OBJECTIVEThe aim of this study was to investigate the role of macrophages in promotion of ovarian tumor cell proliferation mediated by over-expression of antimicrobial peptide LL-37.

METHODSTo co-culture ovarian tumor cells SKOV3, 3AO and HO-8910 with macrophages. The Transwell(®) inserts system was used in the co-culture model. The effect of macrophages promoted ovarian tumor cell proliferation was assessed by BrdU-ELISA and cell number counting. Expressions of mRNA and protein of LL-37 in the macrophages and SKOV3 cells were determined by RT-PCR and Western blot analysis. To observe that LL-37 is responsible for macrophage-promoted ovarian tumor cells growth, LL-37 neutralizing antibody was added to abrogate the LL-37 activation.

RESULTSThe cell number assay showed that after 4 days coincubation with macrophages in the proportion of 1:0.5, the number of SKOV3 cells increased from (6.0 ± 0.5)×10(4) to (11.8 ± 1.3)×10(4), showing a significant difference (P < 0.05). It also showed that the growth of the SKOV3 cells was dependent on the macrophage number (P < 0.05). The number variability of 3AO and HO-8910 cells was as the same as SKOV3 cells upon co-culture with macrophages. As determined by BrdU-ELISA, the resulted proliferation of ovarian tumor cells was similar to the result of cell number counting. RT-PCR and Western blot results showed that the expression of LL-37 mRNA and protein in the macrophages was remarkably enhanced in a time dependent manner upon coincubation with SKOV3 cells, but did not work in SKOV3 cells. BrdU-ELISA assay exhibited that treatment of cells with LL-37 significantly stimulated HO-8910 and 3AO cell proliferation. Addition of LL-37 neutralizing antibody markedly inhibited macrophage-promoted ovarian tumor cell (SKOV3, 3AO and HO-8910 cells) proliferation. The OD values of these three cells were decreased from 2.95 ± 0.11 to 1.45 ± 0.04, from 3.39 ± 0.36 to 1.32 ± 0.09 and from 3.93 ± 0.17 to 1.68 ± 0.23, respectively (P < 0.05).

CONCLUSIONSOver-expression and release of LL-37 from macrophages is responsible for proliferation of ovarian tumor cells in co-culture condition. The data presented indicate that LL-37 may be critical for macrophage-induced tumor progression.

Antibodies, Neutralizing ; pharmacology ; Cathelicidins ; genetics ; metabolism ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coculture Techniques ; Female ; Humans ; Macrophages ; cytology ; physiology ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism

In this study, the synergistic effect of 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2(1H)-quinolinone (cilostazol) and Ginkgo biloba extract (GbE) was examined in apolipoprotein E (ApoE) null mice. Co-treatment with GbE and cilostazol synergistically decreased reactive oxygen species (ROS) production in ApoE null mice fed a high-fat diet. Co-treatment resulted in a significantly decreased atherosclerotic lesion area compared to untreated ApoE mice. The inflammatory cytokines and adhesion molecules such as monocyte chemoattractant-1 (MCP-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and VCAM-1 which can initiate atherosclerosis were significantly reduced by the co-treatment of cilostazol with GbE. Further, the infiltration of macrophages into the intima was decreased by co-treatment. These results suggest that co-treatment of GbE with cilostazol has a more potent anti-atherosclerotic effect than treatment with cilostazol alone in hyperlipidemic ApoE null mice and could be a valuable therapeutic strategy for the treatment of atherosclerosis.
Animals ; Apolipoproteins E/genetics/physiology ; Atherosclerosis/*drug therapy ; Cytokines/metabolism ; Disease Models, Animal ; Drug Synergism ; Ginkgo biloba/*chemistry ; Humans ; Macrophages/cytology/drug effects ; Male ; Mice ; Mice, Nude ; Plant Extracts/*administration & dosage/chemistry ; Reactive Oxygen Species/*metabolism ; Tetrazoles/*administration & dosage

OBJECTIVETo investigate the effect of diclofenac on the expression of Kv1.3 and Kir2.1 channels in human macrophages and the membrane potential and foaming process of the macrophages.

METHODSThe effect of diclofenac on the expression of Kv1.3 and Kir2.1 channels in cultured human monocyte-derived macrophages was investigated using real-time RT-PCR and Western blotting, and its effect on the membrane potential was analyzed with optical mapping of the membrane potential with voltage-sensitive dyes. The ratio of cholesterol ester (CE) in the macrophages following intake of oxidized low-density lipoprotein (OxLDL) was analyzed by an enzymatic fluorometric method.

RESULTSThe expression of Kv1.3 and Kir2.1 channels in the macrophages were down-regulated by diclofenac (1.5 µmol/L and 15 µmol/L). Compared with those in the control group, Kv1.3 mRNA expression was reduced by over 80% and 90% (P<0.05), and Kir2.1 mRNA by over 20% and 30% (P>0.05), respectively; both their protein expression was reduced by over 10% and 60% with a dose- dependent effect (P<0.05). Diclofenac at the two doses dose-dependently reduced the surface fluorescence intensity of the macrophage, and the membrane potential was decreased by 28% and 54%, respectively (P<0.05). Incubation of the macrophages with 30 mg/L OxLDL for 60 h caused an obvious enlargement of the cell volume and deposition of numerous lipid granules in cytoplasm, resulting also in a CE/TC ratio over 50% (P<0.05). Diclofenac at 1.5 and 15 µmol/L both significantly decreased the CE/TC ratio to (23.624∓3.34)% and (13.601∓2.916)% (P<0.05), respectively, but this effect did not show a dose-response relationship (P>0.05).

CONCLUSIONDiclofenac can significant down-regulate the expression of Kv1.3 and Kir2.1 channels in human macrophages, lower their membrane potential and inhibit the process of foam cell formation.

Cells, Cultured ; Diclofenac ; pharmacology ; Foam Cells ; cytology ; drug effects ; Humans ; Kv1.3 Potassium Channel ; metabolism ; Macrophages ; drug effects ; metabolism ; physiology ; Membrane Potentials ; drug effects ; Potassium Channels, Inwardly Rectifying ; metabolism

This study is to investigate the effects of indirubin on ATP-induced immune responses of macrophages. For this, neutral red dye uptake method was used to test phagocytosis, MTT assay was used for measuring cell death, and reactive oxygen species (ROS) was tested with fluorescent probe DHE. The data showed that extracellular ATP attenuated phagocytosis, induced cell death and increased ROS production, and these effects were restored by pre-treating with indirubin. This result suggested that indirubin blockade the effects of ATP on macrophages, because extracellular ATP-induced effects are dependent on P2 receptors, in particular P2X7 receptors. Furthermore, the effects of indirubin on the activation of P2 receptors were tested, in particular P2X7 receptors. The data showed that indirubin significantly decreased ATP-induced, P2 receptors mediated intracellular Ca2+ concentration ([Ca2+]i) rise and inhibited P2X7 receptor-based ethidium bromide (EB) dye uptake. These results suggested the inhibitory effects of indirubin on the activation of P2X7 receptors, which may underlying the effects on ATP induced ROS production, phagocytosis attenuation and cell death of macrophages.
Adenosine Triphosphate ; pharmacology ; Animals ; Calcium ; metabolism ; Cell Death ; drug effects ; Indoles ; pharmacology ; Macrophages ; cytology ; metabolism ; physiology ; Male ; Phagocytosis ; drug effects ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Receptors, Purinergic P2X7 ; metabolism

Endoplasmic reticulum (ER) stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. The purpose of the present study was to investigate the effects of caveolin-1 (Cav-1) on ER stress-induced apoptosis in cultured macrophages and the underlying mechanisms. RAW264.7 cells were incubated with thapsigargin (TG) to establish ER stress model. And Cav-1 expression was detected by Western blot. After being pretreated with filipin(III), a caveolae inhibitor, RAW264.7 cells were assayed with flow cytometry and confocal laser scanning microscopy to detect cell apoptosis. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation and C/EBP homologous protein (CHOP) expression were detected with Western blot. The results showed that Cav-1 expression was markedly increased at early stage of TG treatment (P < 0.05) and then decreased with prolonged or high dose TG treatments. The increasing of Cav-1 expression induced by TG in RAW264.7 cells was abolished under inhibition of caveolae by filipin(III) (P < 0.05). The effect of TG on apoptosis of RAW264.7 cells was further augmented after pretreatment with filipin(III) (P < 0.05). Western blotting showed that MAPK phosphorylation induced by TG was inhibited by filipin(III) in RAW264.7 cells (P < 0.05), whereas CHOP remained unchanged (P > 0.05). These results suggest that Cav-1 may play a critical role in suppressing ER stress-induced macrophages apoptosis in vitro, and one of the mechanisms may be correlated with the activation of p38 MAPK prosurvival pathway.
Animals ; Apoptosis ; drug effects ; Caveolin 1 ; genetics ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; physiology ; Filipin ; pharmacology ; MAP Kinase Signaling System ; Macrophages ; cytology ; drug effects ; Mice ; Thapsigargin ; pharmacology ; Transcription Factor CHOP ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVE: To determine the effect and possible mechanism of an apolipoprotein (apo) A-I mimetic peptide, D-4F, on cholesterol efflux in RAW264.7 macrophages. METHODS: RAW264.7 macrophages were incubated in the medium containing 8-bromo cAMP (8-Br-cAMP, 0.5 mmol/L) and ox-LDL (50 μg/mL) for 24 h. Then various concentrations of D-4F (0-100 μg/mL) or H89 (20 μmol/L, a protein kinase A inhibitor) were added for the purpose of interference. The intracellular cyclic AMP (cAMP) level was determined by enzyme-linked immunoabsobant assay (ELISA). ATP binding cassette transporter A1 (ABCA1) expression in the macrophages was quantitated by real-time PCR and Western blot. RESULTS: D-4F significantly increased the cholesterol efflux in both concentration and time-dependent manner accompanied by the increase in the intracellular cAMP level, ABCA1 mRNA and protein expression. The effect of D-4F on cholesterol efflux ABCA1 expression was enhanced by 8-Br-cAMP. Although H89 did not affect the basal cholesterol efflux and ABCA1 expression, it could attenuate the effect of 8-Br cAMP. CONCLUSION D-4F affects cholesterol efflux, cAMP level, and ABCA1 expression in macrophages, which is likely involved in the pathway of cAMP/PKA/ABCA1.
ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; metabolism ; Apolipoprotein A-I ; chemistry ; pharmacology ; Biological Transport ; drug effects ; physiology ; Biomimetics ; Cell Line ; Cholesterol ; metabolism ; Cyclic AMP ; metabolism ; Humans ; Macrophages ; cytology ; metabolism ; Peptides ; chemistry ; pharmacology

The purpose of the present study is to explore the effect of oxidized low density lipoprotein (ox-LDL) on the induction of endoplasmic reticulum stress (ERS) and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process. RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum, and then treated with ox-LDL (25, 50 and 100 mg/L), anti-CD36 monoclonal antibody+ox-LDL and tunicamycin (TM), respectively. After incubation for 24 h, the cells were collected. The cellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymatic colorimetry. The expression of glucose-regulated protein 94 (GRP94), a molecular marker of ERS, was determined by immunocytochemistry assay. The levels of GRP94 protein, phosphorylated inositol-requiring enzyme 1 (p-IRE1) and X box binding protein 1 (XBP1) in RAW264.7 cells were detected by Western blotting. The results indicated that after incubation with ox-LDL (25, 50 and 100 mg/L) for 24 h, a large amount of lipid droplets were found in the cytoplasm, and the contents of cellular total cholesterol were increased by 2.1, 2.8 and 3.1 folds compared with the control, respectively. Anti-CD36 antibody decreased markedly the cellular lipid accumulation induced by ox-LDL at 100 mg/L. Both ox-LDL and TM, a specific ERS inducer, could up-regulate the protein expression of GRP94 in a dose-dependent manner. Furthermore, p-IRE1 and XBP1, two key components of the unfolded protein response, were also significantly induced by the treatment with ox-LDL. The up-regulations of the three proteins induced by ox-LDL were inhibited significantly when the macrophages were pre-incubated with anti-CD36 antibody. These results suggest that ox-LDL may induce ERS in a dose-dependent way and subsequently activate the unfolded protein response signaling pathway in RAW264.7 macrophages, which is potentially mediated by scavenger receptor CD36.
Animals ; CD36 Antigens ; physiology ; Cell Line ; Cells, Cultured ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum ; drug effects ; Foam Cells ; cytology ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Protein-Serine-Threonine Kinases ; metabolism ; Regulatory Factor X Transcription Factors ; Stress, Physiological ; drug effects ; Transcription Factors ; metabolism ; X-Box Binding Protein 1

OBJECTIVETo investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism.

METHODSThe human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting.

RESULTSS. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells.

CONCLUSIONSS. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

Anthracenes ; pharmacology ; Apoptosis ; physiology ; Caspase 3 ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Mitogen-Activated Protein Kinase 8 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 9 ; antagonists & inhibitors ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Signal Transduction ; physiology ; Staphylococcus aureus ; physiology ; U937 Cells ; bcl-2-Associated X Protein ; metabolism

This study investigated the effect of phloretin (Ph) on the proliferation, activation, and cell-cycle distribution of mouse T lymphocytes and NO production and phagocytosis of macrophages. Carboxyfluorescein diacetatesuccinimidyl ester (CFDA-SE) staining plus flow cytometry assay was employed to obtain the proliferation-related index (PI) of lymphocytes. The expression levels of CD69 and CD25 on T lymphocytes stimulated with Con A were evaluated with flow cytometry after staining with fluorescent monoclonal antibody. Cell-cycle distribution of T lymphocytes was analyzed by propidium iodide staining. Griess kit was used to evaluate the NO production and fluorescent microbeads were used to analyze the phagocytosis ability of macrophages. Our results showed that phloretin (40, 60, and 80 micromol x L(-7)) significantly inhibited the proliferation of T lymphocytes and the PI reduced from 1.41 +/- 0.13 to 1.34 +/- 0.16, 1.19 +/- 0.12 and 1.07 +/- 0.06, respectively. Phloretin significantly inhibited the expression of CD69 and CD25 (P < 0.01). The cell cycle distribution analysis showed that phloretin could induce a cell cycle arrest at G0/G1 phase. NO production of LPS +IFN-gamma group of macrophages was (26.72 +/- 3.57) micromol x L(-1), and was significantly reduced by phloretin (P < 0.01). And phagocytosis rate of macrophages was significantly reduced by phloretin (P < 0.01). The results demonstrate that phloretin might be developed into a new immuosuppressive drug.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Female ; Immunosuppressive Agents ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lectins, C-Type ; metabolism ; Macrophages ; physiology ; secretion ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; secretion ; Phagocytosis ; drug effects ; Phloretin ; pharmacology ; T-Lymphocytes ; cytology ; immunology

BACKGROUNDCardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease. Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum (ER) stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) for five days as an in vitro model, to investigate if acetylated low-density lipoprotein (AcLDL) loading could also induce apoptosis and its underlying mechanisms.

METHODSOil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol (FC) and cholesterol ester (CE) assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed.

RESULTSConfocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 microg/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER.

CONCLUSIONER stress is involved in AcLDL induced apoptosis in THP-1 macrophages with free cholesterol accumulation in the ER.

Apoptosis ; drug effects ; Blotting, Western ; Cell Differentiation ; physiology ; Cell Line ; Cholesterol ; metabolism ; Chromatography, High Pressure Liquid ; Endoplasmic Reticulum ; metabolism ; Humans ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; drug effects ; Microscopy, Confocal ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Transcription Factor CHOP ; genetics