Murine Macrophage-like Cell Line RAW Research Articles

Effect of Ixeris dentata Nakai Extract on Nitric Oxide Production and Prostaglandin E2 Generation in LPS-stimulated RAW264.7 Cells.

Inflammation is the basis of severe acute and chronic diseases. This study investigated the anti-inflammatory property of a crude methanol extract (MeOH-ex) and the solvent fractions of Ixeris dentata Nakai (IDN) in LPS-stimulated murine macrophage-like cell line RAW264.7. Here, we showed that the ethyl acetate fraction (EtOAc-fr) had the most potent inhibitory activity on LPS-induced nitric oxide (NO) production among the tested samples, i.e., IDN MeOH-ex and the three different solvent fractions (chloroform, n-hexane, and EtOAc). We further found that the EtOAc-fr significantly inhibited LPS-induced prostaglandin PGE2 (PGE2) generation in RAW264.7 cells. Furthermore, the treatment with EtOAc-fr effectively suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). These results suggest that the EtOAc-fr of IDN MeOH-ex exhibits an anti-inflammatory activity in vitro by inhibiting LPS-induced NO production and PGE2 generation via suppression of iNOS and COX-2 expression.

Mechanism of macrophage activation induced by polysaccharide from Cordyceps militaris culture broth.

Mushroom-derived polysaccharides have been shown to stimulate immune responses. Our previous report showed that the novel polysaccharide PLCM isolated from the culture broth of Cordyceps militaris could induce nitric oxide production in the murine macrophage-like cell line RAW264.7. In this study, we show that PLCM enhances immunostimulatory activities such as the release of toxic molecules (nitric oxide and reactive oxygen species), secretion of the cytokine tumor necrosis factor (TNF)-α, and phagocytic uptake in RAW264.7 macrophages. In addition, all the specific inhibitors against the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) (SN50, BAY11-7082, PD98059, SP600125 and SB203580) markedly suppressed the nitric oxide production and phagocytic uptake induced by PLCM. Moreover, antibodies specific to the extracellular domain of Toll-like receptor-2, Toll-like receptor-4 or the macrophage receptor Dectin-1 significantly attenuated PLCM-induced secretion of TNF-α. Our results indicate that the C. militaris polysaccharide activates macrophages through the MAPKs and NF-κB signaling pathways via Toll-like receptor 2, Toll-like receptor 4, and Dectin-1.

Anti-inflammatory activity of mycelial extracts from medicinal mushrooms.

Medicinal mushrooms have been essential components of traditional Chinese herbal medicines for thousands of years, and they protect against diverse health-related conditions. The components responsible for their anti-inflammatory activity have yet to be fully studied. This study investigates the anti-inflammatory activity of n-hexane, chloroform, ethyl acetate, and methanol extracts of mycelia in submerged culture from 5 commercially available medicinal mushrooms, namely Cephalosporium sinensis, Cordyceps mortierella, Hericium erinaceus, Ganoderma lucidum, and Armillaria mellea. MTT colorimetric assay was applied to measure the cytotoxic effects of different extracts. Their anti-inflammatory activities were evaluated via inhibition against production of lipopolysaccharide (LPS)-induced nitric oxide (NO) in murine macrophage-like cell line RAW264.7 cells. Of the 20 extracts, n-hexane, chloroform, ethyl acetate, and methanol extracts from C. sinensis, C. mortierella, and G. lucidum; chloroform extracts from H. erinaceus and A. mellea; and ethyl acetate extracts from A. mellea at nontoxic concentrations (<300 μg/mL) dose-dependently inhibited LPS-induced NO production. Among them, the chloroform extract from G. lucidum was the most effective inhibitor, with the lowest half maximal inhibitory concentration (64.09 ± 6.29 μg/mL) of the LPS-induced NO production. These results indicate that extracts from medicinal mushrooms exhibited anti-inflammatory activity that might be attributable to the inhibition of NO generation and can therefore be considered a useful therapeutic and preventive approach to various inflammation-related diseases.

β1‐adrenergic receptor autoantibodies from heart failure patients enhanced TNF‐α secretion in RAW264.7 macrophages in a largely PKA‐dependent fashion

Autoantibodies against the second extracellular loop of β(1) -adrenergic receptor (β(1) -AA) not only contribute to increased susceptibility to heart failure, but also play a causative role in myocardial remodeling through their catecholamine-like effects via binding with the β(1) -adrenergic receptor. The current study was designed to determine whether β(1) -AA isolated from the sera of heart failure patients could cause TNF-α secretion from the murine macrophage-like cell line RAW264.7. Blood samples were collected from 40 patients who had suffered heart failure, as well as from 40 healthy subjects. The titer of β(1) -AA and the level of TNF-α were detected using ELISA. The effect of β(1) -AA on murine macrophage-like cell line RAW264.7 proliferation was detected by CCK-8 kits and CFSE assay. Western blot assay was used to analyze the expression of phospho-VASP. β(1) -AA appeared more frequently in patients with heart failure than in healthy subjects. The β(1) -AA isolated from heart failure patients promoted an increase of TNF-α levels, which could be completely blocked by the selective β(1) -adrenergic receptor antagonist metoprolol and the second extracellular loop of β(1) -adrenergic receptor (β(1) -AR-EC(II) ), but only partially inhibited by PKA inhibitor H89. Furthermore, the β(1) -AA could enhance the proliferation of RAW264.7 cells in vitro. Meanwhile, the expression of phospho-VASP was markedly increased in the presence of β(1) -AA. These results demonstrate for the first time that the β(1) -AA isolated from heart failure patients could bind with β(1) -AR on the surface of RAW264.7 cells, causing the release of TNF-α largely in a PKA-dependent fashion.

Activation of the Classical Complement Pathway by Bacillus anthracis Is the Primary Mechanism for Spore Phagocytosis and Involves the Spore Surface Protein BclA

Interactions between spores of Bacillus anthracis and macrophages are critical for the development of anthrax infections, as spores are thought to use macrophages as vehicles to disseminate in the host. In this study, we report a novel mechanism for phagocytosis of B. anthracis spores. Murine macrophage-like cell line RAW264.7, bone marrow-derived macrophages, and primary peritoneal macrophages from mice were used. The results indicated that activation of the classical complement pathway (CCP) was a primary mechanism for spore phagocytosis. Phagocytosis was significantly reduced in the absence of C1q or C3. C3 fragments were found deposited on the spore surface, and the deposition was dependent on C1q and Ca(2+). C1q recruitment to the spore surface was mediated by the spore surface protein BclA, as recombinant BclA bound directly and specifically to C1q and inhibited C1q binding to spores in a dose-dependent manner. C1q binding to spores lacking BclA (ΔbclA) was also significantly reduced compared with wild-type spores. In addition, deposition of both C3 and C4 as well as phagocytosis of spores were significantly reduced when BclA was absent, but were not reduced in the absence of IgG, suggesting that BclA, but not IgG, is important in these processes. Taken together, these results support a model in which spores actively engage CCP primarily through BclA interaction with C1q, leading to CCP activation and opsonophagocytosis of spores in an IgG-independent manner. These findings are likely to have significant implications on B. anthracis pathogenesis and microbial manipulation of complement.

LPS Induces HMGB1 Relocation and Release by Activating the NF-κB-CBP Signal Transduction Pathway in the Murine Macrophage-Like Cell Line RAW264.7

High mobility group protein B1 (HMGB1) is an important late inflammatory mediator in sepsis. Understanding the mechanisms that regulate HMGB1 release from cells and their downstream signal transduction pathways may lead to the ability to develop anti-HMGB1 therapies to treat inflammation. We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS+ ethylpyruvate (EP) and examined the resulting HMGB1 expression and release. We also studied the expression of related signal transduction factors (NF-κB, p38 MAPK, and CBP). Gene expression of HMGB1 mRNA in RAW264.7 cell showed no significant change at 0-18 h after stimulation with LPS, but increased significantly at 24, 36, and 48 h. HMGB1 mRNA expression in the LPS+EP group was significantly lower than in LPS alone. HMGB1 was distributed mainly in the nucleus; the cytoplasmic level was low before LPS stimulation. After stimulation with LPS, cytoplasmic HMGB1 increased gradually and plateaued at a high level at 12-48 h. Nuclear HMGB1 decreased gradually at 12-24 h, then increased, maintaining a comparatively high level at 36-48 h. EP prevented this pattern significantly. LPS induced p38 MAPK activation and NF-κB signal pathways first, followed by CBP activation. Activated CBP acetylated HMGB1 was stored in a crino-lysosome and secreted activated NF-κB resulted in increased transcription and synthesis of HMGB1, but the expression of up-regulated HMGB1 mRNA was delayed. Extracellular HMGB1 originated from early synthetic reserves present in the nucleus. New HMGB1 protein was synthesized in the nucleus and transferred into the cytoplasm, causing an increase in HMGB1 in the nucleus and cytoplasm. EP inhibits HMGB1 mRNA up-regulation and release from LPS- stimulated macrophages. The molecular function of EP is to attenuate the activation p38 MAPK, NF-κB, and CBP signaling pathways.

Triterpenoid saponins from a cytotoxic root extract of Sideroxylonfoetidissimum subsp. gaumeri

Evaluation of the cytotoxicity of an ethanolic root extract of Sideroxylon foetidissimum subsp. gaumeri (Sapotaceae) revealed activity against the murine macrophage-like cell line RAW 264.7. Systematic bioassay-guided fractionation of this extract gave an active saponin-containing fraction from which four saponins were isolated. Use of 1D ( 1H, 13C, DEPT135) and 2D (COSY, TOCSY, HSQC, and HMBC) NMR, mass spectrometry and sugar analysis gave their structures as 3- O-(β- d-glucopyranosyl-(1 → 6)-β- d-glucopyranosyl)-28- O-(α- l-rhamnopyranosyl-(1 → 3)[β- d-xylopyranosyl-(1 → 4)]-β- d-xylopyranosyl-(1 → 4)-α- l-rhamnopyranosyl-(1 → 2)-α- l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3- O-β- d-glucopyranosyl-28- O-(α- l-rhamnopyranosyl-(1 → 3)[β- d-xylopyranosyl-(1 → 4)]-β- d-xylopyranosyl-(1 → 4)-α- l-rhamnopyranosyl-(1 → 2)-α- l-arabinopyranosyl)-16α-hydroxyprotobassic acid, 3- O-(β- d-glucopyranosyl-(1 → 6)-β- d-glucopyranosyl)-28- O-(α- l-rhamnopyranosyl-(1 → 3)-β- d-xylopyranosyl-(1 → 4)[β- d-apiofuranosyl-(1 → 3)]-α- l-rhamnopyranosyl-(1 → 2)-α- l-arabinopyranosyl)-16α-hydroxyprotobassic acid, and the known compound, 3- O-β- d-glucopyranosyl-28- O-(α- l-rhamnopyranosyl-(1 → 3)[β- d-xylopyranosyl-(1 → 4)]-β- d-xylopyranosyl-(1 → 4)-α- l-rhamnopyranosyl-(1 → 2)-α- l-arabinopyranosyl)-protobassic acid. Two further saponins were obtained from the same fraction, but as a 5:4 mixture comprising 3- O-(β- d-glucopyranosyl)-28- O-(α- l-rhamnopyranosyl-(1 → 3)-β- d-xylopyranosyl-(1 → 4)[β- d-apiofuranosyl-(1 → 3)]-α- l-rhamnopyranosyl-(1 → 2)-α- l-arabinopyranosyl)-16α-hydroxyprotobassic acid and 3- O-(β- d-apiofuranosyl-(1 → 3)-β- d-glucopyranosyl)-28- O-(α- l-rhamnopyranosyl-(1 → 3)[β- d-xylopyranosyl-(1 → 4)]-β- d-xylopyranosyl-(1 → 4)-α- l-rhamnopyranosyl-(1 → 2)-α- l-arabinopyranosyl)-16α-hydroxyprotobassic acid, respectively. This showed greater cytotoxicity (IC 50 = 11.9 ± 1.5 μg/ml) towards RAW 264.7 cells than the original extract (IC 50 = 39.5 ± 4.1 μg/ml), and the saponin-containing fraction derived from it (IC 50 = 33.7 ± 6.2 μg/ml).

Immunostimulatory Characteristics Induced by Linear Polyethyleneimine–Plasmid DNA Complexes in Cultured Macrophages

Intravenously injected plasmid DNA (pDNA) complexed with cationic liposome (lipoplexes) caused NF-kappaB-mediated cytokine production from macrophages, induced by CpG sequence in the pDNA. We have reported that cytokine production caused by linear polyethyleneimine (PEI)-pDNA complexes (PEI polyplexes) was much lower than that caused by lipoplexes (Kawakami, S., Ito, Y., Charoensit, P., Yamashita, F., and Hashida, M. [2006]. J. Pharmacol. Exp. Ther. 317, 1382-1390). As Toll-like receptor-9 recognizing CpG sequence is expressed in the endosomal compartment, we hypothesized that the buffering capacity of PEI enhanced the escape of PEI polyplexes from endosomes, and that consequently cytokine production was decreased. In this study, the mechanism of lower cytokine production induced by PEI polyplexes, compared with lipoplexes, was investigated using the murine macrophage-like cell line RAW 264.7. Although transfection efficacy and cellular association were similar for PEI polyplexes and lipoplexes, tumor necrosis factor-alpha and interleukin-6 production and NF-kappaB activation caused by polyplexes were significantly lower than with lipoplexes. As for intracellular distribution, PEI polyplexes spread into cytosol whereas lipoplexes accumulated in vesicles, suggesting enhancement of escape from endosomes by PEI. Bafilomycin A1, an inhibitor of early endosomes, enhanced cytokine production and NF-kappaB activation by PEI polyplexes but not by lipoplexes; however, chloroquine, an inhibitor of late endosomes, inhibited PEI polyplex-induced cytokine production and NF-kappaB activation, suggesting that the buffering effect of PEI on early endosomes decreases NF-kappaB-mediated cytokine production. In conclusion, we demonstrate that cytokine production and NF-kappaB activation induced by PEI polyplexes are significantly lower than with lipoplexes in cultured macrophages. The significantly low cytokine response of PEI polyplexes may be due to effective transition of PEI polyplexes from endosomes to cytosol.

MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis

Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immune responses to microbial infection. Recent studies have shown that Toll-like receptors (TLRs) play an important role in promoting the clearance of bacteria by up-regulating the phagocytic activity of macrophages. However, information regarding the signaling mechanism of TLR-mediated phagocytosis is still limited. Here, we provide evidence that the stimulation of TLR4 with LPS leads to activation of multiple signaling pathways including MAP kinases, phosphatidylinositide 3-kinase (PI3K), and small GTPases in the murine macrophage-like cell line RAW264.7. Specific inhibition of Cdc42/Rac or p38 MAP kinase, but not PI3K, reduced TLR4-induced phagocytosis of bacteria. Moreover, we have found that either inhibition of actin polymerization by cytochalasin D or the knockdown of actin by RNAi markedly reduced the activation of Cdc42 and Rac by LPS. TLR4-induced activation of Cdc42 and Rac appears to be independent of MyD88. Taken together, our results described a novel actin-Cdc42/Rac pathway through which TLRs can specifically provoke phagocytosis.

Characterization of SodC, a Periplasmic Superoxide Dismutase from Burkholderia cenocepacia

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.

TGF‐β1 suppresses IFN‐γ‐induced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation

TGF-beta1 is a well-known immunosuppressive cytokine; however, little is known of the effect of TGF-beta1 on antigen-presenting cells (APCs). In this report, we investigated the molecular mechanisms of the suppressive effects of TGF-beta1 on APCs including dendritic cells and macrophages. Although TGF-beta1 did not greatly affect the activation of APCs, as assessed by the induction of IL-12 or the upregulation of CD40 in response to LPS, it strongly inhibited IFN-gamma-induced nitric oxide (NO) production from macrophages and dendritic cells. Using murine macrophage-like cell line RAW 264.7, we demonstrated that TGF-beta1 not only reduced the inducible NO synthase (iNOS) protein stability but also suppressed the iNOS gene transcription. We also found that TGF-beta1 directly inhibited IFN-gamma-induced STAT1 activation by reducing STAT1 tyrosine phosphorylation. The IFN-gamma Type I receptor (IFNGR1) was found to be associated with the TGF-beta1 Type I receptor (TGF-betaRI) and was phosphorylated by the TGF-betaRI. Reduced activation of STAT1 by TGF-beta1 was abrogated by the mutation in the IFNGR1 in which the serine residues of potential sites of phosphorylation by TGF-betaRI were replaced by alanine residues. Thus, multiple mechanisms are present for the TGF-beta1-mediated reduction of iNOS production, and we propose a novel mechanism for regulating inflammatory cytokine by an anti-inflammatory cytokine, TGF-beta1; i.e. suppression of IFN-gamma-induced STAT1 activation by an association of the IFNGR1 with the TGF-betaRI.

TGF-β1 enhances degradation of IFN-γ-induced iNOS protein via proteasomes in RAW 264.7 cells

TGF-beta1 is a well-known immunosuppressive cytokine that inhibits inducible nitric oxide synthase (iNOS) gene expression in various cells including macrophages. In this study, we investigated the suppressive mechanisms of TGF-beta1 on IFN-gamma-induced iNOS gene expression using the murine macrophage-like cell line RAW 264.7. TGF-beta1 decreased iNOS protein amount through enhanced degradation, although TGF-beta1 did not affect IFN-gamma-induced iNOS mRNA level or stability. In addition, the enhancement of iNOS protein degradation by TGF-beta1 treatment was almost completely blocked by MG132, a proteasome inhibitor. Furthermore, TGF-beta1 enhanced the trypsin-like activity of proteasomes in the presence of IFN-gamma, although did not enhance the peptidylglutamyl-peptide hydrolyzing and chymotrypsin-like activities of proteasomes. The level of ubiquitinated iNOS protein was not significantly altered by IFN-gamma or IFN-gamma plus TGF-beta1 treatment. Because MG132 inhibited iNOS protein degradation and IFN-gamma plus TGF-beta1 treatment increased the trypsin-like activity of proteasomes, we hypothesized that TGF-beta1 might enhance iNOS protein degradation via the ubiquitin-proteasome pathway in the presence of IFN-gamma. We propose that these mechanisms of TGF-beta1 in the posttranslational regulation of iNOS gene expression may contribute to suppression of excess nitric oxide during inflammatory processes.

Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-α in macrophage cell line and rat Kupffer cells

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-α from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-α release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-α production from RAW cells by 38–63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-α release by 14–22% and 13–22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-α production (by 24–35%). Moreover, the upregulated TNF-α mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-α production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-α production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-α mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-α expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-α may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.

Accumulation of glutathione disulfide mediates NF-kappaB activation during immune stimulation with CpG DNA.

Innate immune cells recognize pathogens by detecting molecular patterns that are distinct from those of the host. One such pattern is unmethylated CpG dinucleotides, which are common in bacterial DNA but not in vertebrate genomes. Macrophages respond to such CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) by inducing NF-kappaB and secreting proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), but the mechanisms regulating this have been unclear. CpG ODN-stimulated cells produce reactive oxygen species (ROS) and have a decreased ratio of intracellular glutathione/glutathione disulfide (GSH/GSSG), indicating a shift to a more oxidized intracellular redox state. To determine whether this may play a role in mediating the CpG-induced macrophage activation, the GSH/GSSG redox state was manipulated in the murine macrophagelike cell line RAW264.7. Treatment of cells with BCNU to inhibit glutathione reductase (GR) enhanced the CpG-induced intracellular oxidation and decreased the GSH/GSSG, with increased activation of NF-kappaB and a doubling in the CpG-induced production of IL-6 and TNF-alpha. Experimental manipulation of the intracellular GSSG concentration during inhibition of cellular prooxidant production demonstrated that increased intracellular GSSG is a primary signal that is directly or indirectly required for CpG-induced NF-kappaB activation but is not in itself sufficient to trigger this in the absence of CpG ODN. These data suggest the existence of a second CpG-induced intracellular signal, independent of GSSG, mediating the activation of innate immunity by bacterial DNA.

Proanthocyanidins and related compounds: antileishmanial activity and modulatory effects on nitric oxide and tumor necrosis factor-alpha-release in the murine macrophage-like cell line RAW 264.7.

A series of 17 proanthocyanidins and structurally related compounds was tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Most of the polyphenols significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.8-10.6 nM) when compared with the antileishmanial drug Pentostam (EC50 10.6 nM), but all were inactive against the extracellular form (EC50 7.8 to >86 nM). Noteworthy is that all compounds exhibited only moderate or no cytotoxicity against the murine host cells (EC50 7.8 to >56 nM; >25 microg/ml). These polyphenols were further evaluated for immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumor necrosis factor-alpha (TNF) and interferon (IFN)-like properties using several functional assays. The results showed that all compounds induced murine RAW 264.7 cells only moderately to release NO (7-26 microM) relative to the reference stimulus IFN-gamma/LPS (119 microM). The TNF-inducing potential of the polyphenols producing 50% lysis in murine L929 cells ranged from absent to 138 U/ml at the host cell subtoxic concentration of 50 microg/ml. The highest TNF-inducing activity was associated with those flavan-3-ols with galloyl groups (98-127 U/ml). For proanthocyanidins, it appeared that an increase in the flavanyl chain length did not enhance the induction of TNF-release (32-86 U/ml and below detection limits for oligomers and polymers, respectively). With interferon-like activities, phylloflavan and a prodelphinidin polymer showed appreciable cytoprotective effects, as reflected by the inhibition of the cytopathic effect of encephalomyocarditis virus on L929 fibroblast cells (38 and 36 U/ml, respectively). All remaining compounds displayed only negligible or moderate protective effects at subtoxic concentrations up to 25 microg/ml (<5 to 12 U/ml). These results indicate that proanthocyanidins and related compounds have favorable antileishmanial activity in vitro and might be considered as beneficial immunological response modifiers provided there are no bioavailability problems.

P-12 - Inhibitory effect of morphine on cell death induced by LPS and interferon-γ in murine macrophage-like cell line RAW264

P-12 - Inhibitory effect of morphine on cell death induced by LPS and interferon-γ in murine macrophage-like cell line RAW264

Inhibitory effect of dehydroevodiamine and evodiamine on nitric oxide production in cultured murine macrophages.

Possible antiinflammatory effects of dehydroevodiamine (1) and evodiamine (2) were examined by assessing their effects on NO production in the murine macrophage-like cell line RAW 264.7. The results indicated that both 1 and 2 inhibited the IFN-gamma/LPS-stimulated NO production in a concentration-dependent manner. However, 1 appeared to inhibit NO production by interfering not only with the priming signal initiated by IFN-gamma but also with iNOS protein synthesis, while 2 affected the former only.

Taxol induces tyrosine phosphorylation of Shc and its association with Grb2 in murine RAW 264.7 cells.

Taxol, a natural product with significant anti-tumor activity, stabilizes microtubules and arrests cells in the G2/M phase of the cell cycle. It has been reported that taxol has additional effects in cells, including an increase in tyrosine phosphorylation of proteins and activation of MAP kinase. We investigated a possible effect of taxol on tyrosine phosphorylation of Shc and on formation of the Shc/Grb-2 complex in the murine macrophage-like cell line RAW 264.7. Shc, an SH2 domain containing adaptor protein, was immunoprecipitated from lysates of taxol-treated cells with anti-phosphotyrosine antibody and its identity determined by Western blotting with anti-Shc antibody. Non-denatured Shc containing protein complexes were immunoprecipitated with anti-Shc antibody, and analysis with an anti-Grb2 antibody revealed the presence of the 24-kDa Grb2 protein. Taxol also activated Raf-1 kinase and ERK1/ERK2 MAP kinases in these cells. These results demonstrate that taxol affects tyrosine phosphorylation of Shc and this may result in the activation of the Raf-1/MAPK cascade.

Macrophage-like RAW 264 cell line and time-resolved fluoroimmunoassay (TRFIA) as tools in screening drug effects on cytokine secretion

A screening system was set up to study the effects of drugs on cytokine secretion by macrophages in vitro. The system is based on the murine macrophage-like cell line RAW 264, which can be activated with lipopolysaccharide (LPS) to produce cytokines. The responsiveness of the RAW 264 cells was outlined by challenging them with different concentrations of LPS for 6 or 24 h. Substantial time- and dose-dependent increases were recorded for interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα) secretions. A general procedure was established to construct time-resolved fluoroimmunoassays (TRFIA) from commercial immunochemicals produced originally for enzyme immunoassays. Practical measuring ranges of the non-competitive assays were 100 pg/ml-10 ng/ml for 1L-1β and TNFα and 10 pg/ml-5 ng/ml for IL-6 and IL-5. The interleukin-5 (IL-5) assay was set up for unrelated human studies, but the others were used in the characterization of RAW 264 cytokine secretion. An immunosuppressive effect with dexamethasone phosphate could be achieved and recorded in the model system. Thus, the system offers a simple and easy-to-use model for screening immunomodulatory effects of drugs on the cytokine secretion of macrophages.

CD14-mediated translocation of nuclear factor-kappa B induced by lipopolysaccharide does not require tyrosine kinase activity.

During the course of serious bacterial infections, lipopolysaccharide (LPS) is believed to interact with macrophage receptors, resulting in the generation of inflammatory mediators and systemic symptoms including hemodynamic instability and shock. CD14, a glycosylphosphatidylinositol-linked antigen, functions as an LPS signaling receptor. A critical issue concerns the mechanism by which CD14, which has no transmembrane domain, transduces its signal following LPS binding. Recently, investigators have hypothesized that CD14-mediated signaling is effected through a receptor-associated tyrosine kinase (TK), suggesting a multicomponent receptor model of LPS signaling. Wild-type Chinese hamster ovary (CHO)-K1 cells can be activated by endotoxin to release arachidonate following transfection with human CD14 (CHO/CD14). Nuclear translocation of cytosolic NF-kappa B is correlated with a number of LPS-inducible responses. We sought to determine if this pathway were present in CHO/CD14 cells and to elucidate the relationship of NF-kappa B activation to the CD14 receptor system. LPS-stimulated translocation of NF-kappa B in CHO/CD14 cells resembled the same response in the murine macrophage-like cell line RAW 264.7. Protein synthesis inhibitors and corticosteroids, which suppress arachidonate release and the synthesis of proinflammatory cytokines, had no effect on translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, demonstrating that NF-kappa B translocation is an early event. Although TK activity was consistently observed by immunoblotting extracts from activated RAW 264.7 cells, LPS-induced phosphotyrosine residues were not observed from similarly treated CHO/CD14 cells. Furthermore, the TK inhibitors herbimycin A and genistein failed to inhibit translocation of NF-kappa B in CHO/CD14 or RAW 264.7 cells, although both of these agents inhibited LPS-induced TK activity in RAW 264.7 cells. These results imply that TK activity is not obligatory for CD14-mediated signal transduction to occur in response to LPS.