• 대한의생명과학회지
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• 제29권4호
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• pp.211-219
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• 2023

Caffeic acid phenethyl ester (CAPE) is an active component of propolis obtained from honeybee hives. CAPE possesses anti-mitogenic, anti-carcinogenic, anti-inflammatory, and immunomodulatory activities in diverse systems, which know as displays antioxidant activity and inhibits lipoxygenase activities, protein tyrosine kinase, and nuclear factor kappa B (NF-κB) activation. This study aimed to investigate the effect of CAPE on lipopolysaccharide (LPS)-induced human neutrophil phagocytosis. Human neutrophils were cultured with various concentrations of CAPE (1, 10, and 100 µM) with or without LPS. The pro-inflammatory proteins (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-6 and IL-8) levels were measured after 4 h incubation. To investigate the intracellular signaling pathway, we measured the levels of mitogen-activated protein kinases (MAPK), including phosphorylation of p38, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Next, to evaluate the potential phagocytosis, neutrophils were labeled with iron particles of superparamagnetic iron oxide nanoparticles (SPIONs, 40 nm) for 1 h in culture medium containing 5 mg/mL of iron. The labeling efficiency was determined by Prussian blue staining for intracellular iron and 3T-wighted magnetic resonance imaging. CAPE decreased the activation of intracellular signaling pathways, including ERK1/2 and c-Jun, and expression of pro-inflammatory cytokines, including TNF-α and IL-6, but had no effect on the signaling pathways of p38 and cytokine IL-8. Furthermore, images obtained after mannan-coated SPION treatment suggested that CAPE induced significantly higher signal intensities than the control or LPS group. Together, these results suggest that CAPE regulates LPS-mediated activation of human neutrophils to reduce phagocytosis.

• International Journal of Oral Biology
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• 제41권1호
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• pp.17-23
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• 2016

Chronic/cyclic neutropenia, leukocyte adhesion deficiency syndrome, Papillon-$Lef{\grave{e}}vre$ syndrome, and $Ch{\grave{e}}diak$-Higashi syndrome are associated with severe periodontitis, suggesting the importance of neutrophils in the maintenance of periodontal health. Various Toll-like receptor (TLR) ligands are known to stimulate neutrophil function, including FcR-mediated phagocytosis. In the present study, the effect of TLR2 activation on the non-opsonic phagocytosis of oral bacteria and concomitant production of reactive oxygen species (ROS) by human neutrophils was evaluated. Neutrophils isolated from peripheral blood were incubated with Streptococcus sanguinis or Porphyromonas gingivalis in the presence of various concentrations of $Pam_3CSK_4$, a synthetic TLR2 ligand, and analyzed for phagocytosis and ROS production by flow cytometry and chemiluminescence, respectively. $Pam_3CSK_4$ significantly increased the phagocytosis of both bacterial species in a dose-dependent manner. However, the enhancing effect was greater for S. sanguinis than for P. gingivalis. $Pam_3CSK_4$ alone induced ROS production in neutrophils and also increased concomitant ROS production induced by bacteria. Interestingly, incubation with P. gingivalis and $Pam_3CSK_4$ decreased the amounts of ROS, as compared to $Pam_3CSK_4$ alone, indicating the possibility that P. gingivalis survives within neutrophils. However, neutrophils efficiently killed phagocytosed bacteria of both species despite the absence of $Pam_3CSK_4$. Although P. gingivalis is poorly phagocytosed even by the TLR2-activated neutrophils, TLR2 activation of neutrophils may help to reduce the colonization of P. gingivalis by efficiently eliminating S. sanguinis, an early colonizer, in subgingival biofilm.

• BMB Reports
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• 제36권6호
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• pp.565-571
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• 2003

The locomotor responses of human peripheral blood neutrophils and lymphocytes were measured by the change from spherical to polarized shapes in the presence of endotoxins (lipopolysaccharide, LPS) of enteric pathogens: S. dysenteriae type 1, V. cholerae Inaba 569B, S. typhimurium, and K. pneumoniae. We reported earlier that these endotoxins are chemotactic factors for the neutrophils since they stimulated cell polarization within a few minutes of incubation. Endotoxins had an inhibitory effect upon neutrophil phagocytosis of opsonized yeast and the cells engulfed fewer yeasts. Interestingly, endotoxins increased neutrophil adhesion to clean glass surfaces, but stimulated the cells to exhibit increased random locomotion (chemokinesis) through cellulose nitrate filters and show an enhanced ability to reduce nitroblue tetrazolium (NBT) dye. Unlike neutrophils, lymphocytes direct from blood do not show polarized morphology towards chemotactic factors but the cells acquire locomotor capacity during 24-72 h culture with mitogens such as phytohemagglutinin (PHA), phorbol myristate acetate or concanavalin A. Stimulation of blood lymphocytes with endotoxins did not induce cell polarization in short-term but long-term culture resulted in an increase in the proportion of polarized cells that acquired locomotor morphologies. The majority of these cells were identified as esterase negative B-lymphocytes that migrated through filters. Despite the optimum time of incubation for each of these cell types being different, we found that lymphocytes respond to much lower concentrations of endotoxins than the neutrophils. These findings suggest that endotoxins of enteric pathogens modulate the functions of human blood neutrophils and lymphocytes.

• 대한약리학회지
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• 제23권1호
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• pp.33-44
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• 1987

면역보체가 결합되어 있는 zymosan 또는 열로 응집된 IgG에 의하여 활성화된 호중구에서의 NADPH oxidase 의존적인 $O_{\overline{2}}$의 생성과 탐식작용은 칼슘의 흡수과정과 일치하였다. 활성화된 백혈구의 반응은 세포외 칼슘 농도에 따라 항진되었으며, 이는 칼슘의 킬레이트제인 EGTA나 EDTA에 의하여 유의하게 억제되었다. 활성화된 백혈구로부터 $O_{\overline{2}}$의 생성은 dantrolene과 chlorpromazine에 의하여 억제되었다. 칼슘 길항제인 bepredil, diltiazem, verapamil, nifedipine, nimodipine은 효과적으로 활성화된 백혈구의 칼슘흡수, $O_{\overline{2}}$ 생성 그리고 탐식 작용을 억제하였고 NADPH oxidase 활성도 또한 억제하였다. 그러므로, 칼슘 길항제는 칼슘 유입을 억제하거나 칼슘의 세포내 재분포 및 NADPH oxidase 반응계에 작용하여 활성화된 백혈구에서의 $O_{\overline{2}}$의 생성과 백혈구의 탐식작용을 억제할 것으로 시사되었다.

• BMB Reports
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• 제35권5호
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• pp.482-487
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• 2002

Early activation of human peripheral blood polymorphonuclear neutrophils is characterized by their morphological changes from spherical to polarized shapes. The endotoxins from enteric pathogens (S. dysenteriae type 1, V. cholerae Inaba 569B, S. typhimurium, and K. pneumoniae) were assessed by their ability to induce morphological polarization of the neutrophils as measures of early activation. Phagocytic activity, adhesion, chemokinetic locomotion, and nitroblue tetrazolium (NBT) dye-reduction ability measured the later activation of the cells. Neutrophils showed distinct morphological polarization in suspension over a wide range of concentrations of these endotoxins when were compared with those that were induced by the standard chemotactic factor, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). It was discovered that all of the endotoxins induced locomotor responses in neutrophils in suspension that were dose- and time-dependent. The optimum concentration for the endotoxins of S. dysenteriae, V. cholerae, and K. pneumoniae was 1 mg/ml in which 71, 69, and 66% of the neutrophils were polarized. However, the S. typhimurium dose was 2 mg/ml in which 50% of the cells responded. Neutrophils that were stimulated with endotoxins also showed increased random locomotion (p<0.005) through cellulose nitrate filters, but an enhanced adhesion of the cells to glass surfaces (p<0.03). These are important functions of these cells to reach and phagocytose damaged cells, as well as invading microorganisms. Interestingly, the endotoxins had a highly-significant inhibitory effect upon the proportions of neutrophils phagocytosing opsonized yeast (p<0.01) with a small number of yeast that were engulfed by the cells (p<0.02). Further, endotoxin-treated cells showed an enhanced ability to reduce NBT dye (p<0.03). Therefore, we concluded that endotoxins of enteric pathogens are neutrophil chemotactic factors.

• Clinical and Experimental Reproductive Medicine
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• 제50권1호
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• pp.44-52
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• 2023

Objective: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. Methods: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. Results: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1β, IL-6, IL-12, interferon α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. Conclusion: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.