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

The study purpose was to explore whether dichloromethylene diphosphonate (Cl(2)MDP)-loaded gelatin particles can induce the depletion of macrophage in reticuloendothelial system of liver and spleen or can depress the immunity of macrophage in SD rat models of immune thrombocytopenic purpura (ITP) to treat the ITP rats. New Zealand rabbits were immunized with platelets of SD rats to prepare rabbit anti-rat platelet serum, and the serum was intravenously injected into SD rats to produce the ITP model. In experimental ITP models, 150 microl of anti-platelet serum was intravenously injected into SD rats per 24 hours. The platelet counts maintained pathological level and were persistently less than 50 x 10(9)/L in the models during experiment process. The MTT test of macrophage RAW264.7 was carried out by means of Cl(2)MDP-loaded gelatin particles in vitro. After intravenous injection of a group dose of Cl(2)MDP-gelatin particles, the platelet counts of the rats were measured at the time of 4 hours, 24 hours, 48 hours, 72 hours and 96 hours, respectively, and bleeding times were detected in 24 hours. The results showed that Cl(2)MDP-loaded gelatin particles increased the platelet counts of ITP models to mean of 180 x 10(9)/L, a physiological level in 24 hours after injection, and kept this platelet level through whole process of 120 hours. Furthermore, rats pre-treated with Cl(2)MDP-loaded gelatin particles avoided the decrease of platelet counts significantly when they were injected anti-platelet serum. It is concluded that Cl(2)MDP-loaded gelatin particles restrain multiplication of macrophage RAW264.7, and promptly, effectively restore platelet counts of ITP models to physiological level in a dose dependent manner. So, the targeting therapy of drug-loaded gelatin particles offers a new idea and approach to treat ITP, and this strategy is worthy of further studies.
Animals ; Clodronic Acid ; administration & dosage ; Drug Carriers ; Drug Delivery Systems ; Gelatin ; administration & dosage ; Liver ; cytology ; drug effects ; Macrophages ; drug effects ; physiology ; Particle Size ; Purpura, Thrombocytopenic, Idiopathic ; therapy ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Spleen ; cytology ; drug effects

Methyl-beta-cyclodextrin, a cyclic oligosaccharide known for its interaction with the plasma membrane induces several events in cells including cell growth and anti-tumor activity. In this study, we have investigated the possible role of cyclooxygenase 2 (COX-2) in cell growth arrest induced by methyl-beta-cyclodextrin in Raw264.7 macrophage cells. Methyl-beta-cyclodextrin inhibited cell growth and arrested the cell cycle, and this cell cycle arrest reduced the population of cells in the S phase, and concomitantly reduced cyclin A and D expressions. Methyl-beta-cyclodextrin in a dose- and time-dependent manner, also induced COX-2 expression, prostaglandin E(2) (PGE(2)) synthesis, and COX-2 promoter activity. Pretreatment of cells with NS398, a COX-2 specific inhibitor completely blocked PGE(2) synthesis induced by methyl-beta-cyclodextrin, however inhibition on cell proliferation and cell cycle arrest was not effected, suggesting non-association of COX-2 in the cell cycle arrest. These results suggest that methyl-beta-cyclodextrin induced cell growth inhibition and cell cycle arrest in Raw264.7 cells may be mediated by cyclin A and D1 expression.
Animals ; Cell Cycle/drug effects/*physiology ; Cell Line ; Cell Proliferation/*drug effects ; Dinoprostone/*metabolism ; Dose-Response Relationship, Drug ; Isoenzymes/genetics/*metabolism ; Macrophages/cytology/*drug effects/physiology ; Mice ; Prostaglandin-Endoperoxide Synthase/genetics/*metabolism ; Research Support, Non-U.S. Gov't ; beta-Cyclodextrins/*pharmacology

Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL. Copyright 2000 Academic Press.
Animal ; Apolipoproteins B/metabolism ; Apoptosis/physiology ; Apoptosis/drug effects ; Cell Death/physiology* ; Cell Division/physiology ; Copper Sulfate/metabolism ; Dose-Response Relationship, Drug ; Human ; Lipid Peroxidation ; Lipids/metabolism ; Lipoproteins, LDL/pharmacology ; Lipoproteins, LDL/metabolism* ; Macrophages/pathology ; Macrophages/drug effects ; Macrophages/cytology* ; Mice ; Necrosis ; Oxidation-Reduction ; Thiobarbituric Acid Reactive Substances/metabolism

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

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

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

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

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

OBJECTIVETo study the effect of the cultured supernatant of human silicotic alveolar macrophages (AM) on the expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in human lung fibroblasts (FB).

METHODSHuman alveolar macrophages were collected from a silicotic patients by bronchoalveolar lavage and exposed to SiO(2), then the cultured supernatant were incubated with human fetal lung fibroblasts for 6, 12, 18, 24, 36, 48 h. The immunocytochemical method was used to detect the level of expression of MMP-1 and TIMP-1 in lung fibroblasts.

RESULTSThe expression of MMP-1 in FB in 24 h incubation was lower in cultured supernatant of silicotic AM unexposed to SiO(2) than in blank control [integrated OD (IOD)]: 0.103 +/- 0.014 vs 0.133 +/- 0.023), while the expression of TIMP-1 was higher (IOD: 0.108 +/- 0.012 vs 0.065 +/- 0.006). The expression of MMP-1 in FB in cultured supernatant of AM exposed to SiO(2) for 24 h was further decreased (IOD: 0.062 +/- 0.008 vs 0.133 +/- 0.023), while that of TIMP-1 was further increased (IOD: 0.143 +/- 0.015 vs 0.065 +/- 0.006).

CONCLUSIONSiO(2) may affect the expression of MMP-1 and TIMP-1 system through AM mediation and participate in the formation of lung fibrosis.

Cells, Cultured ; Coculture Techniques ; Fibroblasts ; drug effects ; metabolism ; Humans ; Lung ; cytology ; Macrophages, Alveolar ; physiology ; Male ; Matrix Metalloproteinase 1 ; metabolism ; Middle Aged ; Silicon Dioxide ; pharmacology ; Silicosis ; pathology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism