| 1. |
- Dahl, Sara, et al.
(författare)
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The host defense peptide LL-37 triggers release of nucleic acids from human mast cells
- 2018
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Ingår i: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 109, s. 39-45
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Tidskriftsartikel (refereegranskat)abstract
- The human host defense peptide LL-37 possesses antimicrobial activity but also affects host cell function and viability. Mast cells are involved in innate immunity but no data have been presented on effects of LL-37 on human mast cell viability and export of nucleic acids. Here, we demonstrated by immunofluorescence microscopy that synthesized LL-37 was internalized by human LAD2 mast cells and detected both in cytoplasm and nucleus. Treatment with high (4 and 10 μM) but not low (1 μM) concentrations of LL-37 for 4 h reduced cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Stimulation with 10 μM LL-37 for 4 h enhanced export of nucleic acids, total protein and lactate dehydrogenase (LDH), suggesting that both nuclear and plasma membranes are permeabilized by LL-37. Although LL-37 triggered release of nucleic acids, no extracellular trap-like structures were observed by laser scanning confocal microscopy of cells incubated with the plasma membrane impermeable nucleic acid fluorophore SYTOX-Green, indicating that LL-37 promotes export of nucleic acids but not formation of extracellular traps. On the other hand, phorbol-12-myristate-13-acetate (PMA), which is a well-known inducer of extracellular traps, stimulated export of nucleic acids and also formation of extracellular trap-like structures. However, PMA had no effect on export of either total protein or LDH. Hence, LL-37 and PMA seem to stimulate export of nucleic acids from LAD2 mast cells through different pathways. In conclusion, we demonstrate that LL-37 triggers release of nucleic acids from human mast cells but not the formation of extracellular trap-like structures.
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| 2. |
- Aidoukovitch, Alexandra, et al.
(författare)
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The host defense peptide LL-37 is internalized by human periodontal ligament cells and prevents LPS-induced MCP-1 production
- 2019
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Ingår i: Journal of Periodontal Research. - : Wiley. - 0022-3484 .- 1600-0765. ; 54:6, s. 662-670
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Anders, Emma, et al.
(författare)
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Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1 : a novel mechanism for regulation of MCP-1 production and function
- 2018
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Ingår i: Biochemical Journal. - 0264-6021. ; 475:4, s. 775-786
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Tidskriftsartikel (refereegranskat)abstract
- The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/ p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.
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| 4. |
- Anders, Emma, et al.
(författare)
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LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis
- 2018
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 501:1, s. 280-285
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Tidskriftsartikel (refereegranskat)abstract
- The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.
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| 5. |
- Svensson, Daniel, et al.
(författare)
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Secretory leukocyte protease inhibitor regulates human periodontal ligament cell production of pro-inflammatory cytokines
- 2017
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Ingår i: Inflammation Research. - : Springer Science and Business Media LLC. - 1023-3830 .- 1420-908X. ; 66:9, s. 823-831
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Svensson, Daniel, et al.
(författare)
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The host defense peptide LL-37 is detected in human parotid and submandibular/sublingual saliva and expressed in glandular neutrophils
- 2018
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Ingår i: European Journal of Oral Sciences. - : Blackwell Munksgaard. - 0909-8836 .- 1600-0722. ; 126:2, s. 93-100
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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