ABSTRACT
Border rows of grain sorghum were planted along two to four sides of an organic Granadero tomato crop in North Florida to reduce fruit injury caused by native and invasive stink bugs. During the 2-yr study, 14 species of stink bugs were encountered, six only in sorghum: Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), Thyanta spp., Oebalus pugnax (Fabricius), Chinavia hilaris (Say), C. pensylvanica (Gmelin), and Mormidea pama (Rolston). There were four species only in tomato: Euschistus obscurus (Palisot de Beauvois), E. tristigmus (Say), E. ictericus (L.), and Arvelius albopunctatus (De Geer). The three most abundant pests in tomato were collected in both crops: Nezara viridula (L.), Euschistus servus (Say), and E. quadrator Rolston, along with Proxys punctulatus (Palisot de Beauvois). Nezara viridula and P. guildinii were the most abundant stink bugs on sorghum. The border rows of sorghum did not reduce the total number of stink bug adults or nymphs in the tomato crop, although many more stink bug adults were captured in sorghum than tomato when the sorghum panicles were in the milk to soft dough stage. Generally, 30% of the females in the sorghum and tomato crops were mated and contained more than 15 eggs, indicating they could generate a considerable number of nymphs. Tomato fruit from the plot with sorghum border rows had significantly more punctures than fruit from the plot without sorghum. The stink bugs frequently probed and blemished tomato fruit in all stages of ripeness but fruit covered with probing sites were nevertheless suitable for human consumption.
Subject(s)
Heteroptera , Solanum lycopersicum , Sorghum , Animals , Female , Crops, Agricultural , Edible Grain , Fruit , Nymph , Population Density , ReproductionABSTRACT
Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1ß (IL-1ß) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1ß secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1ß secretion pathway in human macrophages. This has important implications for understanding UTI in humans.
Subject(s)
Carrier Proteins/immunology , Inflammasomes/drug effects , Interleukin-1beta/immunology , Macrophages/immunology , Uropathogenic Escherichia coli/immunology , Animals , Bacterial Toxins/toxicity , Carrier Proteins/genetics , Cell Death/drug effects , Gene Expression Regulation , Hemolysin Proteins/toxicity , Host-Pathogen Interactions , Humans , Inflammasomes/immunology , Interleukin-1beta/genetics , Macrophages/drug effects , Macrophages/microbiology , Mice , NLR Family, Pyrin Domain-Containing 3 Protein , Primary Cell Culture , Signal Transduction , Species Specificity , Uropathogenic Escherichia coli/pathogenicityABSTRACT
Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome 'specks' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.
Subject(s)
Apoptosis , Carrier Proteins/metabolism , Caspase 8/metabolism , Cytoskeletal Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Apoptosis Regulatory Proteins , CARD Signaling Adaptor Proteins , Caspase 1/metabolism , Caspase 8/genetics , Caspase 9/genetics , DNA-Binding Proteins , Inflammasomes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , RNA Interference , RNA, Small Interfering , Toll-Like Receptor 9/geneticsABSTRACT
Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.
Subject(s)
Adjuvants, Immunologic/metabolism , Adjuvants, Immunologic/pharmacology , CpG Islands/immunology , DNA/immunology , Macrophage Activation/genetics , Macrophages/immunology , Thionucleotides/metabolism , Thionucleotides/pharmacology , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cell Line , Cell Survival/immunology , DNA/antagonists & inhibitors , Dose-Response Relationship, Immunologic , Down-Regulation/immunology , Gene Expression Regulation/immunology , HIV Long Terminal Repeat/immunology , Humans , Interleukin-12/genetics , Macrophages/metabolism , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Nitric Oxide/biosynthesis , Nucleic Acid Conformation , Oligodeoxyribonucleotides/metabolism , Oligodeoxyribonucleotides/pharmacology , Organophosphates/immunology , Organophosphates/metabolism , Phosphorylation , Promoter Regions, Genetic/immunology , Receptor, Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
The major cause of death in cystic fibrosis (CF) is chronic lung disease associated with persistent infection by the bacterium, Pseudomonas aeruginosa. S100A8, an S-100 calcium-binding protein with chemotactic activity, is constitutively expressed in the lungs and serum of CF patients. Levels of S100A8 mRNA were found to be three to four times higher in the lungs of mice carrying the G551D mutation in CF transmembrane conductance regulator compared with littermate controls. Intravenous injection of bacterial LPS induced S100A8 mRNA in the lung to a greater extent in G551D mice than in wild-type littermates. Localization of S100A8 mRNA and protein in the lung indicate that it is a marker for neutrophil accumulation. Bone marrow-derived macrophages from G551D mice were shown to also exhibit hypersensitivity to LPS, measured by induction of TNF-alpha. These results provide evidence that the pathology of CF relates to abnormal regulation of the immune system.
Subject(s)
Amino Acid Substitution/genetics , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Lung/pathology , Macrophages/pathology , Mutation, Missense , Animals , Aspartic Acid/genetics , Biomarkers , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Movement , Cystic Fibrosis/immunology , Cystic Fibrosis/metabolism , Disease Models, Animal , Glycine/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Injections, Intravenous , Lipopolysaccharides/administration & dosage , Lung/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Mutant Strains , Neutrophils/pathology , RNA, Messenger/biosynthesisABSTRACT
Macrophage colony-stimulating factor (CSF-1) binds to a receptor (CSF-1R) encoded by the c-fms proto-oncogene and activates transcription of the urokinase plasminogen activator (uPA) gene in murine bone-marrow-derived macrophages. This article demonstrates that the murine macrophage cell line RAW264 responds to CSF-1 with inducible phosphorylation of cytoplasmic proteins on tyrosine residues but fails to induce transcription of uPA. The defect was correlated with a selective failure to maintain CSF-1Rs on the cell surface, whereas all RAW264 cells contained abundant CSF-1Rs within the presumptive Golgi/endoplasmic reticulum compartment. Transfection with a CSF-1R expression plasmid permitted CSF-1-dependent activation of the signalling pathway targeting an Ets/AP1 (activator protein 1) element in the uPA promoter that has been shown previously to be a target of oncogenic ras and protein kinase C pathways. Mutation of the expressed CSF-1R at either Y807 or Y559, sites of receptor tyrosine phosphorylation implicated in signal transduction, reduced but did not abolish uPA promoter activation by CSF-1. Activation by mutant CSF-1R plasmids was additive; there was no evidence of mutual complementation. The results indicate that maintenance of elevated uPA transcription by CSF-1 requires new receptors emerging continuously on the cell surface. Parallel, partly redundant, signalling pathways arising from phosphorylated tyrosines on the CSF-1R activate multiple cis-acting elements on the complex uPA promoter.
Subject(s)
Gene Expression Regulation, Enzymologic , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/enzymology , Receptor, Macrophage Colony-Stimulating Factor/physiology , Transcription, Genetic , Urokinase-Type Plasminogen Activator/genetics , Amino Acid Substitution , Animals , Cell Line , Gene Expression Regulation, Enzymologic/drug effects , Mice , Mutagenesis, Site-Directed , Phosphorylation , Phosphotyrosine/metabolism , Promoter Regions, Genetic , Receptor, Macrophage Colony-Stimulating Factor/genetics , Recombinant Proteins/metabolism , Signal Transduction , Transcription, Genetic/drug effects , TransfectionABSTRACT
Macrophage/dendritic cells and B cells remain the only cell types where direct responses to CpG DNA are well established. The role of macrophages in vivo in DNA clearance and the potent cytokine induction in macrophages and dendritic cells places them in the central role in the in vivo response to foreign DNA. Although responses to DNA are unlikely to evolve and be retained if they are not significant in the immune response to infection, the relative contributions of DNA and other stimulators of the innate immune recognition of foreign organisms is difficult to assess. Although CpG DNA and LPS have similar actions, significant differences are emerging that make the use of DNA as a therapeutic immunostimulatory molecule feasible. The macrophage response to DNA generates cytokines favouring the development of Th1-type immunity, and active oligonucleotides now show promise as Th1-promoting adjuvants and as allergy treatments.
Subject(s)
CpG Islands/immunology , DNA/immunology , Macrophage Activation/immunology , Animals , Dendritic Cells/immunology , HumansABSTRACT
Unmethylated CpG motifs within bacterial DNA constitute a pathogen-associated molecular pattern recognized by the innate immune system. Many of the immunomodulatory functions of bacterial DNA can be ascribed to the ability to activate macrophages and dendritic cells. Here we show stimulatory DNA, like LPS, caused growth arrest of murine bone marrow-derived macrophages proliferating in CSF-1. Stimulatory DNA caused selective down-modulation of CSF-1 receptor surface expression. Flow cytometric analysis of CSF-1-deprived bone marrow-derived macrophages revealed that in contrast to the synchronous reduction of CSF-1 receptor upon CSF-1 addition, activating DNA (both bacterial DNA and CpG-containing oligonucleotide) caused rapid removal of receptor from individual cells leading to a bimodal distribution of surface expression at intermediate times or submaximal doses of stimulus. Despite causing growth arrest, both stimulatory DNA and LPS promoted factor-independent survival of bone marrow-derived macrophages, which was associated with phosphorylation of the mitogen-activated protein kinase family members, extracellular-regulated kinase 1 and 2. CSF-1 receptor down-modulation may polarize the professional APC compartment to the more immunostimulatory dendritic cell-like phenotype by suppressing terminal macrophage differentiation mediated by CSF-1.
Subject(s)
Bone Marrow Cells/metabolism , CpG Islands/immunology , DNA, Bacterial/immunology , Down-Regulation/immunology , Growth Inhibitors/immunology , Macrophages/metabolism , Receptor, Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptor, Macrophage Colony-Stimulating Factor/biosynthesis , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/enzymology , Cell Division/immunology , Cell Membrane/immunology , Cell Membrane/metabolism , Cell Survival/immunology , Down-Regulation/genetics , Escherichia coli/genetics , Lipopolysaccharides/immunology , Macrophages/cytology , Macrophages/enzymology , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Molecular Mimicry/immunology , Phosphorylation , Receptor, Macrophage Colony-Stimulating Factor/physiologyABSTRACT
Murine macrophages are able to distinguish bacterial from mammalian DNA. The response is mimicked by single-stranded oligonucleotides containing unmethylated CG dinucleotides ("CpG" motifs) in specific sequence contexts. The dose-response curve for activation is influenced by variation in the sequence flanking the core CpG motif. CpG or bacterial DNA activates several signaling pathways in common with bacterial lipopolysaccharide (LPS), leading to induction of cytokine genes such as tumor necrosis factor alpha. Pretreatment with LPS causes desensitization to subsequent activation by CpG DNA. Both stimuli also cause cell cycle arrest in macrophages proliferating in response to the macrophage growth factor colony-stimulating factor-1 (CSF-1), but prevent apoptosis caused by growth factor removal. In part, cell cycle arrest by CpG DNA and LPS may be linked to rapid down-modulation of the CSF-1 receptor from the cell surface, a response that occurs in an all-or-nothing manner. The response of macrophages to CpG DNA has aspects in common with the DNA damage response in other cell types, which may provide clues to the underlying mechanism.
Subject(s)
CpG Islands/immunology , DNA, Bacterial/immunology , Macrophage Activation , Macrophages/immunology , Animals , Cell Differentiation , Cytokines/immunology , Down-Regulation , Humans , Lipopolysaccharides/immunology , Macrophage Activation/immunology , Mice , Receptor, Macrophage Colony-Stimulating Factor/biosynthesis , Th1 Cells/cytology , Th1 Cells/immunologyABSTRACT
Bacterial lipopolysaccharide (LPS) stably induced the protein kinase C substrate, MacMARCKS, in murine resident peritoneal macrophages; initial induction of MacMARCKS mRNA was detected within 15 min and was protein synthesis-independent. This response was observed in the macrophage cell line RAW264, and occurred also in response to plasmid DNA, a partial mimetic of other responses to LPS. In murine bone marrow-derived macrophages, MacMARCKS was expressed constitutively due to induction by macrophage colony-stimulating factor. Nuclear run-on transcription revealed that, like tumor necrosis factor alpha (TNF-alpha), MacMARCKS was transcribed constitutively in RAW264 cells. The MacMARCKS promoter was sequenced to -1.7 kb and the transcription start site determined. Transient transfections of RAW264 cells revealed that the 113-bp GC-rich proximal promoter contained all the elements required for both high basal activity and 15- to 20-fold activation by LPS.
Subject(s)
Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Membrane Proteins/genetics , Animals , Base Sequence , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Calmodulin-Binding Proteins , Gene Expression Regulation, Neoplastic/drug effects , Intracellular Signaling Peptides and Proteins , Macrophages/metabolism , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Membrane Proteins/biosynthesis , Mice , Microfilament Proteins , Molecular Sequence Data , Neoplasm Proteins/biosynthesis , Organ Specificity , Promoter Regions, Genetic , Protein Kinase C/metabolism , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transcription, Genetic/drug effects , Transfection , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolismABSTRACT
We investigate the regulation of plasminogen activator inhibitor-2 (PAI-2) in murine macrophages. PAI-2 mRNA was inducible by bacterial lipopolysaccharide (LPS) in primary cells and macrophage-like cell lines. Evidence is presented for a role for autocrine factors, including cyclooxygenase products but not the cytokines tumor necrosis factor alpha or interferon-beta (IFN-beta). PAI-2 mRNA levels generally varied inversely from those of its target, urokinase-type plasminogen activator (uPA), and the macrophage growth factor CSF-1, which induces uPA, inhibited PAI-2 expression in cells treated subsequently with LPS. Expression of PAI-2 was distinct from that of other LPS-inducible genes in terms of induction time course, LPS dose response, and sensitivity to co-stimulation with IFN-gamma. Induction of PAI-2 mRNA in subclones of the cell line RAW 264 was not uniform, reflecting heterogeneous expression in the parent line. The expression pattern of PAI-2 is discussed in terms of a possible role in LPS-induced pathology such as septicemia.
Subject(s)
Bacterial Toxins/metabolism , Gene Expression Regulation , Lipopolysaccharides/metabolism , Macrophages/metabolism , Plasminogen Activator Inhibitor 2/genetics , Animals , Bacterial Toxins/pharmacology , Blotting, Northern , Cell Line , Cells, Cultured , Humans , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Male , Mice , Mice, Inbred BALB C , RNA, MessengerABSTRACT
Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote T helper 1 (Th1) responses, an adjuvant activity that is desirable for vaccination against leishmaniasis. To test this, susceptible BALB/c mice were vaccinated with soluble leishmanial antigen (SLA) with or without CpG ODN as adjuvant and then challenged with Leishmania major metacyclic promastigotes. CpG ODN alone gave partial protection when injected up to 5 weeks prior to infection, and longer if the ODN was bound to alum. To demonstrate an antigen-specific adjuvant effect, a minimum of 6 weeks between vaccination and infection was required. Subcutaneous administration of SLA alone, SLA plus alum, or SLA plus non-CpG ODN resulted in exacerbated disease compared to unvaccinated mice. Mice receiving SLA plus CpG ODN showed a highly significant (P < 5 x 10(-5)) reduction in swelling compared to SLA-vaccinated mice and enhanced survival compared to unvaccinated mice. The modulation of the response to SLA by CpG ODN was maintained even when mice were infected 6 months after vaccination. CpG ODN was not an effective adjuvant for antibody production in response to SLA unless given together with alum, when it promoted production of immunoglobulin G2a, a Th1-associated isotype. Our results suggest that with an appropriate antigen, CpG ODN would provide a stable, cost-effective adjuvant for use in vaccination against leishmaniasis.
Subject(s)
Adjuvants, Immunologic/pharmacology , Leishmania major/immunology , Leishmaniasis, Cutaneous/prevention & control , Oligodeoxyribonucleotides/pharmacology , Protozoan Vaccines/immunology , Animals , Antibodies, Protozoan/blood , Female , Immunoglobulin G/classification , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , VaccinationABSTRACT
An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA-Binding Proteins , Macrophage Colony-Stimulating Factor/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases , Proto-Oncogene Proteins/metabolism , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Repressor Proteins , Trans-Activators/metabolism , Transcription Factors , 3T3 Cells , Animals , Blotting, Western , Cell Line , Enzyme Activation , Humans , Kinetics , Luciferases/biosynthesis , Macrophages/metabolism , Mice , Mitogen-Activated Protein Kinase 3 , Phosphorylation , Phosphothreonine/analysis , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Protein c-ets-2 , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/chemistry , RNA, Messenger/biosynthesis , Receptor, Macrophage Colony-Stimulating Factor/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Trans-Activators/biosynthesis , Trans-Activators/chemistry , Transcription, Genetic , TransfectionABSTRACT
The HIV-1 promoter was used as a model to identify transcription factors involved in LPS-dependent transcription in RAW 264 murine macrophages. Expression plasmids for Ets-2 and PU.1 trans-activated the HIV-1 LTR and recombinant PU.1 and an Ets-2 DNA binding domain/GST fusion protein bound to the 5' kappa B site of the HIV-1 enhancer. Ets-2 mRNA was LPS-inducible in RAW 264 cells and LPS stimulated phosphorylation of threonine 72 residue within the Ets-2 pointed domain. Induction of Ets-2 and other LPS-responsive transcription factors was also observed upon addition of plasmid DNA, which complicates interpretation of transient transfections. The proximal promoter region, containing two Sp1 sites, was also LPS-responsive. We propose that the kappa B elements and the tandem Sp1 sites act as LPS response elements and that kappa B-mediated LPS action involves Ets and rel factors.
Subject(s)
DNA-Binding Proteins , HIV Long Terminal Repeat/genetics , Macrophages/metabolism , Proto-Oncogene Proteins/pharmacology , Repressor Proteins , Sp1 Transcription Factor/pharmacology , Trans-Activators/pharmacology , Transcription Factors , Animals , Binding Sites , Cell Line , DNA/metabolism , Gene Expression , Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , Mice , NF-kappa B/pharmacology , Plasmids , Promoter Regions, Genetic , Proto-Oncogene Protein c-ets-2 , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcriptional Activation , TransfectionABSTRACT
Macrophages recognize and are activated by unmethylated CpG motifs in bacterial DNA. Here we demonstrate that production of nitric oxide (NO) from murine RAW 264 macrophages and bone marrow-derived macrophages (BMM) in response to bacterial DNA is absolutely dependent on interferon-gamma (IFN-gamma) priming. Similarly, arginine uptake and expression of the inducible nitric oxide synthase (iNOS) gene in response to bacterial DNA in BMM occurred only after IFN-gamma priming. In contrast, mRNA for the cationic amino acid transporter, CAT2, was induced by plasmid DNA alone, and priming with IFN-gamma had no effect on this response. Tumor necrosis factor-alpha (TNF-alpha) release from RAW 264 and BMM in response to bacterial DNA was augmented by IFN-gamma pretreatment. In a stably transfected HIV-1 long terminal repeat (LTR) luciferase RAW 264 cell line, IFN-gamma and bacterial DNA synergized in activation of the HIV-1 LTR. Bacterial DNA has been shown to induce IFN-gamma production in vivo as an indirect consequence of interleukin-12 (IL-12) and TNF-alpha production from macrophages. The results herein suggest the existence of a self-amplifying loop that may have implications for therapeutic applications of bacterial DNA.
Subject(s)
DNA, Bacterial/genetics , Interferon-gamma/pharmacology , Macrophages/drug effects , Nitric Oxide Synthase/genetics , Animals , Arginine/metabolism , Catalase/genetics , Cell Line , HIV-1/genetics , Mice , Mice, Inbred Strains , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II , RNA, Messenger/biosynthesis , Repetitive Sequences, Nucleic AcidABSTRACT
Recent evidence suggests that bacterial DNA activates immune responses. Here we showed that TNF-alpha mRNA was induced in bone marrow-derived macrophages and the macrophage cell line RAW 264 by plasmid DNA, but not by DNaseI-digested plasmid, plasmid methylated on CpG dinucleotides, or by vertebrate genomic DNA, which is naturally largely methylated on these sequences. Synthetic polynucleotides poly d(I-C) and poly I x poly C also induced TNF-alpha. IL-1 beta and plasminogen activator inhibitor-2 mRNAs were induced by plasmid DNA, and IFN-gamma-pretreated macrophages responded to DNA with induction of inducible nitric oxide synthase. The HIV-1 long terminal repeat was activated by exogenous DNA in a manner similar to TNF-alpha, and was also activated by a CpG-containing oligonucleotide. Transcription factor nuclear factor-kappa B (NF-kappa B) is involved in regulation of the HIV-1 long terminal repeat and many inflammatory response genes. NF-kappa B binding activity was increased by plasmid DNA. An important question is whether these effects involve DNA binding to a cell surface receptor that signals to the interior, or whether internalization is necessary. Here we found that plasmid was taken up by RAW 264 cells and remained sufficiently intact to code for luciferase protein. Results suggest that DNA is taken up by macrophages and characteristic bacterial DNA sequences, which include an unmethylated CpG sequence, activate a signaling cascade leading to activation of NF-kappa B and inflammatory gene induction. Relevance to DNA vaccination, gene therapy, antisense, and transfection studies is discussed.
Subject(s)
DNA, Bacterial/metabolism , DNA, Bacterial/pharmacology , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/immunology , Phagocytosis/drug effects , Animals , Bone Marrow Cells , Cell Line , Macrophage Activation/genetics , Macrophages/microbiology , Mice , NF-kappa B/biosynthesis , NF-kappa B/genetics , Phagocytosis/genetics , RNA, Messenger/biosynthesisABSTRACT
The Ras oncogene products regulate the expression of genes in transformed cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are the targets of Ras signaling pathways, the abilities of several Ets factors to activate Ras-responsive enhancer (RRE) reporters in the presence of oncogenic Ras were examined. In transient transfection assay, reporters containing RREs composed of Ets-AP-1 binding sites could be activated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage-like line RAW264 by the combination of Ets1 or Ets2 and Ras but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. Phosphoamino acid analysis of radiolabeled Ets2 revealed that Ras induced normally absent threonine-specific phosphorylation of the protein. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors.
Subject(s)
DNA-Binding Proteins , Proto-Oncogene Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Repressor Proteins , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcriptional Activation , ras Proteins/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Conserved Sequence , Enhancer Elements, Genetic , Molecular Sequence Data , Mutation , Phosphorylation , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Protein c-ets-2 , Proto-Oncogene Proteins c-ets , Signal Transduction , Threonine/geneticsABSTRACT
The mouse urokinase-type plasminogen activator (uPA) gene was used as a model macrophage colony-stimulating factor 1 (CSF-1)-inducible gene to investigate CSF-1 signalling pathways. Nuclear run-on analysis showed that induction of uPA mRNA by CSF-1 and phorbol myristate acetate (PMA) was at the transcriptional level in bone marrow-derived macrophages. CSF-1 and PMA synergized strongly in the induction of uPA mRNA, showing that at least some components of CSF-1 action are mediated independently of protein kinase C. Promoter targets of CSF-1 signalling were investigated with NIH 3T3 cells expressing the human CSF-1 receptor (c-fms). uPA mRNA was induced in these cells by treatment with CSF-1, and a PEA3/AP-1 element at -2.4 kb in the uPA promoter was involved in this response. Ets transcription factors can act through PEA3 sequences, and the involvement of Ets factors in the induction of uPA was confirmed by use of a dominant negative Ets-2 factor. Expression of the DNA binding domain of Ets-2 fused to the lacZ gene product prevented CSF-1-mediated induction of uPA mRNA in NIH 3T3 cells expressing the CSF-1 receptor. Examination of ets-2 mRNA expression in macrophages showed that it was also induced synergistically by CSF-1 and PMA. In the macrophage cell line RAW264, the uPA PEA3/AP-1 element mediated a response to both PMA and cotransfected Ets-2. uPA promoter constructs were induced 60- to 130-fold by Ets-2 expression, and the recombinant Ets-2 DNA binding domain was able to bind to the uPA PEA3/AP-1 element. This work is consistent with a proposed pathway for CSF-1 signalling involving sequential activation of fms, ras, and Ets factors.
Subject(s)
Macrophage Colony-Stimulating Factor/pharmacology , Transcription, Genetic/drug effects , Urokinase-Type Plasminogen Activator/genetics , 3T3 Cells , Animals , Base Sequence , Gene Expression Regulation, Enzymologic , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Phorbol Esters/pharmacology , RNA, Messenger/biosynthesis , Signal Transduction , Urokinase-Type Plasminogen Activator/biosynthesisABSTRACT
Urokinase-type plasminogen activator (uPA) mRNA is induced in macrophages by the lineage specific growth factor CSF-1. Upon removal of CSF-1 from bone marrow-derived macrophages (BMM), uPA mRNA decayed with a half-life of 2 h. If RNA synthesis inhibitors actinomycin D, 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB) or alpha-amanitin were added at the time as CSF-1 removal, the uPA message was stabilised. This was not a general effect on CSF-1 responsive mRNAs, as c-myc mRNA decayed with normal kinetics in the presence of inhibitors. The requirement for ongoing RNA synthesis for the degradation of uPA mRNA in BMM suggests that a component of the degradative pathway may be induced following removal of CSF-1.
Subject(s)
Macrophages/metabolism , RNA, Messenger/metabolism , RNA/biosynthesis , Urokinase-Type Plasminogen Activator/biosynthesis , Amanitins/pharmacology , Animals , Base Sequence , Bone Marrow , Cell Differentiation , Dactinomycin/pharmacology , Dichlororibofuranosylbenzimidazole/pharmacology , Enzyme Induction/drug effects , Humans , Kinetics , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/cytology , Macrophages/drug effects , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Oligodeoxyribonucleotides , RNA/antagonists & inhibitors , Recombinant Proteins/pharmacologyABSTRACT
The process of tumorigenesis is frequently associated with resistance to growth inhibition by physiological regulators of normal cells. Murine macrophage-like cell lines BAC1.2F5, RAW264, J774.1A and PU5/1.8 were resistant to growth inhibition by bacterial lipopolysaccharide (LPS) and pertussis toxin, agents that blocked growth of primary bone marrow-derived macrophages (BMDM) in the presence of macrophage colony-stimulating factor (CSF-1). The resistance of the CSF-1-dependent cell line BAC1.2F5 to growth inhibition by pertussis toxin argues against the possibility that pertussis toxin-sensitive G proteins are essential for the pathway of growth stimulation by CSF-1. Conversely, these data add further weight to the argument that LPS mediates some of its biological activities by mimicking the action of pertussis toxin and inhibiting G protein function. The resistance of cell lines to LPS and pertussis toxin was not correlated with any alteration in the expression of mRNA encoding any of three pertussis-toxin sensitive G protein alpha subunits. The pattern of G protein expression was consistent between primary cells and tumour cells, suggesting that this is a differentiation marker. In particular, Gi alpha 2 mRNA was expressed at remarkably high levels in all of the cells. The specificity of LPS resistance was investigated by studying down-regulation of CSF-1 binding and induction of protooncogene c-fos and tumour necrosis factor (TNF) mRNA. BAC1.2F5 cells were LPS-resistant in each of these assays. In CSF-1 binding, RAW264 and J774.1A responded in the same way as bone marrow-derived macrophages but required higher doses of LPS, whereas c-fos and TNF mRNA were induced in these cells at concentrations that did not inhibit growth. In PU5/1.8 cells, CSF-1 binding was already very low and was not further down-regulated, but c-fos and TNF mRNA was inducible by LPS. By contrast to primary macrophages, the cell lines did not respond to LPS with down-regulation of c-fms mRNA, which encodes the CSF-1 receptor. Hence, the resistance of macrophage-like tumour cells to LPS and pertussis toxin was specific to the pathways controlling growth, and was correlated with altered regulation of the CSF-1 receptor.