Abstract
ObjectiveRegulation of immune-like cell properties of periodontal ligament (PDL) cells is not understood. We investigate the importance of secretory leukocyte protease inhibitor (SLPI) for production of pro-inflammatory cytokines in human PDL cells.Materials and methodsPDL cells were isolated from teeth extracted for orthodontic reasons. Cellular location of SLPI was investigated by immunocytochemistry. Cytokine transcript and protein expression were assessed by quantitative real-time RT-PCR and Western blotting. SLPI gene activity was knocked-down by siRNA. NF-κB signaling was assessed by measuring IκBα, and phosphorylated p65 and p105 protein expression.ResultsPDL cells showed cytoplasmic expression of SLPI. Cellular expression level of SLPI negatively correlated to LPS-induced stimulation of IL-6 and MCP-1. Both SLPI gene activity and protein were reduced by about 70% in PDL cells treated with SLPI siRNA compared to cells treated with non-coding construct. Treatment with SLPI siRNA was associated with up-regulation of both basal and LPS-stimulated IL-6, MCP-1 and TLRs mRNA expression. The up-regulation of MCP-1 transcript in SLPI siRNA-treated cells was confirmed on protein level. SLPI siRNA-treatment enhanced the phosphorylated NF-κB p105 protein expression.ConclusionsSLPI regulates PDL cell pro-inflammatory cytokine expression and modulates NF-κB signaling, suggesting that SLPI governs the immune cell-like properties of PDL cells.
Highlights
Periodontal ligament (PDL) cells are fibroblast-like cells, but they show osteoblastic features such as expressing bone marker proteins and alkaline phosphatase activity and mineralized nodule formation [1, 2]
The tissue explants of periodontal ligament were washed in phosphate-buffered saline (PBS), seeded in culture flasks in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics and fetal calf serum (10%), and the culture flasks were placed in a water-jacketed cell incubator at 37 °C under 5% CO2 in air
We investigated the cellular expression of secretory leukocyte protease inhibitor (SLPI) protein in periodontal ligament (PDL) cells by immunocytochemistry (Fig. 1)
Summary
Periodontal ligament (PDL) cells are fibroblast-like cells, but they show osteoblastic features such as expressing bone marker proteins and alkaline phosphatase activity and mineralized nodule formation [1, 2]. The PDL cells produce pro-inflammatory cytokines upon stimulation with bacterial endotoxins such as lipopolysaccharides (LPS), suggesting that they may act as immune cells and contribute to initiation and progress of periodontal inflammation via this mechanism [3]. The nutrient and hormone vitamin D have recently been shown to attenuate PDL cell production of pro-inflammatory cytokines, suggesting that it may influence the development of periodontal inflammation through this mechanism [4, 5]. Pro-inflammatory cytokines such as the interleukin-6 (IL-6) and IL-1b are regarded as osteolytic factors in periodontitis, there are reports suggesting that IL6 and IL-1b may drive osteogenic differentiation of PDL cells [6, 7]. PDL cell production of cytokines may both promote the inflammatory reaction and enhance healing and tissue regeneration. Since the regulatory mechanisms which determine the functional properties of PDL cells are poorly understood, more information is needed to clarify this issue
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