Lipid A binding sites in membranes of macrophage tumor cells

Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.     

Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes

Lipopolysaccharide (LPS) endotoxin is implicated as the bacterial product responsible for the clinical syndrome of Gram-negative septicemia. Although the lipid A domain of LPS appears to be responsible for the toxicity of endotoxin, lipid A from the photosynthetic bacterium Rhodobacter sphaeroides (RSLA) and a disaccharide precursor of lipid A from enteric bacteria, termed lipid IVA, have little activity on human cells. Using the human promonomyelocytic cell line THP-1 and human monocytic cells, we now show that both lipid IVA and RSLA are antagonists of LPS. Complete, apparently competitive, inhibition of LPS activity is possible at a 10-100-fold excess of antagonist, as judged by measuring the release of cytokines and prostaglandin E2. Both antagonists prevent monocyte stimulation by endotoxin extracted from a variety of Gram-negative bacteria. Cells pretreated with either inhibitor and subsequently washed still show attenuated responses to LPS. Stimulation of monocytes by whole Gram-negative bacteria is also antagonized in a dose-dependent manner. Lipid X has no inhibitory effect in the same dose range as lipid IVA and RSLA. These findings rule out LPS sequestration as the explanation for the observed antagonism. Neither inhibitor alters monocyte stimulation by phorbol 12-myristate 13-acetate, Staphylococcus aureus, or purified protein derivative, demonstrating specificity for LPS. Although RSLA appears to inhibit LPS when tested with macrophages from both humans and mice, lipid IVA had the unique ability to act as an LPS antagonist with human-derived cells but to exhibit LPS-like effects with murine-derived cells. Like LPS, lipid IVA stimulated the release of both tumor necrosis factor alpha and arachidonic acid from murine-derived RAW 264.7 macrophage tumor cells. The range of concentrations necessary for lipid IVA to induce LPS-like effects in murine cells was similar to that necessary to antagonize the actions of LPS in human monocytes. The agonist activities of lipid IVA were completely inhibitable by RSLA. This unique species-dependent pharmacology observed with lipid IVA may reflect differences between human and murine LPS receptors. RSLA and lipid IVA may be useful in defining the role of LPS in Gram-negative bacterial infections and may prove to be prototypical therapeutic agents for the treatment of Gram-negative septicemia.     

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.     

The soluble variant surface glycoprotein of African trypanosomes is recognized by a macrophage scavenger receptor and induces I kappa B alpha degradation independently of TRAF6-mediated TLR signaling

The GPI residues of soluble variant surface glycoprotein (sVSG) molecules released from the membrane of African trypanosomes during infection induce macrophage activation events. In this study, we demonstrate that the trypanosome sVSG molecule binds to the membrane of murine RAW 264.7 macrophages and activates the NF-kappaB cascade independently of a TLR-mediated interaction. The binding of fluorochrome-labeled sVSG molecules to macrophage membranes was saturable, was inhibited by the scavenger receptor-specific ligand maleylated BSA, and was followed by rapid intracellular uptake of the molecules and subsequent internalization to lysosomal compartments. Inhibition of cellular phagocytic and endocytic uptake processes by cytochalasin B and monodansylcadaverine, respectively, revealed that sVSG internalization was necessary for IkappaBalpha degradation and occurred by an actin-dependent, clathrin-independent process. Activation of RAW 264.7 cells by sVSG following treatment of the cells with the TRAF6 inhibitory peptide DIVK resulted in enhanced NF-kappaB signaling, suggesting both that TRAF6-dependent TLR activation of the pathway alone is not required for signaling and that TLR pathway components may negatively regulate expression of sVSG-induced signaling. These results demonstrate that stimulation of macrophages by sVSG involves a complex process of receptor-mediated binding and uptake steps, leading to both positive and negative signaling events that ultimately regulate cellular activation.     

Lipid profiling reveals arachidonate deficiency in RAW264.7 cells: Structural and functional implications

Glycerophospholipids containing arachidonic acid (20:4) serve as the precursors for an array of biologically active lipid mediators, most of which are produced by macrophages. We have applied mass spectrometry-based lipid profiling technology to evaluate the glycerophospholipid structure and composition of two macrophage populations, resident peritoneal macrophages and RAW264.7 cells, with regard to their potential for 20:4-based lipid mediator biosynthesis. Fatty acid analysis indicated that RAW264.7 cells were deficient in 20:4 (10 +/- 1 mol %) compared to peritoneal macrophages (26 +/- 1 mol %). Mass spectrometry of total glycerophospholipids demonstrated a marked difference in the distribution of lipid species, including reduced levels of 20:4-containing lipids, in RAW264.7 cells compared to peritoneal macrophages. Enrichment of RAW264.7 cells with 20:4 increased the fatty acid to 20 +/- 1 mol %. However, the distribution of the incorporated 20:4 remained different from that of peritoneal macrophages. RAW264.7 cells pretreated with granulocyte-macrophage colony stimulating factor followed by lipopolysaccharide and interferon-gamma mobilized similar quantities of 20:4 and produced similar amounts of prostaglandins as peritoneal macrophages treated with LPS alone. LPS treatment resulted in detectable changes in specific 20:4-containing glycerophospholipids in peritoneal cells, but not in RAW264.7 cells. 20:4-enriched RAW264.7 cells lost 88% of the incorporated fatty acid during the LPS incubation without additional prostaglandin synthesis. These results illustrate that large differences in glycerophospholipid composition may exist, even in closely related cell populations, and demonstrate the importance of interpreting the potential for lipid-mediator biosynthesis in the context of overall glycerophospholipid composition.     

Inhibition of reactive oxygen species down-regulates protein synthesis in RAW 264.7

In order to examine the endoplasmic reticulum responses in macrophages, we stimulate macrophage cell line RAW 264.7 by LPS. We found the phosphorylation of eukaryotic initiation factor eIF2α and the expression of ATF4, GADD34, and GADD153 in RAW 264.7 cells in late time by the relatively large amount of LPS stimulation. Unexpectedly LPS in the presence of ROS inhibitor N-acetyl-l-cysteine rapidly induced phosphorylation of eIF2α and induction of GADD34 expression. We measured intra-cytoplasmic TNFα production in LPS stimulated RAW 264.7 cells. TNFα production induced by LPS stimulation was greatly suppressed by N-acetyl-l-cysteine. This suppression occurred relatively early, which correlated with early eIF2α phosphorylation indicating ER stress mediated shutoff of protein synthesis.     

A role of mitogen and stress-activated protein kinase 1/2 in survival of lipopolysaccharide-stimulated RAW 264.7 macrophages

The effect of inhibition of mitogen and stress-activated protein kinases 1/2 (MSK1/2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was investigated. Pretreatment with Ro 31-8220, an inhibitor of MSK1/2, induced cell death in LPS-stimulated RAW 264.7 cells. In contrast, calphostin C, another inhibitor of protein kinase C, did not cause cell death. Cell death was not mediated by the release of pro-inflammatory mediators from LPS-stimulated RAW 264.7 cells. Cell death was accompanied by DNA fragmentation and annexin V binding, suggesting apoptotic cell death. Further, several caspase inhibitors did not prevent LPS-induced cell death of Ro 31-8220-pretreated RAW 264.7 cells. Nuclear translocation of apoptosis-inducing factor (AIF) was detected in Ro 31-8220-pretreated cells after LPS stimulation. Cell death was due to mitochondrial damage. Ro 31-8220 exclusively inhibited the phosphorylation of cAMP-responsive element binding protein (CREB), a substrate of MSK1/2. RAW 264.7 cells transfected with the dominant-negative MSK1 clones underwent cell death in response to LPS. Hence, it was suggested that MSK1/2 might play a critical role in the survival of LPS-stimulated RAW 264.7 cells.     

Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis

Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.     

Interleukin-10 inhibits tumor necrosis factor-alpha production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)-alpha production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF-alpha production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-kappaB, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF-alpha production via reduced MyD88 expression.     

Heparin stimulation of plasminogen activator secretion by macrophage-like cell line RAW264.7: role of the scavenger receptor

Secretion of urokinase-type plasminogen activator (uPA) by RAW264.7 cells was stimulated by heparin in a dose- and time-dependent manner. Secretion of uPA was not detected when cells were exposed to heparin at 4 degrees C, indicating that heparin was not simply releasing receptor-bound uPA. Furthermore, prior removal of membrane-associated uPA did not influence heparin's ability to stimulate the release of uPA from the macrophage-like line. Low molecular weight and weakly anticoagulant heparins stimulated uPA secretion but less effectively than other heparin fractions. The observed stimulation in macrophage uPA secretion by heparin is similar to that previously reported for polyanions recognized by the scavenger receptor including fucoidan, polyinosinic acid, dextran sulfate, and acetyl-LDL (Falcone and Ferenc: J. Cell. Physiol., 135:387-396, 1988). Evidence that heparin's binding to RAW264.7 cells is mediated by the scavenger receptor is derived from experiments in which fucoidan blocked the specific binding of [3H]-heparin to RAW264.7 cells. However, heparin partially inhibited the stimulation of cholesteryl [3H]-oleate synthesis observed in these cells upon incubation with acetyl-LDL and weakly inhibited cellular binding of 125I-acetyl-LDL at 4 degrees C. These data indicate that heparin's binding to RAW264.7 cells is mediated, only in part, by the scavenger receptor. Nonetheless, neither heparin nor fucoidan was able to stimulate the release of plasminogen activator activity from monocyte-like U937 cells which are devoid of scavenger receptor activity.     

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.     

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.     

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Lipopolysaccharide-induced monocyte chemotactic protein-1 is enhanced by suppression of nitric oxide production, which depends on poor CD14 expression on the surface of skeletal muscle

It is known that lipopolysaccharide (LPS)-induced monocyte chemotactic protein (MCP)-1 secretion from tissues recruits monocytes from the circulation, but the mechanism of the LPS-induced MCP-1 production in skeletal muscle is largely unexplained. To clarify the effect of LPS on MCP-1 production in skeletal muscle cells, C2C12 cells from a mouse skeletal muscle cell line, and RAW 264.7 cells from a mouse macrophage cell line, were used to assess production of LPS-induced MCP-1, nitric oxide (NO) and interferon (IFN)-beta. In addition, we evaluated inducible NO synthases (iNOS) mRNA expression using RT-PCR, and cell surface expression of CD14 and toll-like receptor (TLR) 4 using flow cytometry. In C2C12 cells, LPS stimulation increased MCP-1 production (p < 0.01), but combined treatment with LPS and NO inducer, diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (NONOate), significantly inhibited its production (p < 0.01). LPS stimulation neither induced production of NO nor of IFN-beta, which is an NO inducer. Recombinant IFN-beta stimulation, on the other hand, enhanced LPS-induced NO production (p < 0.01). Interestingly, we found that surface expression of CD14, which regulates IFN-beta production, in C2C12 cells was much lower than that in RAW 264.7 cells, although TLR4 expression on C2C12 cells was similar to that on RAW 264.7 cells. These data suggest that the reduced NO production in response to LPS may depend on low expression of CD14 on the cell surface of skeletal muscle, and that it may enhance LPS-induced MCP-1 production. Together, these functions of skeletal muscle could decrease the risk of bacterial infection by recruitment of monocytes.     

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.