ABSTRACT
Several cystic fibrosis (CF) mouse models demonstrate an increased susceptibility to Pseudomonas aeruginosa lung infection, characterized by excessive inflammation and high rates of mortality. Here we developed a model of chronic P. aeruginosa lung disease in mice homozygous for the murine CF transmembrane conductance regulator G551D mutation that provides an excellent model for CF lung disease. After 3 days of infection with mucoid P. aeruginosa entrapped in agar beads, the G551D animals lost substantially more body weight than non-CF control animals and were less able to control the infection, harboring over 40-fold more bacteria in the lung. The airways of infected G551D animals contained altered concentrations of the inflammatory mediators tumor necrosis factor-alpha, KC/N51, and macrophage inflammatory protein-2 during the first 2 days of infection, suggesting that an ineffective inflammatory response is partly responsible for the clearance defect.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Cystic Fibrosis/microbiology , Lung Diseases/microbiology , Mutation/physiology , Pseudomonas Infections/complications , Alleles , Animals , Body Weight , Bronchoalveolar Lavage Fluid/chemistry , Chronic Disease , Colony Count, Microbial , Cytokines/metabolism , Homozygote , Inflammation Mediators/metabolism , Lung/microbiology , Lung Diseases/metabolism , Lung Diseases/pathology , Mice , Mice, Mutant Strains , Pneumonia, Bacterial/microbiology , Pneumonia, Bacterial/pathology , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Pseudomonas Infections/pathology , Pseudomonas aeruginosa/isolation & purification , Reference ValuesABSTRACT
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
The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A variant mRNA was identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount in situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.
