LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-IR rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK 1/2 phosphorylation. Using toll-like receptor (th-)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R. (C) 2005 Elsevier GmbH. All rights reserved.

CpG DNA activates survival in murine macrophages through TLR9 and the phosphatidylinositol 3-kinase-Akt pathway

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently. of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-alpha production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.

Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Aims: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results: Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway.

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in a subset of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomerular mesangial cells of the kidney and pancreatic acinar cells. We have identified novel TRAP mRNAs that differ in their 5-untranslated region (5'-UTR) sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'-UTRs represent alternative first exons located upstream of the known 5'-UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'-UTR (Exon 1A) is restricted to adult bone and spleen tissue. Exon 1B is expressed primarily in tissues containing TRAP-positive nonhaematopoietic cells. The known TRAP 5'-UTR (Exon 1) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon 1C promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon 1C transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression patterns for each of the TRAP 5'-UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue- and cell-restricted promoters. (C) 2003 Elsevier Science B.V. All rights reserved.

L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or N-G-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.

Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Immunohistological analysis of Tannerella forsythia-induced lesions in a murine model

Tannerella forsythia has been implicated as a defined periodontal pathogen. In the present study a mouse model was used to determine the phenotype of leukocytes in the lesions induced by subcutaneous injections of either live (group A) or nonviable (group B) T. forsythia. Control mice (group C) received the vehicle only. Lesions were excised at days 1, 2, 4, and 7. An avidin-biotin immunoperoxidase method was used to stain infiltrating CD4(+) and CD8(+) T cells, CD14(+) macrophages, CD19(+) B cells, and neutrophils. Hematoxylin and eosin sections demonstrated lesions with central necrotic cores surrounded by neutrophils, macrophages and lymphocytes in both group A and group B mice. Lesions from control mice exhibited no or only occasional solitary leukocytes. In both groups A and B, neutrophils were the dominant leukocyte in the lesion 1 day after injection, the numbers decreasing over the 7-day experimental period. There was a relatively low mean percent of CD4(+) and CD8(+) T cells in the lesions and, whereas the percent of CD8(+) T cells remained constant, there was a significant increase in the percent of CD4(+) T cells at day 7. This increase was more evident in group A mice. The mean percent of CD14(+) macrophages and CD19(+) B cells remained low over the experimental period, although there was a significantly higher mean percent of CD19(+) B cells at day 1. In conclusion, the results showed that immunization of mice with live T. forsythia induced a stronger immune response than nonviable organisms. The inflammatory response presented as a nonspecific immune response with evidence of an adaptive (T-cell) response by day 7. Unlike Porphyromonas gingivalis, there was no inhibition of neutrophil migration.

Reduction of the in vitro pro-inflammatory response by macrophages to poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

This study evaluates the pro-inflammatory response to the thermoplastic biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through the analysis of cellular responses in vitro. The murine macrophage RAW264.7 cell line was cultured on solvent cast PHBV films, which was found to induce pro-inflammatory activity that required direct contact between the material and the macrophages. The identity of the pro-inflammatory stimulus was determined by culturing bone marrow-derived macrophages from bacterial lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice and CpG non-responsive TLR9-/- mice on PHBV. The lack of a pro-inflammatory response by the C3H/HeJ cells indicates that the pro-inflammatory agent present within PHBV is predominately LPS while the TLR9-/- macrophages confirmed that CpG-containing bacterial DNA is unlikely to contribute to the activity. A series of purification procedures was evaluated and one procedure was developed that utilized hydrogen peroxide treatment in solution. The optimized purification was found to substantially reduce the pro-inflammatory response to PHBV without adversely affecting either the molecular structure or molecular weight of the material thereby rendering it more amenable for use as a biomaterial in vivo. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Regulation of the murine TRACP gene promoter

The activity of the TRACP promoter has been investigated as a model of gene regulation in osteoclasts. The murine TRACP gene promoter contains potential binding sites for a number of transcription factors in particular, candidate sites for the Ets factor PU.1 and for the microphthalmia transcription factor (MiTF). These are of relevance to osteoclast biology because the PU.1 knockout mouse has an osteopetrotic phenotype, and MiTF, when mutated in the mi/mi mouse, also results in osteopetrosis. The binding sites for both of these factors have been identified, and they have been determined to be functional in regulating TRACP expression. A novel assay system using the highly osteoclastogenic RAW/C4 subclone of the murine macrophage cell line RAW264.7 was used to perform gene expression experiments on macrophage and osteoclast cell backgrounds. We have shown that TRACP expression is a target for regulation by the macrophage/osteoclast transcription factor PU.1 and the osteoclast commitment factor MiTF and that these factors act synergistically in regulating this promoter. This directly links two controlling factors of osteoclast differentiation to the expression of an effector of cell function.

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at similar to3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 mug/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NSO) cells. Four homogeneous NSO cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab clatasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (C) 2004 Wiley Periodicals, Inc.

Uptake of antigen encapsulated in polyethylcyanoacrylate nanoparticles by D1-dendritic cells

Polyethylcyanoacrylate (PECA) nanoparticles were prepared by interfacial polymerization of a water-in-oil microemulsion. Nanoparticles were isolated from the polymerization template by sequential ethanol washing and centrifugation. A nanocapsule preparation yielding the original particle size and distribution following redispersion in an aqueous solution was achieved by freeze-drying the isolated nanoparticles in a solution of 5% w/v sugar. The cytotoxicity and uptake of nanocapsules by dendritic cells was investigated using a murine-derived cell line (D1). PECA nanoparticles were found to adversely effect cell viability at concentrations greater than 10 mug/ml of polymer in the culture medium. In comparison to antigen in solution, cell uptake of antigen encapsulated within nanoparticles was significantly higher at both 4 and 37 degreesC. Following a 24 h incubation period, the percentage of cells taking-up antigen was also increased when antigen was encapsulated in nanoparticles as compared to antigen in solution. The uptake of nanoparticles and the effect of antigen formulation on morphological cell changes indicative of cell maturation were also investigated by scanning electron microscopy (SEM). SEM clearly demonstrated the adherence of nanoparticles to the cell surface. Incubation of D1 dendritic cells with nanoparticles containing antigen also resulted in morphological changes indicative of cell maturation similar to that observed when the cells were incubated with lipopolysaccharide. In contrast, cells incubated with antigen solution did not demonstrate such morphological changes and appeared similar to immature cells that had not been exposed to antigen.