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.
Objective The human host defense peptide LL‐37 both shows antimicrobial effects and modulates host cell properties. Here, we assess the effects of synthesized LL‐37 on lipopolysaccharide (LPS)‐induced inflammation in human periodontal ligament (PDL) cells and investigates underlying mechanisms. Background LL‐37 has been detected in the periodontal tissues, but its functional importance for PDL cell innate immune responses is not known. Methods Human PDL cells were obtained from premolars extracted on orthodontic indications. Cellular pro‐inflammatory monocyte chemoattractant protein‐1 (MCP‐1) mRNA expression was determined using quantitative real‐time RT‐PCR. MCP‐1 protein production was assessed by western blot and ELISA. Internalization of LL‐37 by PDL cells was visualized by immunocytochemistry. Nuclear factor kappa‐light‐chain‐enhancer of activated B‐cell (NF‐κB) activity was assessed by western blot of phosphorylated p65, phosphorylated p105, and IκBα proteins. Binding of LL‐37 to PDL cell DNA was determined by isolation and purification of DNA and dot blot for LL‐37 immunoreactivity. Results Treatment with LL‐37 (1 µmol/L) for 24 hours prevented LPS‐induced stimulation of MCP‐1 expression analyzed both on transcript and on protein levels. Stimulation with LL‐37 (1 µmol/L) for 24 hours had no effect on toll‐like receptor (TLR)2 and TLR4 transcript expression, suggesting that LL‐37 acts downstream of the TLRs. Preincubation with LL‐37 for 60 minutes followed by stimulation with LPS for 24 hours in the absence of LL‐37 completely prevented LPS‐evoked MCP‐1 transcript expression, implying that LL‐37 acts intracellularly and not via binding and neutralization of LPS. In PDL cells stimulated with LL‐37 for 60 minutes, the peptide was internalized as demonstrated by immunocytochemistry, suggesting an intracellular mechanism of action. LL‐37 immunoreactivity was observed both in the cytosol and in the nucleus. Downregulation of LPS‐induced MCP‐1 by LL‐37 was not mediated by reduction in NF‐κB activity as shown by unaltered expression of phosphorylated p65, phosphorylated p105, and IκBα NF‐κB proteins in the presence of LL‐37. Immunoreactivity for LL‐37 was observed in PDL cell DNA treated with but not without 0.1 and 1 µmol/L LL‐37 for 60 minutes in vitro. Conclusion LL‐37 abolishes LPS‐induced MCP‐1 production in human PDL cells through an intracellular, NF‐κB‐independent mechanism which probably involves direct interaction between LL‐37 and DNA.
The human host defense peptide, LL-37, is an important player in the first line of defense against invading microorganisms. LL-37 and its precursor, hCAP18, have been detected in unstimulated whole saliva but no reports showing hCAP18/LL-37 in isolated, parotid, and/or submandibular/sublingual saliva have been presented. Here, we measured the levels of hCAP18/LL-37 in human parotid and submandibular/sublingual saliva and investigated the expression of hCAP18/LL-37 in parotid and submandibular gland tissue. Parotid and submandibular/sublingual saliva was collected from healthy volunteers, and the levels of hCAP18/LL-37 in saliva were analyzed by dot blot, ELISA, and western blotting. Cellular expression of hCAP18/LL-37 in human parotid and submandibular glands was investigated by immunohistochemistry. Immunoreactivity for hCAP18/LL-37 was detected in both parotid and submandibular/sublingual saliva of all individuals. The concentration of hCAP18/LL-37 was similar in parotid and submandibular/sublingual saliva, and was determined by densitometric scanning of each dot and normalization to the total protein concentration of each sample, and by ELISA. Double immunohistochemistry revealed that intravascular neutrophils of both parotid and submandibular glands express hCAP18/LL-37. For the first time, we demonstrate hCAP18/LL-37 in isolated human parotid and submandibular/sublingual saliva and expression of hCAP18/LL-37 in glandular intravascular neutrophils, indicating that neutrophils of the major salivary glands contribute to the LL-37 content of whole saliva.
The antimicrobial peptide LL‐37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL‐37 is formed from its intracellular pro‐form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell‐containing and cell‐free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL‐37 in these fractions using ELISA. Cellular expression of hCAP18/LL‐37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell‐free saliva contained hCAP18/LL‐37. Western blot analysis of cell‐pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL‐37. Phase‐contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL‐37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL‐37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL‐37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.
Objective: Odontoblasts are thought to be involved in innate immunity but their precise role in this process is not fully understood. Here, we assess effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA), produced by Gram-negative and Gram-positive bacteria, respectively, on matrix metalloproteinase-8 (MMP-8), interleukin-6 (IL-6) and cathelin-related antimicrobial peptide (CRAMP) expression in odontoblast-like MDPC-23 cells. Material and methods: Gene activity and protein production was determined by quantitative real-time RT-PCR and ELISA, respectively. Cellular expression of CRAMP was determined by immunocytochemistry. Results: Stimulation with LTA (5 and 25 mg/ml) but not LPS (1 and 5 mg/ml) for 24 h enhanced IL-6 mRNA expression. The LTA-induced up-regulation of IL-6 mRNA levels was associated with increased IL-6 protein levels. Stimulation with either LPS or LTA for 24 h lacked effect on both MMP-8 transcript and protein expression. Immunocytochemistry disclosed that MDPC-23 cells expressed immunoreactivity for CRAMP. MDPC-23 cells showed mRNA expression for CRAMP, but stimulation with either LPS or LTA did not modulate CRAMP transcript expression. Conclusions: We show that MDPC-23 cells possess immune-like cell properties such as LTA-induced IL-6 production and expression of the antimicrobial peptide CRAMP, suggesting that odontoblasts may modulate innate immunity via these mechanisms.
Objective. The aim of this study was to investigate if desquamated oral epithelial cells (DOECs) express the epidermal growth factor (EGF) and if these cells thereby may contribute to salivary EGF contents. Background. DOECs have recently been shown to harbor the antimicrobial peptide LL-37, proposing that they may also store other biologically important salivary peptides/proteins. The EGF peptide is a growth factor which plays a critical role to maintain epithelial integrity and promote epithelial healing. The EGF is produced by salivary glands, but it is not known whether DOECs contain the EGF and thereby contribute to salivary EGF levels. Materials and Methods. DOECs were isolated from unstimulated whole saliva collected from four healthy volunteers. EGF protein expression was determined in cell lysates by dot blot and ELISA. Cellular distribution of cytokeratin, the proliferation marker Ki67, and EGF immunoreactivity were assessed by immunocytochemistry. EGF gene expression was investigated by qPCR. Expression of EGF transcript and protein in DOECs was compared to that in the human cultured keratinocyte cell line (HaCaT) cells. Results. EGF protein expression was detected in DOEC cell lysates by both dot blot and ELISA. Strong cytoplasmic EGF immunoreactivity was observed in DOECs, although some cells showed only a weak immunoreactive signal for EGF. Moreover, DOECs, besides containing EGF protein, also expressed transcript for EGF. Interestingly, ELISA analysis revealed that EGF protein contents were higher in DOECs than in HaCaT cells. ELISA analysis also disclosed that EGF concentration was about 10 times higher in whole saliva compared to DOECs. EGF transcript expression was about 50% lower in HaCaT cells stimulated with high (10%) compared to low (0.1%) concentration of fetal bovine serum, representing growth-stimulated and growth-restricted conditions, respectively, implying that growth-stimulus exerts negative feedback on EGF gene activity in HaCaT cells. Conclusion. Here, we show for the first time that DOECs express the EGF, arguing that these cells contribute to salivary EGF contents and hence may play a role in gingival epithelial repair and wound healing.
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