β-葡聚糖对小鼠单核巨噬细胞系RAW264.7的免疫刺激作用

目的研究β-葡聚糖的应用对Balb/c小鼠巨噬细胞株RAW264.7的刺激作用。方法将不同浓度（0～150μg/ml）的β-葡聚糖与Balb/c小鼠来源的巨噬细胞株RAW264.7作用1～7d后,以四甲基偶氮唑蓝（MTT）法检测细胞的增殖情况并绘制细胞生长曲线。结果β-葡聚糖在50～75μg/ml的浓度范围内能够明显地刺激细胞发生增殖。结论适当剂量的β-葡聚糖作用足够时间,RAW264.7细胞系可以发生显著的生长促进效应。     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Evidence for lipopolysaccharideinduced differentiation of RAW264.7 murine macrophage cell line into dendritic like cells

Effect of lipopolysaccharide (LPS) on RAW264.7 macrophage cell line was studied. LPS-treated RAW264.7 cells increased in cell size and acquired distinct dendritic morphology. At the optimal dose of LPS (1 mg/ml), almost 70% RAW264.7 cells acquired dendritic morphology. Flow cytometric studies indicate that the cell surface markers known to be expressed on dendritic cells and involved in antigen presentation and T cell activation (B 7.1, B 7.2, CD40, MHC class II antigens and CD1d) were also markedly upregulated on LPS-treated RAW 264.7 cells. Our results suggest the possibility that LPS by itself could constitute a sufficient signal for differentiation of macrophages into DC-like cells.     

CEL-I, an invertebrate N-acetylgalactosamine-specific C-type lectin, induces TNF-alpha and G-CSF production by mouse macrophage cell line RAW264.7 cells

Our previous studies demonstrated that CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea) showed potent cytotoxicity to several cell lines such as HeLa, MDCK and XC cells. In this study, we found that CEL-I induced increased secretion of tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony stimulation factor (G-CSF) by mouse macrophage cell line RAW264.7 cells in a dose-dependent manner, whereas this cell line was highly resistant to CEL-I cytotoxicity. The cytokine-inducing activity of CEL-I was stronger than that of phytohaemagglutinin (PHA-L). A binding study using FITC-labelled CEL-I (F-CEL-I) indicated that the amount of bound F-CEL-I on RAW264.7 cells was greater than that of F-PHA-L, suggesting that the greater activity of CEL-I to induce cytokine secretion by RAW264.7 cells is partly due to the higher binding ability. Since the cell binding and cytokine-inducing activity of CEL-I were partly but significantly inhibited by the specific sugar (GalNAc), it is considered that the binding of CEL-I to cell-surface-specific saccharide moieties, which may be recognized by CEL-I with higher affinity than GalNAc, is essential for the induction of cytokine secretion. The secretion of TNF-alpha and G-CSF from CEL-I-treated RAW264.7 cells were almost completely prevented by brefeldin A (BFA), whereas increase in mRNA levels of these cytokines were not affected by BFA. Bio-Plex beads assay suggested that temporal increase in phosphorylation of extracellular-regulated kinase (ERK), c-jun NH(2)-terminal kinase (JNK) and p38 MAP kinase occurred at relatively early time following CEL-I treatment. Furthermore, the secretion of TNF-alpha and G-CSF were inhibited by specific inhibitors for these MAP kinases. These results suggest that the intracellular signal transduction through the activation of MAP kinase system is involved in CEL-I-induced cytokine secretion.     

Phosphatidic acid positively regulates LPS-induced differentiation of RAW264.7 murine macrophage cell line into dendritic-like cells

Phosphatidic acid (PA) is an important second messenger produced by the activation of numerous cell surface receptors. Recent data have suggested that PA regulates multiple cellular processes. In this study, we found that PA positively regulates the lipopolysaccharide (LPS)-induced differentiation of RAW264.7 murine macrophage cells into dendritic-like cells. Co-treatment of PA with LPS further increased dendritic cell surface marker expressions (CD80, CD86, CD40, MHC class I, and class II antigens) and reduced the phagocytic activity of LPS-treated cells. Moreover, PA up regulated allostimulatory activity and the secretion of IL-12 in LPS-treated RAW264.7 cells. Taken together, these data indicate that PA might play a role in the LPS-mediated differentiation of macrophage cells into dendritic-like cells.     

Melanocortin receptor signaling in RAW264.7 macrophage cell line

Melanocortin peptides modulate cytokine release and adhesion molecule expression. Here we have investigated the early cell-signaling pathway responsible for the induction of interleukin-10 (IL-10) in RAW264.7 cells. Cell incubation with ACTH(1-39) or MTII (melanotan II) did not alter ERK1/2 and JNK phosphorylation, while p38 phosphorylation and intracellular cAMP accumulation occurred within minutes. ACTH(1-39) and MTII provoked a time-dependent accumulation of IL-10 that was abrogated by the PKA inhibitor H-89 and only partially blocked by the p38 MAPK inhibitor SB203580. Thus, in RAW264.7 cells, IL-10 induction by the melanocortins is via the PKA pathway, and this mechanism could contribute to their anti-inflammatory profile.     

Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material

Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with l-N⁶-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A₂ (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. l-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA₂ but not PI-3K-dependent fashion.     

Inhibitory effect of resveratrol dimerized derivatives on nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells

Four types of resveratrol dimerized analogues were synthesized and evaluated in vitro on LPS-induced NO production in RAW 264.7 cells. The results showed that several compounds, especially those containing 1,2-diphenyl-2,3-dihydro-1H-indene core (type I), exhibited good inhibitory activities. Among 25 analogues, 12b showed a significant inhibitory activity (49% NO production at 10 μM, IC₅₀ = 3.38 μM). Further study revealed that compound 12b could suppress LPS-induced iNOS expression, NO production, and IL-1β release in a concentration-dependently manner. The mechanism of action (MOA) involved for its anti-inflammatory responses was through signaling pathways of p38 MAPK and JNK1/2, but not ERK1/2.     

Pyrrolidinone diterpenoid from Isodon excisus and inhibition of nitric oxide production in lipopolysaccharide-induced macrophage RAW264.7 cells

A new pyrrolidinone diterpenoid, excisusin F (1), was isolated from the aerial parts of Isodon excisus (Lamiaceae), together with four known compounds, and their structures were determined mainly by NMR (1D and 2D) and mass spectrometry. Excisusin F (1) and inflexarabdonin E (3) showed potent inhibitory effects of LPS-induced nitric oxide production in RAW264.7 cells with the IC₅₀ value of 10.4 and 3.8 μM, respectively.     

Inhibitory effect of DCDC on lipopolysaccharide-induced nitric oxide synthesis in RAW 264.7 cells

In the present study we have examined the effect of DCDC (2',5'-dihydroxy-4-chloro-dihydrochalcone) on lipopolysaccharide (LPS)-induced responses in murine macrophage cell line RAW 264.7. Exposure of LPS-stimulated cells to DCDC inhibited the nitrite accumulation in culture medium. DCDC also concentration-dependently inhibited LPS-stimulated increase of iNOS expression; however, it had little effect on iNOS enzyme activity, suggesting that the inhibitory action to DCDC is mainly due to the inhibition on iNOS expression rather than iNOS enzyme activity. DCDC significantly inhibited LPS-evoked degradation of IkappaB-alpha and the nuclear translocation of NF-kappaB; it also exhibited the activity of scavenging the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). DCDC also inhibited cyclooxygenase-2 activity in RAW 264.7 cells with an IC50 of 3.0 microM; furthermore, it also significantly decreased LPS-induced mortality rate in mice. Taken together, we demonstrate that DCDC exhibits inhibitory effects on nitric oxide production through the inhibition of IkappaB-alpha degradation and NF-kappaB activation, and therefore the suppression of iNOS expression. DCDC also shows the antioxidant activity and COX-2 inhibitory action. Moreover, it improves survival in a murine model of endotoxaemia suggesting that DCDC may be potential in the therapy of septic shock.     

Myo-inositol restores the inflammation-induced down-regulation of taurine transport by the murine macrophage cell line,RAW 264.7

The role of myo-inositol in the regulation of taurine transport in activated murine macrophage cell line, RAW 264.7, was studied. Challenge of RAW 264.7 murine macrophages for 24 hr with phorbol ester 12-myristate 13-acetate (PMA) (10 ng/ml), a PKC activator, resulted in a 62% decrease in taurine transport activity. Among the various monosaccharides (1 mM) tested in the presence of PMA, myo-inositol was most effective in restoring the PMA-induced down-regulation of taurine transport in murine macrophages (82% increase compared to the value for cells treated with PMA Alone, p < 0.01). The protective role of myo-inositol against stress-induced down-regulation of taurine transport by macrophages was further investigated in conditions mimicking bacterial infection, inflammation, and immune-suppressed circumstances. A challenge of murine macrophages with lipopolysaccharide (LPS) (0.1 and 10 microg/ml) resulted in a 60% decrease in taurine transport activity compared to the value for untreated control cells (p < 0.01). When cells were co-treated with myo-inositol (100 nM approximately 10 mM) in the presence of LPS for 24 hrs, taurine transport activity increased in a dose-dependent manner compared to the value for cells treated with LPS only. Taurine transport activity in cells treated with LPS (10 microg/ml) plus interferon-gamma (IFN-gamma) (150 unit/ml) for 24 hrs was 13% of the value for untreated control cells (p < 0.01). Again, this inflammation-induced down-regulation of taurine transport activity was completely antagonized with co-administration of 100 nM or higher levels of myo-inositol in the culture medium. Similarly, myo-inositol effectively restored the taurine transport activity suppressed by cyclosporin A (0.5 and 50 nM) in murine macrophages (p < 0.01). From these results, myo-inositol appears to be a common accelerator of taurine transport by murine macrophages in diverse conditions of down-regulated taurine transport.     

Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.     

5-Aminoimidazole-4-carboxamide riboside suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of phosphatidylinositol 3-kinase/Akt activation in RAW 264.7 murine macrophages

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.     

Angiopoietin-1/Tie2 signaling pathway inhibits lipopolysaccharide-induced activation of RAW264.7 macrophage cells

Angiopoietin-1 (Ang1) is a ligand for the endothelial-specific tyrosine kinase receptor Tie2 and has been shown to play an essential role in embryonic vasculature development. There have been many studies about the anti-inflammatory effects of Ang1, most of which focus on endothelium cells. In the present study, we explore the role of Ang1-Tie2 signaling in the activation of macrophages upon lipopolysaccharide (LPS) stimulation. We found that Tie2 receptor is expressed on macrophages and Ang1 could inhibit LPS-induced activation of macrophages, as evidenced by cell migration and TNF-α production, specifically through Tie2 receptor. We further investigated the mechanism and found that Ang1-Tie2 could block LPS-induced activation of NF-κB which has been shown to be necessary for macrophage activation with LPS treatment. Thus, we described, for the first time, the role of Ang1-Tie2 signaling in macrophage activation and the possible mechanisms in response to immune stimulation.     

Induction of gene expression for nitric oxide synthase by immunomodulating drugs in the RAW264.7 murine macrophage cell line

 We have elucidated the direct effects of PSK (a protein-bound polysaccharide) and OK-432 (a streptococcal preparation), both immunomodulating drugs, on the gene expression for an inducible nitric oxide synthase and on the production of nitric oxide (NO) in the RAW264.7 murine macrophage cell line. As determined by northern blot analysis, both immunomodulating drugs were potent inducers of gene expression for inducible NO synthase when cells were costimulated with interferon-γ (IFNγ). Expression of mRNA for the enzyme occurred in a dose-dependent manner after 3 h, when 10 – 50 μg/ml PSK or 0.001 – 1 KE/ml OK-432 was used. Furthermore, NO was also produced in response to these drugs, as detected by the Griess reagent reaction. The enhancement of NO synthesis was thought to be mediated, in part, through tumor necrosis factor α (TNFα) induction by these agents, since a neutralizing antibody to TNFα significantly suppressed NO production in RAW264.7 cells stimulated with PSK or OK432 in combination with IFNγ. We speculate that NO production may play a role in tumoricidal and microbicidal activities of PSK or OK-432 in vivo. Received: 9 August 1995 / Accepted: 1 April 1996     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.