1.Effect of Enterococcus faecalis on apoptosis rate and the release of matrix metalloproteinase-8 of polymorphonuclear leukocytes.
West China Journal of Stomatology 2009;27(4):440-446
OBJECTIVETo evaluate the release of matrix metalloproteinase-8 (MMP-8) and apoptosis rate of polymorphonuclear leukocytes (PMNs) after PMNs was triggered by Enterococcus faecalis (E. faecalis) in vitro.
METHODSThe activated E. faecalis suspension was prepared and added to PMNs suspension as experiment group. As a positive control, phorbol myristate acetate (PMA) was used. As negative control, PMNs suspension was incubated with PBS. The release of MMP-8 was measured at 0, 20, 60, 120 min by ELISA method. E. faecalis lysate acted on PMNs as experiment group, PMNs suspension was incubated with PBS as negative control, samples in two groups were incubated at 37 degrees C for 2, 5, 10, 15 h. The apoptosis rate of PMNs was tested by Flow Cytometry.
RESULTSAt 0 min, there was no significant difference of MMP-8 release in the experiment group and positive control (P>0.01); whereas at 60, 120 min, E. faecalis induced a significant lower MMP-8 release compared with the positive control (P<0.01). The apoptosis rate of PMNs in both groups increased along with time, and apoptotic rate in experiment group was higher than that in the control group at 2, 5, 10, 15 h (P<0.01).
CONCLUSIONAfter E. faecalis act on PMNs, no significant release of MMP-8 from PMNs was observed. E. faecalis don't induce PMNs apoptosis delay.
Apoptosis ; Enterococcus faecalis ; Humans ; Matrix Metalloproteinase 8 ; Neutrophils ; Tetradecanoylphorbol Acetate
2.Differential Regulation of Antioxidant Enzymes during Monocyte Differentiation.
Young Sup LEE ; Shin Sung KANG ; Eun Jie KIM
Korean Journal of Immunology 1997;19(1):121-128
Antioxidant enzymes levels were determined in monocytes during phorbol myristate acetate (PMA)-induced differentiation. PMA induced the differentiation of a human monocytic leukemia cell line THP-1 into macrophage-like cells as indicated by activity of acid phosphatase and morphological changes. The level of Mn-superoxide dismutase (SOD) was selectively increased in PMA-treated THP-1 cells after one day of culture, while the levels of Cu/Zn-SOD and catalase were progressively decreased by Western blot analysis. In contrast, levels of Cu/Zn-SOD and catalase protein and enzyme activitiy remained unchanged in THP-1 cells after transforming growth factor-p, treatment. Cu/Zn-SOD is oxidatively inactivated by exposure to H,O, which is produced by PMA-treated THP-1 cells, and then the inactivated enzyme undergoes proteolysis and fragmentation as analyzed by radiolabeled method. Thus monocytes have a coordinated system for synthesis and degradation of antioxidant enzymes during PMA-induced differentiation.
Acid Phosphatase
;
Blotting, Western
;
Catalase
;
Cell Line
;
Humans
;
Leukemia
;
Monocytes*
;
Proteolysis
;
Superoxide Dismutase
;
Tetradecanoylphorbol Acetate
3.Effect of Betulinic Acid on MUC5AC and MUC5B Expression in Airway Epithelial Cells.
Hoon sung KIM ; Yoon Seok CHOI ; Jun Hyeok LEE ; Na Kyung PARK ; Chang Hwi PARK ; Young Ha LEE ; Gui Ok KIM ; Si Youn SONG ; Chang Hoon BAE ; Seung Ho LEE ; Yong Woon KIM ; Yong Dae KIM
Korean Journal of Otolaryngology - Head and Neck Surgery 2014;57(8):526-532
BACKGROUND AND OBJECTIVES: MUC5AC and MUC5B are representative secretory mucin genes in the human airway, whose expressions are increased by a variety of inflammatory mediators. Betulinic acid, a naturally occurring pentacyclic triterpenoid, is known to have an anti-inflammatory property. However, the effects of betulinic acid on mucin secretion of airway epithelial cells still have not been reported. Therefore, in this study, the effect of betulinic acid on inflammatory mediators-induced MUC5AC and MUC5B expressions was investigated in human airway epithelial cells. SUBJECTS AND METHOD: In the mucin-producing human NCI-H292 airway epithelial cells, the effects of betulinic acid on interleukin-1beta (IL-1beta)-, lipopolysaccharide (LPS)-, and phorbol myristate acetate (PMA)-induced MUC5AC and MUC5B expressions were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Betulinic acid attenuated IL-1beta-, LPS-, and PMA-induced MUC5B mRNA and glycoprotein expression in NCI-H292 cells. On the other hand, betulinic acid did not attenuate IL-1beta-, and LPS-, but induced PMA-induced MUC5AC mRNA and glycoprotein expressions in NCI-H292 cells. CONCLUSION: These results suggest that betulinic acid attenuates IL-1beta-, LPS-, and PMA-induced MUC5B expression in the airway epithelial cells. Therefore, betulinic acid may modulate a control of mucus-hypersecretion in airway inflammatory diseases.
Enzyme-Linked Immunosorbent Assay
;
Epithelial Cells*
;
Glycoproteins
;
Hand
;
Humans
;
Interleukin-1beta
;
Mucins
;
RNA, Messenger
;
Tetradecanoylphorbol Acetate
4.The characteristics of neutrophil extracellular traps produced by all-trans retinoic acid-induced dHL-60 under PMA stimulation.
Wang LIU ; Jinhua FANG ; Tiantian HONG ; Jiaqi HUANG ; Baisong ZHAO ; Ying FANG ; Jianhua WU ; Jiangguo LIN
Journal of Biomedical Engineering 2022;39(5):909-918
Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.
Humans
;
Extracellular Traps
;
Tetradecanoylphorbol Acetate/pharmacology*
;
Neutrophils
;
HL-60 Cells
;
Tretinoin/pharmacology*
5.Chemokine Lkn-1/CCL15 enhances matrix metalloproteinase-9 release from human macrophages and macrophage-derived foam cells.
Sang Hee KWON ; Seong A JU ; Ji Hye KANG ; Chu Sook KIM ; Hyeon Mi YOO ; Rina YU
Nutrition Research and Practice 2008;2(2):134-137
Atherosclerosis is characterized by a chronic inflammatory disease, and chemokines play an important role in both initiation and progression of atherosclerosis development. Leukotactin-1 (Lkn-1/CCL15), a new member of the human CC chemokine family, is a potent chemoattractant for leukocytes. Our previous study has demonstrated that Lkn-1/CCL15 plays a role in the initiation of atherosclerosis, however, little is currently known whether Lkn-1/CCL15 is associated with the progression of atherosclerosis. Matrix metalloproteinases (MMPs) in human coronary atherosclerotic lesions play a crucial role in the progression of atherosclerosis by altering the vulnerability of plaque rupture. In the present study, we examined whether Lkn-1/CCL15 modulates MMP-9 release, which is a prevalent form expressed by activated macrophages and foam cells. Human THP-1 monocytic cells and/or human peripheral blood monocytes (PBMC) were treated with phorbol myristate acetate to induce their differentiation into macrophages. Foam cells were prepared by the treatment of THP-1 macrophages with human oxidized LDL. The macrophages and foam cells were treated with Lkn-1/CCL15, and the levels of MMP-9 release were measured by Gelatin Zymography. Lkn-1/CCL15 significantly enhanced the levels of MMP-9 protein secretion from THP-1 monocytic cells-derived macrophages, human PBMC-derived macrophages, as well as macrophage-derived foam cell in a dose dependent manner. Our data suggest that the action of Lkn-1/CCL15 on macrophages and foam cells to release MMP-9 may contribute to plaque destabilization in the progression of atherosclerosis.
Atherosclerosis
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Chemokines
;
Foam Cells
;
Gelatin
;
Humans
;
Leukocytes
;
Lipoproteins, LDL
;
Macrophages
;
Matrix Metalloproteinase 9
;
Matrix Metalloproteinases
;
Monocytes
;
Phorbols
;
Rupture
;
Tetradecanoylphorbol Acetate
6.Interleukin-8 production and interleukin-8 mRNA expression induced by lipopolysaccharides from Prevotella intermedia and Prevotella nigrescens in monocyte-derived macrophages.
The Journal of the Korean Academy of Periodontology 2009;39(2):177-184
PURPOSE: Interleukin-8 (IL-8) is an important mediator of immune and inflammatory reactions and is produced by a variety of different cell types. This study was undertaken to investigate the effects of lipopolysaccharides (LPSs) from Prevotella intermedia and Prevotella nigrescens, the major causes of inflammatory periodontal disease, on the production of IL-8 and the expression of IL-8 mRNA in differentiated THP-1 cells, a human monocytic cell line. METHODS:LPSs from P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 were prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. RESULTS: We found that LPS preparations from P. intermedia and P. nigrescens can induce IL-8 mRNA expression and stimulate the release of IL-8 in differentiated THP-1 cells without additional stimuli. CONCLUSIONS: There are no previous reports of the ability of P. intermedia and P. nigrescens LPS to stimulate the release of IL-8, and the present study clearly shows, for the first time, that LPSs from P. intermedia and P. nigrescens fully induced IL-8 mRNA expression and IL-8 production in differentiated human monocytic cell line THP-1. The ability of P. intermedia and P. nigrescens LPS to promote the production of IL-8 may be important in the pathogenesis of inflammatory periodontal disease.
Cell Line
;
Humans
;
Interleukin-8
;
Lipopolysaccharides
;
Macrophages
;
Periodontal Diseases
;
Phorbols
;
Prevotella
;
Prevotella intermedia
;
Prevotella nigrescens
;
RNA, Messenger
;
Tetradecanoylphorbol Acetate
7.Changes of Telomerase Activity by Protein Kinase C Modulators in Human Ovarian Cancer Cell Lines.
Soo Young HUR ; Joon Mo LEE ; Sung Eun NAMKOONG ; Jin Woo KIM
Journal of the Korean Cancer Association 2000;32(4):724-733
PURPOSE: This study was designed to find out whether protein kinase C (PKC) may affect telomerase activity in human ovarian cancers. MATERIALS AND METHODS: To determine whether PKC modulators influence PKC activities, NIH: OVCAR-3 and CUMO-2, cells were treated with PKC inhibitors, G 6976 and bisindolyl maleimide I, and PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA). Telomerase acti vity was determined by telomeric repeat amplification protocol (TRAP). Analysis of the expres sion of each telomerase subunits, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT), was performed by RT-PCR. We also examined the alternative splicing of hTERT. RESULTS: G 6976 and bisindolylmaleimide I inhibited PKC activity. Telomerase activities appeared to be affected in a time-dependent manner by these two PKC inhibitors. PKC activities were increased in parallel with telomerase activity by TPA at the low dose (10 nM), but their activities were down-regulated at the high dose (1 micrometer). RT-PCR demonstrated the presence of hTR and hTERT mRNA before and after the treatment of PKC modulators, respectively, and showed the presence of one alternatively spliced transcript and full-length hTERT transcripts. CONCLUSION: These results showed that telomerase activity was affected by PKC and suggested PKC modulation may serve as an useful tool in the regulation of telomerase activity.
Alternative Splicing
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Cell Line*
;
Humans*
;
Ovarian Neoplasms*
;
Protein Kinase C*
;
Protein Kinases*
;
RNA
;
RNA, Messenger
;
Telomerase*
;
Tetradecanoylphorbol Acetate
8.A New Endothelial Molecule Involved in Melanoma Cell Binding to Human Dermal Microvascular Endothelial Cells.
Kwang Hoon LEE ; Kee Yang CHUNG ; Thomas J LAWLEY ; Robert A SWERLICK
Annals of Dermatology 1994;6(1):9-16
BACKGROUND: We have previously demonstrated that phorbol myristate acetate (PMA)-induced increases in melanoma cell binding to endothelial cells derived from human dermis (HDMEC) are not mediated via known cell adhesion molecules and may be affected through microvessel-specific novel proteins not previously described on endothelial cells. OBJECTIVE: This study was performed to identify new molecules which may play a role in HDMEC-melanoma cells binding. METHODS: We have generated a monoclonal antibody(Mab) against PMA-stimulated HDMEC. A Mab was evaluated functionally through melanoma cell-endothelial cell adherence assay and characterized by Western immunoblot. RESULTS: Mab EM-71 recognized a molecule with expression levels in vitro that could be upregulated by PMA(EM-71 molecule). The expression of EM-71 molecule on HDMEC was increased in a dose-dependent manner by PMA only, but not affected by interleukin 1 alpha(IL-lα) or tumor necrosis factor alpha(TNFα) PMA augmented melanoma cell adherence to HDMEC, which is coincident with an increase in EM-71 molecule expression on HDMEC by PMA. Mab EM-71 partially inhibited up to 59% of the increased melanoma cell binding to PMA-stimulated HDMEC and failed to block melanoma cell binding to IL-lα or TNFα-stimulated HDMEC. Western immunoblots of lysates of HDMEC demonstrated a 200 kDa protein on HDMEC. CONCLUSION: This study demonstrates that EM-71 molecule may play a partial role in melanoma binding to PMA-stimulated HDMEC.
Blotting, Western
;
Cell Adhesion Molecules
;
Dermis
;
Endothelial Cells*
;
Humans*
;
In Vitro Techniques
;
Interleukin-1
;
Melanoma*
;
Tetradecanoylphorbol Acetate
;
Tumor Necrosis Factor-alpha
9.Inhibition of Interleukin-1 Signal by Annexin-1 in Phorbol Myristate Acetate Stimulated Lymphocytes.
Hae Jin RHEE ; Kun Koo PARK ; Doe Sun NA ; Ha Won KIM
Korean Journal of Immunology 1999;21(2):147-152
Annexin-1 (ANX1) is a 37 kDa protein that is induced and secreted by glucocorticosteroid hormone. The secreted ANX1 has been believed to exert its function by binding to its putative rnembrane receptor. In this report we demonstrate that ANXl receptor (ANX1R) signal blocks the interleukin-1B (IL-1B) receptor signal pathway in human peripheral blood mononuclear cells (PBMCs). When PBMCs were treated with both IL-1B (100 ng/ml) and PMA (10 ng/ml) in the absence or presence of dexamethasone for 5 days, dexamethasone (100 nM) suppressed lymphocyte proliferation to 24% of the control. However addition of anti-ANX1 polyclonal antibody of 1:200 and 1:1,000 dilution to this system induced recovery of proliferation to 80% and 40%, respectively, when compared to the control. In the mixed lymphocyte reaction, dexamethasone suppressed lymphocyte proliferation to 9% of that of control when stimulated with IL-1B (100 ng/ml) and phorbol myristate acetate (10 ng/ml). Addition of anti-ANX1 polyclonal antibody (1:1,000) to this system also recovered the proliferation to 20% of that of the control system. In the ANX1 receptor induction experiment using flow cytometry, ANX1 receptor expression on lymphocytes, CD4+ T cells, CD8+ T cells and monocytes increased depending on the externally added IL-1B ranging from 10 to 1,000 ng/ml. From these results, it is evident that dexamethasone induces ANX1 secretion into the culture medium and anti-ANX1 polyclonal antibody abolishes the effects of dexamethasone. Furthermore these results imply that extracellular ANX1 exerts its effects by binding to the receptor on the cell membrane and the activated signal(s) of ANX1R block IL-1B receptor signal in the lymphocytes.
Cell Membrane
;
Dexamethasone
;
Flow Cytometry
;
Humans
;
Interleukin-1*
;
Lymphocyte Culture Test, Mixed
;
Lymphocytes*
;
Monocytes
;
Signal Transduction
;
T-Lymphocytes
;
Tetradecanoylphorbol Acetate*
10.Effects of As2O3 in combination with TPA on K562 cells.
Fang-Fang YUAN ; Xu-Hua ZHANG ; Rui-Hua MI ; Rui-Hua FAN ; Qing-Song YIN ; Xu-Dong WEI
Journal of Experimental Hematology 2014;22(4):943-949
This study was aimed to investigate the effects of arsenic trioxide (As2O3) combined with TPA on cell cycle, cell differentiation and apoptosis of K562 cell line, and their possible mechanisms. K562 cells were treated with 200 nmol/L TPA, 2 µmol/L As2O3 alone and 200 nmol/L TPA combined with 2 µmol/L As2O3. The proliferative inhibition rates were determined with CCK-8. Annexin V and agarose gel electrophoresis were adopted to detect apoptosis. Colony formation test was used to determine the colony-formation efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38 proteins. The results showed that combination treatment had synergistic effects on the proliferative inhibition and apoptosis, which were much higher than those treated alone. As2O3 could decrease the colony formation ability of K562 cells. The cells treated with both TPA and As2O3 expressed far more CD11b antigens compared with cells exposed to As2O3 alone. K562 cells treated with TPA were arrested in G1 phase compared with the control group, As2O3 increased the percentage of K562 cells in the G2 phase. The combination treatment increased the expression of p-P38 of K562 cells compared with the cells exposed As2O3 alone. It is concluded that TPA can enhance the effect of As2O3 on inducing apoptosis and adjusting cell cycle , which will expect to provide a new therapeutic program.
Apoptosis
;
drug effects
;
Arsenicals
;
pharmacology
;
Cell Cycle
;
drug effects
;
Drug Synergism
;
Humans
;
K562 Cells
;
Oxides
;
pharmacology
;
Tetradecanoylphorbol Acetate
;
pharmacology