| 1. |
- Säll, Johanna, et al.
(author)
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The antimicrobial peptide LL-37 alters human osteoblast Ca2+ handling and induces Ca2+-independent apoptosis
- 2013
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In: Journal of Innate Immunity. - : S. Karger. - 1662-811X .- 1662-8128. ; 3:5, s. 290-300
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Journal article (peer-reviewed)abstract
- The human antimicrobial peptide cathelicidin LL-37 has, besides its antimicrobial properties, also been shown to regulate apoptosis in a cell type-specific manner. Mechanisms involved in LL-37-regulated apoptotic signaling are not identified. Here, we show that LL-37 reduces the human osteoblast-like MG63 cell number and cell viability in the micromolar concentration range with an IC50 value of about 5 µM. Treatment with 4 µM LL-37 increased the number of annexin V-positive cells and stimulated activation of caspase 3 showing that LL-37 promotes apoptosis. Treatment with 4 µM LL-37 caused an acute and sustained rise in intracellular Ca(2+) concentration assessed by laser-scanning confocal microscopy of Fluo-4-AM-loaded MG63 cells. LL-37 increased Ca(2+) also in the presence of the respective L- and T-type voltage-sensitive Ca(2+) channel blockers nifedipine and NiCl2. LL-37 had no effect on Ca(2+) in cells incubated with Ca(2+)-free solution. LL-37 (4 and 8 µM) reduced the MG63 cell number both in the presence and absence of Ca(2+) in the medium. In conclusion, LL-37 reduces the osteoblast cell number by promoting apoptosis, and furthermore, LL-37 stimulates Ca(2+) inflow via a mechanism independent of voltage-sensitive Ca(2+) channels. Interestingly, LL-37-induced lowering of the cell number seems to be mediated via a mechanism independent of Ca(2+).
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| 2. |
- Nebel, Daniel, et al.
(author)
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1α,25-dihydroxyvitamin D3 promotes osteogenic activity and downregulates proinflammatory cytokine expression in human periodontal ligament cells
- 2015
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In: Journal of Periodontal Research. - : John Wiley & Sons. - 0022-3484 .- 1600-0765. ; 50:5, s. 666-673
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Journal article (peer-reviewed)abstract
- Background and ObjectiveThe aim of this study was to assess the impact of 1,25-dihydroxyvitamin D3 (vitamin D3) on osteogenic and inflammatory properties of human periodontal ligament (PDL) cells and investigate underlying mechanisms. Material and MethodsHuman PDL cells, obtained from four subjects, were stimulated with vitamin D3 for 4-48h. The bone markers osteopontin and osteocalcin and proinflammatory cytokine/chemokine expression was determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Cytokine and chemokine expression was determined after stimulation with the inflammation promoter lipopolysaccharide (LPS) in the presence or absence of vitamin D3. Alkaline phosphatase activity was assessed using p-nitrophenylphosphate substrate. ResultsTreatment with 30ng/mL of vitamin D3, corresponding to an optimal plasma concentration of vitamin D, for 24h had no effect on PDL cell number and morphology but increased PDL cell osteopontin and osteocalcin mRNA expression by about 70 and 40%, respectively, and, moreover, treatment with vitamin D3 for 48h enhanced PDL cell alkaline phosphatase activity by about two times showing that vitamin D3 exerts pro-osteogenic effects in human PDL cells. Stimulation with LPS (1g/mL) for 4h increased PDL cell interleukin (IL)-6 cytokine and chemokine ligand 1 (CXCL1) chemokine mRNA expression several fold. The LPS-induced increase in IL-6 and CXCL1 transcripts was attenuated by vitamin D3 (30ng/mL). Treatment with vitamin D3 (3-300ng/mL) for 24h reduced the LPS-evoked increase in PDL cell IL-6 protein by about 50%. Vitamin D3 (30ng/mL) had no effect on LPS-induced IL-1 and MCP-1 mRNA expression. ConclusionsVitamin D3 promotes osteogenic differentiation but also downregulates inflammation promoter-induced IL-6 cytokine and CXCL1 chemokine expression in human PDL cells, suggesting that vitamin D3 both stimulates bone regeneration and antagonizes inflammation in human periodontal tissue.
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| 3. |
- Svensson, Daniel, et al.
(author)
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Human endogenous peptide p33 inhibits detrimental effects of LL-37 on osteoblast viability
- 2015
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In: Journal of Periodontal Research. - : John Wiley & Sons. - 0022-3484 .- 1600-0765. ; 50:1, s. 80-88
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Journal article (peer-reviewed)abstract
- Background and Objective: High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types. Material and Methods: Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [3H]-thymidine incorporation. Cell number was assessed by counting cells in a Bu€rker chamber. Intracellular Ca2+ was monitored by recording Fluo 4-AM fluorescence using a laser scanning confo- cal microscope. Cellular expression of p33 was determined by western blotting. Results: LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50) of 4 lM and a rapid and sustained rise in the intracellular Ca2+ concentration of osteoblasts, sug- gesting that LL-37 forms pores in the cell membrane. p33 (10 lM) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca2+ con- centration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonsti- mulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts. Conclusions: The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteo- blasts from LL-37-induced cell damage in patients suffering from chronic peri- odontitis associated with high levels of LL-37 locally.
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| 4. |
- Svensson, Daniel, et al.
(author)
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Secretory leukocyte protease inhibitor regulates human periodontal ligament cell production of pro-inflammatory cytokines
- 2017
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In: Inflammation Research. - : Springer Science and Business Media LLC. - 1023-3830 .- 1420-908X. ; 66:9, s. 823-831
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Journal article (peer-reviewed)abstract
- Objective: Regulation 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 methods: PDL 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. Results: PDL 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. Conclusions: SLPI regulates PDL cell pro-inflammatory cytokine expression and modulates NF-κB signaling, suggesting that SLPI governs the immune cell-like properties of PDL cells.
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