lipid-a and herbimycin

Activation of macrophages by bacterial lipopolysaccharide (LPS) induces transcription of genes that encode for proinflammatory regulators of the immune response. Previous work has suggested that activation of the transcription factor activator protein 1 (AP-1) is one LPS-induced event that mediates this response. Consistent with this notion, we found that LPS stimulated AP-1-mediated transcription of a transfected reporter gene in the murine macrophage cell line RAW 264.7. As AP-1 activity is regulated in part by activation of the c-Jun N-terminal kinase (JNK), which phosphorylates and subsequently increases the transcriptional activity of c-Jun, we examined whether LPS treatment of macrophages resulted in activation of this kinase. LPS treatment of RAW 264.7 cells, murine bone marrow-derived macrophages, and the human monocyte cell line THP-1 resulted in rapid activation of the p46 and p54 isoforms of JNK. Treatment with wild-type and rough mutant forms of LPS and synthetic lipid A resulted in JNK activation, while pretreatment with the tyrosine kinase inhibitor herbimycin A inhibited this response. Binding of LPS-LPS binding protein (LBP) complexes to CD14, a surface receptor that mediates many LPS responses, was found to be crucial, as pretreatment of THP-1 cells with the monoclonal antibody 60b, which blocks this binding, inhibited JNK activation. These results suggest that LPS activation of JNK in monocyte/macrophage cells is a CD14- and protein tyrosine phosphorylation-dependent event that may mediate the early activation of AP-1 in regulating LPS-triggered gene induction.

Topics: Animals; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Escherichia coli; Humans; JNK Mitogen-Activated Protein Kinases; Kinetics; Lactams, Macrocyclic; Lipid A; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mitogen-Activated Protein Kinases; Monocytes; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; Salmonella; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transfection

Porphyromonas gingivalis 381 lipid A possesses 1-phospho beta(1-6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2'-positions, respectively. P. gingivalis lipid A indicated lower activities in inducing interleukin-1 beta (IL-1 beta) mRNA expression, pro-IL-1 beta protein synthesis and IL-1 beta production than those of synthetic Escherichia coli lipid A (compound 506) in human peripheral blood mononuclear cells (PBMC). The induction of IL-6 mRNA and IL-6 synthesis by P. gingivalis lipid A were comparable to those of compound 506. Herbimycin A, H-7 and H-8, inhibitors of tyrosine kinase, protein kinase C and cyclic nucleotide-dependent protein kinase, inhibited P. gingivalis lipid A- and compound 506-induced IL-1 beta and IL-6 synthesis. W-7, an inhibitor of calmodulin (CaM) kinase, inhibited only P. gingivalis lipid A-induced IL-1 beta production. The result suggests that the CaM kinase-dependent cascade is involved in the down-regulation of IL-1 beta production by P. gingivalis lipid A. P. gingivalis lipid A and compound 506 also functioned in the induction of tyrosine and serine/threonine phosphorylation of several proteins in PBMC. P. gingivalis lipid A inhibited specific binding of fluorescein-labelled E. coli LPS to the PBMC. The nontoxic lipid A of P. gingivalis, having a chemical structure different from toxic compound 506, appears to induce the up- and down-regulation of the differential cytokine-producing activities following the activation of various intracellular enzymes including the CaM kinase through the common receptor sites of LPS.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Benzoquinones; Blotting, Northern; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Down-Regulation; Enzyme Inhibitors; Escherichia coli; Flow Cytometry; Humans; Interleukin-1; Interleukin-6; Lactams, Macrocyclic; Leukocytes, Mononuclear; Lipid A; Lipopolysaccharide Receptors; Molecular Structure; Porphyromonas gingivalis; Quinones; Rifabutin; RNA, Messenger; Signal Transduction; Sulfonamides; Up-Regulation

The synthetic low-toxicity lipid A analog DT-5461a induces endogenous TNF production in mice. The activity of TNF so induced is probably the main contributor to the antitumor effect of this compound. In the present study, we investigated the mechanism by which DT-5461a induces TNF production in murine macrophage RAW 264 cells. DT-5461a mimicked the ability of LPS to induce TNF production in a dose-dependent manner. DT-5461a at higher concentrations inhibited specific binding of [3H]LPS to the cells and reduced LPS-induced TNF production to the level induced by DT-5461a alone. In addition, DT-5461a, as well as LPS, induced tyrosine phosphorylation of MAP kinases, the early signal transduction pathway of this production. Herbimycin A, an inhibitor of tyrosine kinase, inhibited the LPS- and DT-5461a-induced tyrosine phosphorylation, expression of TNF mRNA, and subsequent TNF secretion. These results suggest that DT-5461a and LPS induce TNF production in murine macrophages through the common receptor sites and the similar early signaling pathway.

Topics: Animals; Benzoquinones; Binding, Competitive; Blotting, Western; Cell Line, Transformed; Disaccharides; Lactams, Macrocyclic; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Mice; Protein Kinases; Protein-Tyrosine Kinases; Quinones; Receptors, Immunologic; Rifabutin; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

We have examined the differentiation-inducing effects of ONO-4007, a new synthetic lipid A derivative with low endotoxic activities, on a rat myelomonocytic cell line, c-WRT-7, in vitro and in vivo. ONO-4007 induced the differentiation of c-WRT-7 cells into macrophage-like cells and inhibited the proliferation of c-WRT-7 cells in vitro. Stimulation with ONO-4007 induced messenger RNA expression of interleukin-1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha (TNF-alpha), which have been reported to induce differentiation of several leukemia cell lines. However, autocrine production of these cytokines may not be involved in the mechanisms of differentiation induced by ONO-4007, because the treatment with IL-1 alpha, IL-6, or TNF-alpha does not induce the differentiation of c-WRT-7 cells. In vivo treatment by intravenous administration of ONO-4007 resulted in a significant prolongation of survival time of the rats inoculated intravenously with c-WRT-7 cells compared with that of untreated rats. These results suggest that ONO-4007 can be therapeutically useful for the treatment of leukemia.

Topics: Alkaloids; Animals; Base Sequence; Benzoquinones; Calmodulin; Cell Division; Cell Transformation, Neoplastic; In Vitro Techniques; Interleukin-1; Interleukin-6; Lactams, Macrocyclic; Leukemia, Myeloid; Lipid A; Molecular Sequence Data; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; RNA, Messenger; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, stimulates immune responses by activating macrophages, B lymphocytes, and other cells of the immune system. The mechanisms by which LPS activates these cells are poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signaling event that mediates cellular responses, we examined whether LPS alters tyrosine phosphorylation in macrophages. We found that Escherichia coli K235 LPS increased tyrosine phosphorylation of several proteins in the RAW 264.7 murine macrophage cell line and in resident peritoneal macrophages from C3H/HeSNJ mice. Changes in tyrosine phosphorylation were detectable by 4-5 min, reached a maximum by 15 min, and declined after 30-60 min. Protein tyrosine phosphorylation increased following stimulation with LPS at 100 pg/ml and was maximal with 10 ng/ml. Similar changes in tyrosine phosphorylation were induced by Salmonella minnesota R595 LPS and by the biologically active domain of LPS, lipid A, but not by the inactive lipid A derivative N2-monoacylglucosamine 1-phosphate. Phorbol 12-myristate 13-acetate also stimulated protein tyrosine phosphorylation, but some of the modulated proteins were different than those phosphorylated by LPS. Treatment of RAW 264.7 cells with a tyrosine kinase inhibitor, herbimycin A, inhibited both LPS-stimulated tyrosine phosphorylation and LPS-stimulated release of arachidonic acid metabolites. Thus, increased protein tyrosine phosphorylation is a rapid LPS-activated signaling event that may mediate release of arachidonic acid metabolites in RAW 264.7 cells.

Topics: Animals; Benzoquinones; Cell Line, Transformed; Escherichia coli; Female; Kinetics; Lactams, Macrocyclic; Lipid A; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred C3H; Peritoneal Cavity; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rifabutin; Salmonella; Tetradecanoylphorbol Acetate; Tyrosine