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.     

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.     

Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA

Bacterial DNA can stimulate the production of cytokines and nitric oxide (NO), while mammalian DNA can block these responses. If mammalian DNA is transfected into macrophages, however, it can stimulate NO production, without inducing IL-12. To define further this activity, signaling pathways induced by transfected calf thymus (CT) DNA were studied. Using RAW264.7 cells as a model, CT DNA in the transfection agent FuGENE 6 activated cells through the NF-kappaB and MAPKs pathways, similar to bacterial DNA and LPS. The role of these pathways was further investigated using specific inhibitors, with studies indicating that NO production is blocked by inhibitors of NF-kappaB and p38 but not other MAPKs. These data indicate that the immune activity of DNA is influenced by context or intracellular location and that, when transfected into cells, mammalian DNA can activate cells through signaling pathways similar to those of bacterial DNA.     

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.     

Oxidative stress reprograms lipopolysaccharide signaling via Src kinase-dependent pathway in RAW 264.7 macrophage cell line

Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-kappa B translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-kappa B translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-kappa B translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-kappa B translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-kappa B translocation in oxidant-treated macrophages, this strategy had no effect on NF-kappa B translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion.     

Uptake of histamine by mouse peritoneal macrophages and a macrophage cell line,RAW264.7

We have previously demonstrated that dietary histamine is accumulated in the spleens of L-histidine decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis. To characterize the clearance system for dietary histamine in mice, we investigated the cell type and mechanism responsible for histamine uptake in the spleens of HDC-deficient mice. Immunohistochemical analyses using an antihistamine antibody indicated that a portion of the CD14⁺ cells in the spleen is involved in histamine storage. Peritoneal macrophages obtained from Balb/c mice and a mouse macrophage cell line, RAW264.7, had potential for histamine uptake, which was characterized by a low affinity and high capacity for histamine. The histamine uptake by RAW264.7 cells was observed at physiological temperature and was potently inhibited by pyrilamine, chlorpromazine, quinidine, and chloroquine, moderately inhibited by N-methylhistamine, dopamine, and serotonin, and not affected by tetraethylammonium and 1-methyl-4-phenylpyridinium. Intracellular histamine was not metabolized in RAW264.7 cells and was released at physiological temperature in the absence of extracellular histamine. These results suggest that histamine uptake by macrophages may be involved in the clearance of histamine in the local histamine-enriched environment. cation transporter; chlorpromazine; pyrilamine; quinidine     

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.     

Accumulation of lysophosphatidylinositol in RAW 264.7 macrophage tumor cells stimulated by lipid A precursors

N2,O3-Diacylglucosamine 1-phosphate (lipid X), a monosaccharide precursor of Escherichia coli lipid A, was used to stimulate RAW 264.7 macrophage tumor cells, and the effects on macrophage phospholipid metabolism were examined. The addition of E. coli lipid X to the medium of cells that had been uniformly labeled with 32Pi resulted in a 4-8-fold increase in the level of lysophosphatidylinositol. This effect was maximal at 5 microM lipid X. Lysophosphatidylinositol levels reached a maximum 45 min after stimulation, followed by a gradual decline to near normal levels within 2 h. The formation of lysophosphatidylinositol was dependent upon extracellular calcium and was almost completely inhibited when cycloheximide was added at the time of stimulation. The addition of the disaccharide lipid A precursor IVA, commercial lipopolysaccharide (1 microgram/ml), phorbol 12-myristate 13-acetate (10(-7) M), or calcium ionophore A23187 (10(-6) M) to these cells resulted in a similar increase in lysophosphatidylinositol levels, but phosphatidic acid was inactive. The stimulation by IVA and phorbol myristate acetate was blocked by cycloheximide, but the stimulation by lipopolysaccharide was only partially blocked. The stimulation by A23187 was unaffected by cycloheximide. The increase in lysophosphatidylinositol levels might be related to the stimulation of arachidonate release and prostaglandin synthesis that is also observed in cells treated with lipid A precursors. The disaccharide precursor, IVA, was at least 100 times more effective than lipid X at stimulating lysophosphatidylinositol formation and prostaglandin release. The relative ability of lipid X and IVA to stimulate these cells correlated well with their effects on other lipopolysaccharide-responsive systems. Macrophage tumor cells also had the ability to inactivate lipid X by dephosphorylating it.     

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.     

Identification of multiple-molecular-weight forms of thymocyte comitogenic activity from the monocyte/macrophage cell line RAW 264.7

The cloned monocyte/macrophage cell line RAW 264.7 was investigated for interleukin 1 (IL-1) production. Of the inducers tested, bacterial lipopolysaccharide was found to be the most effective. The cyclic nucleotide analogs 8-BrcAMP and 8-BrcGMP were also tested, with only 8-BrcGMP being capable of inducing a small amount of IL-1 activity. Gel filtration studies revealed thymocyte mitogenic and comitogenic activity in three molecular-weight peaks: > 70,000, 30,000 to 40,000, and 12,000 to 18,000 Da. The multiple-molecular-weight forms were present when samples were prepared under serum-free conditions and also when samples were prepared and chromatographed in high ionic strength NaCl or under disulfide reducing conditions. Molecular charge heterogeneity was observed when proteins were chromatographed using column chromatofocusing (PBE 94). The intermediate-molecular-weight form eluted from the column over a pH range of 5.0 to 5.4; while the low-molecular-weight form eluted at three separate pH's: ?7.4 (unbound material), 5.2, and 4.8. The low-molecular-weight and intermediate-molecular-weight forms exhibited different dose-response curves when assayed under conditions used by other investigators (1 × 10⁷ cells/ml; phytohemagglutinin, 1 μg/ml), but very similar dose-response curves when assayed under conditions used by our laboratory (2 × 10⁶ cells/ml; concanavalin A, 0.25 μg/ml) in a thymocyte comitogen assay. The possible relationship of these multiple-molecular-weight species of thymocyte comitogenic activity from RAW 264.7 to other biological activities from cloned and noncloned cellular sources is discussed.     

Macropinocytosis contributes to the macrophage foam cell formation in RAW264.7 cells

The key event in the atherosclerosis development is the lipids uptake by macrophage and the formation of foam cell in subendothelial arterial space. Besides the uptake of modified low-density lipoprotein （LDL） by scavenger receptor-mediated endocytosis, macrophages possess constitutive macropinocytosis, which is capable of taking up a large quantity of solute. Macrophage foam cell formation could be induced in RAW264.7 cells by increasing the serum concentration in the culture medium. Foam cell formation induced by serum could be blocked by phosphoinositide 3-kinase inhibitor, LY294002 or wortmannin, which inhibited macropinocytosis but not receptor-mediated endocytosis. Further analysis indicated that macropinocytosis took place at the gangliosides-enriched membrane area. Cholesterol depletion by β-methylcyclodextrin-blocked macropinocytosis without affecting scavenger receptormediated endocytosis of modified LDLs. These results suggested that macropinocytosis might be one of the important mechanisms for lipid uptake in macrophage. And it made significant contribution to the lipid accumulation and foam cell formation.     

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。     

Characterization of TRPM8-like channels activated by the cooling agent icilin in the macrophage cell line RAW 264.7

Icilin is recognized as a chemical agonist of nociceptors and can activate TRPM8 channels. However, whether this agent has any effects on immune cells remains unknown. In this study, the effects of icilin on ion currents were investigated in RAW 264.7 murine macrophage-like cells. Icilin (1–100 μM) increased the amplitude of nonselective (NS) cation current (I _NS) in a concentration-dependent manner with an EC₅₀ value of 8.6 μM. LaCl₃ (100 μM) or capsazepine (30 μM) reversed icilin-induced I _NS; however, neither apamin (200 nM) nor iberiotoxin (200 nM) had any effects on it. In cell-attached configuration, when the electrode was filled with icilin (30 μM), a unique population of NS cation channels were activated with single-channel conductance of 158 pS. With the use of a long-lasting ramp pulse protocol, increasing icilin concentration produced a left shift in the activation curve of NS channels, with no change in the slope factor of the curve. The probability of channel opening enhanced by icilin was increased by either elevated extracellular Ca²⁺ or application of ionomycin (10 μM), while it was reduced by BAPTA-AM (10 μM). Icilin-stimulated activity is associated with an increase in mean open time and a reduction in mean closed time. Under current-clamp conditions, icilin caused membrane depolarization. Therefore, icilin interacts with the TRPM8-like channel to increase I _NS and depolarizes the membrane in these cells.     

Phospholipid sources for adrenic acid mobilization in RAW 264.7 macrophages. Comparison with arachidonic acid

Cells metabolize arachidonic acid (AA) to adrenic acid (AdA) via 2-carbon elongation reactions. Like AA, AdA can be converted into multiple oxygenated metabolites, with important roles in various physiological and pathophysiological processes. However, in contrast to AA, there is virtually no information on how the cells regulate the availability of free AdA for conversion into bioactive products. We have used a comparative lipidomic approach with both gas chromatography and liquid chromatography coupled to mass spectrometry to characterize changes in the levels of AA- and AdA-containing phospholipid species in RAW 264.7 macrophage-like cells. Incubation of the cells with AA results in an extensive conversion to AdA but both fatty acids do not compete with each other for esterification into phospholipids. AdA but not AA, shows preference for incorporation into phospholipids containing stearic acid at the sn-1 position. After stimulation of the cells with zymosan, both AA and AdA are released in large quantities, albeit AA is released to a greater extent. Finally, a variety of phosphatidylcholine and phosphatidylinositol molecular species contribute to AA; however, AdA is liberated exclusively from phosphatidylcholine species. Collectively, these results identify significant differences in the cellular utilization of AA and AdA by the macrophages, suggesting non-redundant biological actions for these two fatty acids.     

Monitoring the polysaccharide quality of Agaricus blazei in submerged culture by examining molecular weight distribution and TNF-alpha release capability of macrophage cell line RAW 264.7

A method for monitoring the biological activity of broth polysaccharides of Agaricus blazei (AB-BP) in a submerged culture is described. The TNF-alpha releasing capability of AB-BP on the macrophage cell line, RAW 264.7, correlated with the molecular weight of AB-BP. The quality of polysaccharide in a submerged culture of Agaricus blazei was indirectly monitored by analyzing the distribution of its molecular weight within 1 h. The harvest time of the maximum polysaccharide production did not coincide with that of the maximum biological characteristics in the batch culture.