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.     

Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S-nitrosothiol-induced inhibition of glutathione reductase

We investigated whether endogenously or exogenously produced nitric oxide (NO) can inhibit cellular glutathione reductase (GR) via the formation of S-nitrosothiols to decrease cellular glutathione (GSH) and increase oxidative stress in RAW 264.7 cells. The specificity of this inhibition was demonstrated by addition of a NO-synthase inhibitor, and met- or oxyhemoglobin. Using isolated GR we found that only certain NO donors inhibit this enzyme via S-nitrosothiol. Furthermore, we found that cellular GSH decrease is paralleled by an increase of superoxide anion production. Our results show that the GR enzyme is a potential target of S-nitrosothiols to decrease cellular GSH levels and to induce oxidative stress in macrophages.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

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.     

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.     

Nucleotide-binding oligomerization domain-containing protein 2 regulates suppressor of cytokine signaling 3 expression in Burkholderia pseudomallei-infected mouse macrophage cell line RAW 264.7

Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular Gram-negative bacterium that can survive and multiply inside the macrophages. Toll-like receptors are one class of pattern recognition receptors (PRRs) that have been documented to play significant role in B. pseudomallei infection. In the present study, we investigated a potential role of nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1 and NOD2), cytoplasmic pattern recognition receptors, in B. pseudomallei-infected mouse macrophage cell line RAW 264.7. Both live and heat-killed B. pseudomallei were able to up-regulate NOD1 and NOD2 expression in a time-dependent manner. Marked reduction of a negative regulator, suppressor of cytokine signaling 3 (SOCS3), expression was observed only in B. pseudomallei-infected NOD2-depleted macrophages and not in NOD1-depleted macrophages. The decrease in SOCS3 expression also led to an increase in IFN-γ responsiveness as judged by an enhanced STAT-1 phosphorylation on tyrosine 701 in the B. pseudomallei-infected macrophages. Together, these results suggested that, in addition to using other PRRs to evade macrophage defense, B. pseudomallei may also use NOD2 to regulate a negative regulator like SOCS3.     

Falcarindiol inhibits LPS-induced inflammation via attenuating MAPK and JAK-STAT signaling pathways in murine macrophage RAW 264.7 cells

The purpose of the study was to investigate the mechanism of total flavone of Desmodium styracifolium (TFDS) in regulating the formation of urinary calculi. Protein levels of KIM-1, LC3-II, p-p38 were measured by Western blot. The effect of different COM concentrations, different TFDS concentrations, SB203580 (specific inhibitor of p38/MAPK), and overexpression of KIM-1 on cell viability were detected by WST-1 assay. The apoptotic cells and FITC positive cells were detected by flow cytometry. HK-2 cell viability decreased with the increase of COM concentration, and the protein levels of KIM-1, LC3-II, and p-p38 increased with the time. Blocking the p38/MAPK pathway or co-cultured with TFDS inhibited the effects of COM on apoptosis and autophagy of HK-2 cells. In addition, blocking the p38/MAPK pathway inhibited the expression of KIM-1. In COM-induced cells, after treated with SB203580, overexpression of KIM-1 could reverse the protection effect of SB203580 on COM-induced cell damage and the inhibition of SB203580 on COM-induced excessive autophagy, suggesting p38/MAPK regulated KIM-1 to regulate COM-induced cell apoptosis and autophagy. Finally, we proved that TFDS inhibited p38/MAPK pathway. And the protection effect of COM-induced cell injury increased with the increase of TFDS concentration, and the adhesion between COM and cells decreased with the increase of TFDS concentration. With the increase of the concentration of TFDS, p38/MAPK pathway was gradually inhibited, and KIM-1 and autophagy related proteins were decreased. TFDS inhibited HK-2 cell apoptosis and autophagy by regulating KIM-1 via p38/MAPK pathway.     

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.     

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     

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。     

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.     

Parthenolide inhibits TRIF-dependent signaling pathway of toll-like receptors in RAW264.7 macrophages

Toll-like receptors (TLRs) play an important role in induction of innate immune responses for host defense against invading microbial pathogens. Microbial component engagement of TLRs can trigger the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent downstream signaling pathways. Parthenolide, an active ingredient of feverfew (Tanacetum parthenium), has been used for centuries to treat many chronic diseases. Parthenolide inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-κB kinase. However, it is not known whether parthenolide inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of parthenolide, its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly [I:C]) was examined. Parthenolide inhibited nuclear factor-κB and interferon regulatory factor 3 activation induced by LPS or poly[I:C], and the LPS-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10. These results suggest that parthenolide can modulate TRIF-dependent signaling pathways of TLRs, and may be the basis of effective therapeutics for chronic inflammatory diseases.     

Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway

The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium (⁸⁶Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at 60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens. Src kinase; receptors     

On the importance of plasmalogen status in stimulated arachidonic acid release in the macrophage cell line RAW 264.7

We examined the dependence of stimulated arachidonic acid release on plasmalogens using the murine, macrophage cell line 264.7 and two plasmalogen-deficient variants, RAW.12 and RAW.108. All three strains responded to unopsinized zymosan to release arachidonic acid from phospholipid stores. Arachidonic acid release appeared to be dependent on calcium-independent phospholipase A(2) activation (iPLA(2)); bromoenol lactone, a specific inhibitor of calcium-independent iPLA(2), blocked arachidonic acid release with an IC(50) of approximately 2 x 10(-7)M. Propanolol, an inhibitor of phosphatidate phosphatase, and RHC-80267, an inhibitor of diglyceride lipase, had no effect on arachidonic acid release. Arachidonic acid release in the variants displayed similar magnitude, kinetics of response and sensitivity to the inhibitors when compared to the parent strain. Arachidonic acid was released from all major phospholipid head group classes with the exception of sphingomyelin. In wild-type cells, arachidonic acid released from the ethanolamine phospholipids was primarily from the plasmalogen form. However, in the plasmalogen-deficient cells release from the diacyl species, phosphatidylethanolamine, was increased to compensate. Restoration of plasmalogens by supplementation of the growth medium with the bypass compounds sn-1-hexadecylglycerol and sn-1-alkenylglycerol had no effect on arachidonic acid release. In summary, plasmalogen status appears to have no influence on the zymosan A stimulated release of arachidonic acid from the RAW 264.7 cell line.     

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.