to the gingival margin, that is, supragingival calculus, which is located coronal to the gingiva, or subgingival calculus, which is located apical to the gingival margin.1 It has been demonstrated that the main crystal forms found in calculus are hydroxyapatite (HA), dicalcium phosphate dihydrate, octacalcium phosphate, and ?-tricalcium phosphate (TCP) or whitlockite.2 Saliva is the mineral source for the calcification of supragingival calculus, whereas the gingival crevicular fluid provides minerals for the mineralization of subgingival calculus. Calculus formation begins with the deposition of precursor phases of calcium phosphate, such as octacalcium phosphate and dicalcium phosphate dihydrate, which are gradually hydrolyzed and transformed into less soluble HA and TCP or whitlockite mineral phases during the formation and maturation of calculus.3 It has been demonstrated that there is a clear association between the presence of dental calculus and the initiation/progression of periodontitis.4-9 However, calculus may be the result rather than the cause of periodontitis, as calcification of subgingival plaque requires high influx of gingival crevicular fluid, which is part of the serum transudate/inflammatory exudate and provides the minerals for subgingival calculus formation.10 It is currently assumed that dental calculus is not a direct causative factor in the pathogenesis of periodontal diseases, but acts as a reservoir for bacterial plaque because it is covered with a layer of viable and metabolically active bacteria on the porous surface.1,11 However, the evidence for the role of calculus in the initiation/progression of periodontitis is still inconclusive.

    It has been reported that the deposition of calcium phosphate particles occurs in various tissues depending on local environmental conditions, and it has been implicated in various pathologies, such as osteoarthritis12 and cardiovascular disease.13,14 Calcium phosphate particles of ?1 ?m or less in diameter have been reported to elicit secretion of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-?, interleukin (IL)-1?, and IL-8, from monocytes-macrophages,15,16 as well as cell death in vascular smooth muscle cells.17 Calcifying nanoparticles, which were originally recognized as self-propagating and cultivable macromolecular complexes18 and have now been revealed to be oxidized lipids with calcium apatite crystals,19 are a potential etiologic factor involved in various pathologic calcification diseases20 such as kidney stones and calcified arteries. Recently, it has been reported that calcifying nanoparticles are detected in gingival crevicular fluid and dental calculus in periodontitis,21 suggesting their possible involvement in the formation of dental calculus. Furthermore, it has been demonstrated that cell-independent deposition of calcium phosphate particles is induced by 1 mM excess phosphate (Pi) or hyperphosphatemia,22,23 defined as a Pi concentration of 2 mM compared with 1 mM in healthy donors.24 Considering that concentrations of calcium and Pi in gingival crevicular fluid as well as saliva are higher than those in serum,25-28 it is conceivable that calcium phosphate particles may be formed at an early phase of dental calculus formation and also liberated into periodontal pockets by dissolution due to their solubility29 or by a procedure to remove dental calculus as a periodontal treatment.

    Gingival epithelial cells, or rather junctional epithelial cells, which are located anatomically closest to dental calculus in the gingival crevice, represent the first line of defense against infection. The interactions between periodontal bacteria and gingival epithelial cells lead to cell activation and induce the expression of a broad range of pro-inflammatory cytokines.30 These lines of evidence led the authors to examine whether nano/microsized calcium phosphate particles may elicit a pro-inflammatory response to gingival epithelial cells. This study demonstrates, for the first time, that nano/microsized calcium phosphate particles stimulate human gingival epithelial cells to induce the expression of IL-8, a CXC chemokine, at gene and protein levels via the nuclear factor (NF)-?B signaling pathway.