Cultured Mouse Peritoneal Macrophages Research Articles

Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity.

The lethal toxin of Bacillus anthracis is central to the pathogenesis of anthrax. Using primary cultures of mouse peritoneal macrophages, we have demonstrated that intracellular calcium release inhibitors protect against anthrax lethal toxin-induced cytotoxicity. The cytolytic effect of anthrax lethal toxin was markedly reduced by dantrolene, an inhibitor of calcium release from intracellular calcium stores. Pretreatment of macrophages with cyclosporin A, which has been shown to be a potent inhibitor of calcium release from mitochondria, also protected cells against cytotoxicity. These results indicate that calcium release from intracellular store may be an essential step for the propagation of anthrax lethal toxin-induced cell damage in macrophages. Thus our findings suggest that dantrolene, cyclosporin A, and possibly other drugs affecting intracellular calcium pools might be effectively preventing the toxicity from anthrax lethal toxin.

Melanocortin-1 and melanocortin-5 receptors are expressed in cultured mouse peritoneal macrophages

Melanocortin-1 and melanocortin-5 receptors are expressed in cultured mouse peritoneal macrophages

Inhibitory effect of resveratrol on interleukin 6 release by stimulated peritoneal macrophages of mice.

In the present investigation, interleukin 6 (IL-6) activity in the supernatant of cultured mouse peritoneal macrophages was monitored using a sensitive bioassay involving the IL-6-dependent murine hybridoma B9 cell line. The effects of resveratrol on Il-6 release by mouse peritoneal macrophages stimulated with calcium ionophore A23187 and fMLP were explored. Resveratrol, at a concentration range from 5 x 10(-6) to 4 x 10(-5) mol.l-1, was found to dose-dependently inhibit IL-6 release by cultured macrophages induced by A23187 and fMLP, and showed no direct cytotoxic effect, but induced proliferation of cultured mouse thymus cells. Resveratrol, at a concentration range from 10(-8) to 10(-5) mol.l-1, was shown to dose-dependently inhibit calcium ion influx into the cells with the stimulation of fMLP (10(-6) mol.l-1). These results suggest that the blocking of calcium ion influx into cells by reveratrol is one of the possible mechanisms of the IL-6 biosynthesis inhibitory action of resveratrol.

Involvement of phospholipase A2 activation in anthrax lethal toxin-induced cytotoxicity.

The molecular mechanism of cytotoxic effect exerted by the lethal toxin (LeTx) of Bacillus anthracis is not well understood. In the present study, using primary culture of mouse peritoneal macrophages, we have investigated possible cytotoxic mechanisms. LeTx was not found to induce high levels of nitric oxide (NO) production for NO-mediated toxicity. Fragmentation of DNA, a biochemical marker of apoptosis, was not observed in LeTx-treated cells. Pretreatment of cells with antioxidants such as melatonin and dehydroepiandrosterone (DHEA) did not protect the LeTx-induced cytotoxicity. However, addition of phospholipase A2 (PLA2) inhibitors (quinacrine, p-bromophenacyl bromide, manoalide, butacaine) to the culture medium resulted in the inhibition of cytotoxicity of LeTx in a dose-dependent manner. LeTx-induced cytotoxicity was also inhibited by the tyrosine-specific protein kinase inhibitor genistein, but not by the protein kinase C inhibitors staurosporine or H-7. The results of these studies indicate a role for PLA2 and protein kinase in the cytotoxic mechanism of macrophages by anthrax lethal toxin.

Hepatoprotective and nitric oxide production inhibitory activities of coumarin and polyacetylene constituents from the roots of Angelica furcijuga.

The methanolic extract from the roots of Angelica furcijuga KITAGAWA was found to exhibit protective effects on liver injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). From the methanolic extract, seventeen coumarins, two phenylpropanoids, and two polyacetylenes were isolated and examined their in vitro and in vivo hepatoprotective effects and inhibitory activity of NO production in macrophages. A acylated khellactone, isoepoxypteryxin, showed protective activity against D-GalN-induced cytotoxicity in primary cultured rat hepatocytes. On the other hand, six acylated khellactones (hyuganins A, B, C, and D, anomalin, isopteryxin) and two polyacetylenes [(-)-falcarinol and falcarindiol] strongly inhibited NO production induced by LPS in cultured mouse peritoneal macrophages, and also other acylated khellactones (isoepoxypteryxin, pteryxin, and suksdorfin) and a coumarin glycosides (praeroside II) were found to show the activity. By comparison of the inhibitory activities for acylated khellactones with those for other coumarins, acyl groups were found to be essential to exerting potent activity.

Arginine-vasopressin effect on the content of cyclic nucleotides, cellular protein phosphorylation, and adhesion to plastic of peritoneal mouse macrophages

Intracellular concentration of cAMP and phosphorylation of 15 kD protein in cultured mouse peritoneal macrophages are increased in the presence of arginine-vasopressin. Vasopressin, decreases adhesion of macrophages to plastic.

Involvement of Specific Mechanism in Plasmid DNA Uptake by Mouse Peritoneal Macrophages

The binding and uptake of plasmid DNA encoding luciferase reporter gene (pCMV-Luc) were studied in vitro using cultured mouse peritoneal macrophages. A significant and time-dependent cellular association of [32P]pCMV-Luc with resident macrophages was observed at 37 °C and this decreased at 4 °C. The binding at 4 °C was saturable and a Scatchard plot gave a maximum binding capacity of 0.81 μg/mg-protein and a dissociation constant of 0.30 μg/ml. The binding of [32P]pCMV-Luc was inhibited by polyinosinic acid, dextran sulfate and salmon sperm DNA, but not by polycytidylic acid, dextran and EDTA. A confocal microscopic study demonstrated that fluorescein-labeled pCMV-Luc was internalized at 37 °C while only cell surface binding occurred at 4 °C. No significant luciferase gene expression was obtained after incubation with a high concentration (100 μg/ml) of pCMV-Luc. These data suggest that plasmid DNA is taken up by macrophages via a mechanism mediated by a receptor like the macrophage scavenger receptor.

Increase in gentamicin uptake by cultured mouse peritoneal macrophages and rat hepatocytes by its binding to polybutylcyanoacrylate nanoparticles

Polybutylcyanoacrylate nanoparticles of 3H labelled gentamicin were used to investigate the possibility of gentamicin nanoparticles as the drug delivery system for intracellular chemotherapy. 3H labelled gentamicin nanoparticles were incubated with mouse peritoneal macrophages or rat hepatocytes for various lengths of time. The cells were then separated from the nanoparticles and the radioactivity of 3H in the cells was measured by a liquid scintillation counter. By comparison with the gentamicin solution, the binding to nanoparticles produced a 5.34-fold increase in the uptake of gentamicin by the macrophages in 30-min incubation and 26.74-, 8.03- and 7.36-fold increase in uptake by the hepatocytes in 1-, 12- and 24-h incubation. The stabilizers used in the preparation of nanoparticles, particle size, surfactant coating and the gentamicin concentration were each found to have an effect on the uptake of the nanoparticles by both types of cells.

Inhibitory effect and action mechanism of sesquiterpenes from Zedoariae Rhizoma on D-galactosamine/lipopolysaccharide-induced liver injury.

Hepatoprotective sesquiterpenes were isolated from the aqueous acetone extract of Zedoariae Rhizoma, the rhizome of Curcuma zedoaria ROSCOE (Zingiberaceae). Principal sesquiterpenes, furanodiene, germacrone, curdione, neocurdione, curcumenol, isocurcumenol, aerugidiol, zedoarondiol, and curcumenone and curcumin were found to show potent protective effect on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver injury in mice. Plausible action mechanisms for their hepatoprotective activity were clarified on the basis of the inhibitory effect on D-GalN-induced cytotoxicity in primary cultured rat hepatocytes, LPS-induced NO production in cultured mouse peritoneal macrophages, and D-GalN/tumor necrosis factor-alpha (TNF-alpha)-induced liver injury in mice.

Medicinal foodstuffs. XIV. On the bioactive constituents of moroheiya. (2): New fatty acids, corchorifatty acids A, B, C, D, E, and F, from the leaves of Corchorus olitorius L. (Tiliaceae): structures and inhibitory effect on NO production in mouse peritoneal macrophages.

Following the characterization of the glycosidic constituents in a medical foodstuff "moroheiya," the leaves of Corchorus olitorius L., four higher fatty acids with a trienone function, corchorifatty acids, A, B, C, and D, an undecanoic acid, corchorifatty acid E, and a trihydroxyfatty acid, corchorifatty acid F, were isolated from the less polar fraction of "moroheiya". The structures and optical purity of corchorifatty acids were determined on the basis of chemical and physicochemical evidence. Corchorifatty acids A, B, and C showed an inhibitory effect on lipopolysaccharide-induced NO production in cultured mouse peritoneal macrophages.

Inhibition of LPS-induced TNF-α production by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

The purpose of this study was to examine whether rCGRP has effects on TNF-α produced by mouse resident peritoneal macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5 × 10 5 cells per well and allowed to adhere for 2 hr. Pretreatment with rCGRP (10 nM − 1 μM) for 24 hr, the macrophages were cultured with LPS 1 μg/ml for another 24 h. The medium was harvested for measuring TNF-α by ELISA kits. The results showed that rCGRP had no direct effects on TNF-α production, but it inhibited LPS-induced TNF-α production in a concentration-dependent manner. When rCGRP was at a concentration of 1 μM, the LPS-induced TNF-α production was inhibited by 39%. The effect of rCGRP was reversed by hCGRP8-37 (10 μM), an antagonist of CGRP 1 receptor. The LPS-induced TNF-α production from macrophages was also inhibited by forskolin 3 μM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 μM or Rp-cAMPS 100 μM, the inhibitors of cAMP-dependent protein kinase, the effect of rCGRP was abolished. These data suggest that the LPS-induced TNF-α production is inhibited by rCGRP via activation of cAMP responses in mouse resident peritoneal macrophages.

Cholesterol deposition in macrophages: foam cell formation mediated by cholesterol-enriched oxidized low density lipoprotein.

Oxidized low density lipoprotein (LDL) is thought to mediate the transformation of macrophages to cholesterol-rich foam cells. Yet convincing evidence for this process is lacking in vitro. We suggest that oxidized LDL-mediated foam cell formation is not seen in vitro because the cholesteryl ester content of LDL particles (oxidized in the presence of transition metals) is dramatically reduced. Thus, if oxidized LDL could be cholesterol-enriched prior to its addition to macrophages, this lipoprotein would be made more capable of inducing the cellular deposition of cholesteryl esters. When we enriched cupric sulfate-oxidized LDL with cholesterol by incubation of this lipoprotein with unesterified cholesterol/phosphatidylcholine liposomes and added it to mouse peritoneal macrophage cultures, we found that: a) the enrichment of oxidized LDL with cholesterol did not alter the extent of oxidized LDL degradation; b) the cells accumulated massive amounts of cholesteryl ester (148 microg/mg cell protein) and unesterified cholesterol (260 microg/mg cell protein) after 24 h of incubation; and c) Sephacryl S-1000 chromatography of the cholesterol-enriched oxidized LDL verified the formation of large oxidized LDL-unesterified cholesterol/phosphatidylcholine complexes. These results demonstrate that oxidized LDL, when cholesterol-enriched, can mediate the formation of macrophage foam cells in culture

Enhancement of phagocytosis by corticostatin I (CSI) in cultured mouse peritoneal macrophages

Corticostatin I (CSI) is one of the corticostatic peptides which inhibit ACTH-stimulated steroidogenesis. To clarify the function of CSI on the immune system, the effect of CSI on phagocytosis by peritoneal macrophages was examined by means of flow cytofluorometry. In the presence of Ca2+ and Mg 2+, CSI enhanced phagocytosis of latex beads in a dose-dependent manner. Unstimulated phagocytosis in physiological solution consisted of Ca2+ and Mg2+-dependent and -independent phagocytosis. Divalent cations-independent phagocytosis was sensitive to CSI. Present results suggest that the enhancement of phagocytosis by CSI may be one of the mechanisms modulating the immune response regarding infection and inflammation. Present study also showed that one of defensin HNP-1 enhanced phagocytosis.

Urethan anesthesia protects rats against lethal endotoxemia and reduces TNF-α release

Urethan is a commonly used animal anesthetic for nonrecovery laboratory surgery. However, urethan has diverse biological effects that may complicate the interpretation of experimental findings. This study examined the effect of urethan on the response to an intravenous bolus of lipopolysaccharide (LPS; 30 mg/kg) in rats. In instrumented rats, urethan (1.2 gm/kg i.p.) completely prevented the fall in arterial pressure immediately after LPS administration but did not prevent late cardiovascular collapse. In uninstrumented rats, urethan also attenuated indexes of organ injury measured 4 h after LPS administration, including mural bowel hemorrhage, hemoconcentration, hypoglycemia, metabolic acidosis, and lung myeloperoxidase activity, a measure of neutrophil sequestration. The peak increase in tumor necrosis factor-alpha (TNF-alpha) 90 min after LPS administration was reduced 88% by urethan (2,060 +/- 316 vs. 16,934 +/- 847 pg/ml; P < 0.001). In uninstrumented animals, urethan at 1.2 gm/kg reduced the 90% mortality rate of a lethal dose of LPS to 0-10% when given up to 24 h before LPS administration but did not reduce mortality when given 2 h after LPS. Urethan neither directly bound LPS by Limulus assay nor inhibited LPS-stimulated TNF-alpha mRNA expression in cultured mouse peritoneal macrophages, but TNF-alpha mRNA expression was suppressed by serum from a urethan-treated rat. Moreover, rauwolscine, which shares alpha 2-adrenoceptor-blocking activity with urethan, also prevented death from a subsequent 90% lethal dose LPS bolus. We conclude that urethan or its metabolites protect against LPS, in part, by reducing TNF-alpha release and speculate that this may be mediated by alpha 2-adrenoceptors. These actions of urethan make it an undesirable anesthetic agent for in vivo studies of sepsis or LPS.

Induction of cytokine granulocyte-macrophage colony-stimulating factor and chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legionella pneumophila or Salmonella typhimurium attachment requires different ligand-receptor systems.

The attachment of bacteria to macrophages is mediated by different ligands and receptors and induces various intracellular molecular responses. In the present study, induction of cytokines and chemokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 2 (MIP-2), was examined, following bacterial attachment, with regard to the ligand-receptor systems involved. Attachment of Legionella pneumophila or Salmonella typhimurium to cultured mouse peritoneal macrophages increased the steady-state levels of cellular mRNAs for the cytokines interleukin 1beta (IL-1beta), IL-6, and GM-CSF as well as the chemokines MIP-1beta, MIP-2, and KC. However, when macrophages were treated with alpha-methyl-D-mannoside (alphaMM), a competitor of glycopeptide ligands, induction of cytokine mRNAs was inhibited, but the levels of chemokine mRNAs were not. Pretreatment of the bacteria with fresh mouse serum enhanced the level of GM-CSF mRNA but not the level of MIP-2 mRNA. In addition, serum treatment reduced the inhibitory effect of alphaMM on GM-CSF mRNA. These results indicate that bacterial attachment increases the steady-state levels of the cytokine and chemokine mRNAs tested by at least two distinct receptor-ligand systems, namely, one linked to cytokine induction and involving mannose or other sugar residues and the other linked to chemokine induction and relatively alphaMM insensitive. Furthermore, opsonization with serum engages other pathways in the cytokine response which are relatively independent of the alphaMM-sensitive system. Regarding bacterial surface ligands involved in cytokine mRNA induction, evidence is presented that the flagellum may be important in stimulating cytokine GM-CSF message but not chemokine MIP-2 message. Analysis of cytokine GM-CSF and chemokine MIP-2 signaling pathways with protein kinase inhibitors revealed the involvement of calmodulin and myosin light-chain kinase in GM-CSF but not MIP-2 mRNA induction, adding further evidence that several distinct receptor systems are engaged during the process of bacterial attachment and induction of cytokines and chemokines, such as GM-CSF and MIP-2, respectively.

Myelin-stimulated macrophages release neurotrophic factors for adult dorsal root ganglion neurons in culture.

1. Our previous study demonstrated that cultured macrophages release neurotrophic factors spontaneously. In a histological study of Wallerian degeneration, macrophages phagocytosed myelin debris and expressed activated markers. 2. To investigate the role of myelin-stimulated macrophages on neurite regeneration, we prepared conditioned media from cultured mouse peritoneal macrophages which had phagocytosed a myelin fraction. This conditioned media enhanced both neurone survival and neurite regeneration of adult dorsal root ganglia (DRG) neurons compare to conditioned media from macrophage cultures without myelin. 3. The production of the neurotrophic supernatant was dose-dependent on myelin fraction and specific for myelin because supernatants from macrophages incubated with LPS (lipoplysaccharide), MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) or latex beads were not neurotrophic. 4. The neurotrophic factors from myelin-stimulated macrophages were different from spontaneously released macrophage factors as they differed in heat-sensitivity. 5. These results suggest that myelin-stimulated macrophages contribute to axon regeneration after Wallerian degeneration.

Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages.

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. To clarify the function of CGRP on the immune system, the effect of CGRP on phagocytosis by peritoneal macrophages was examined by means of flow cytofluorometry. CGRP enhanced phagocytosis of latex beads in a dose-dependent manner. Because the phosphodiesterase inhibitor 3-isobutyl, 1-methylxanthine (IBMX) enhanced the CGRP-induced enhancement of phagocytosis, the enhancement might be mediated by cAMP. In the presence of mannan, the phagocytosis was suppressed and the CGRP-induced enhancement was also blocked, suggesting that mannose receptors on macrophages were involved in mediating the phagocytosis of latex beads, and CGRP enhanced the mannose receptor-mediated phagocytosis. The present results indicate that CGRP can modulate the function of macrophages in nerve terminals of sensory neurons during the development and maintenance of inflammation.

The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux

The influence of apolipoprotein A-II (apoA-II) molecular structure on the capacity of high density lipoproteins (HDL) to promote cellular cholesterol efflux was investigated in cultured mouse peritoneal macrophages (MPM). Conversion by reduction and carboxamidomethylation of the naturally occurring dimeric apoA-II to its monomeric form in both native or reconstituted HDL did not change apolipoprotein secondary structure and lipoprotein size/composition. All particles containing monomeric apoA-II, i.e., native HDL 3 or reconstituted HDL with or without apoA-I, showed a higher ability to promote cholesterol efflux originating from plasma membrane and intracellular stores, compared to particles containing dimeric apoA-II. These findings indicate that apolipoprotein molecular structure is a major determinant of HDL capacity to promote cholesterol efflux from cells.

Lipolysis-induced partitioning of free fatty acids to lipoproteins: effect on the biological properties of free fatty acids.

Free fatty acids (FFA) released during the lipolysis of triglyceride (TG)-rich lipoproteins in vivo are generally believed to be bound to serum albumin. When hypertriglyceridemic (HTG) sera were lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL), there was an 11- to 18-fold increase in serum FFA levels, and a major portion (> 80%) of the FFA in serum was partitioned to lipoprotein fractions. The greatest portion (33%) of FFA in lipolyzed HTG serum was associated with newly formed flocculent remnants that banded just below low density lipoproteins (LDL) in the density gradient tube. Very low density lipoprotein (VLDL), LDL, and high density lipoprotein (HDL) fractions in lipolyzed HTG serum contained 18- to 29-times more FFA molecules than those in prelipolysis serum. Analysis of the fatty acyl chain composition of FFA in lipolyzed HTG serum showed that the extent of partitioning of saturated FFA into the lipoprotein fractions relative to that of polyunsaturated FFA was about 4.5- to 11-times greater than that partitioned into the free protein fraction; most (84%) of FFA partitioned into flocculent remnants were saturated fatty acids. In vivo lipolysis of TG-rich lipoproteins in HTG subjects, induced by heparinization, resulted in only a small (2.8-fold) increase in serum FFA and little or no increase in the partitioning of FFA to lipoproteins. However, in vitro incubation of the postheparin serum at 37 degrees C for 90 min resulted in a 2.9- to 6.8-fold increase in the serum FFA level and the partitioning of > 66% of total serum FFA into lipoprotein fractions. Studies of the interaction of various plasma fractions from control and in vitro lipolyzed HTG serum with cultured mouse peritoneal macrophages (MPM) showed that FFA partitioned to lipoprotein fractions were highly cytotoxic to cultured MPM, whereas FFA partitioned to albumin at a 10 x greater concentration were not cytotoxic. The cytotoxic potencies of FFA bound to lipoproteins and albumin were further compared after in vitro incorporation of FFA (oleic acids) into LDL and to albumin. FFA bound to LDL but not to albumin were cytotoxic to cultured MPM; the cytotoxicity of FFA bound to LDL was more closely related to the FFA to LDL-cholesterol molar ratio than to the total FFA concentration in the culture dish. The ability of FFA bound to LDL and albumin to induce foam cell formation was studied in THP-1 monocyte-derived macrophages, which were less susceptible to cytotoxicity produced by FFA bound to LDL than MPM.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of stress proteins in mouse peritoneal macrophages by the antirheumatic agents gold sodium thiomalate and auranofin

Gold sodium thiomalate and auranofin, anti-rheumatic gold-containing compounds, induced some stress proteins in cultured mouse peritoneal macrophages. The enhanced synthesis of two proteins, heme oxygenase (a 34-kDa protein) and a 23-kDa protein, was particularly prominent. The 23-kDa protein induced by the gold compounds was identical to that found in macrophages exposed to oxidative stress and was suggested to have antioxidant activity. Intraperitoneal injection of gold sodium thiomalate and oral administration of auranofin to mice induced enhanced synthesis of these proteins in peritoneal macrophages analyzed ex vivo. These data suggest that increased synthesis of these proteins may have a role in mediating the pharmacologic effect of these agents.