Our previous work has provided strong evidence that the proteasome is central to most of the genes induced in mouse macrophages in response to LPS stimulation. In the studies presented here, we evaluated the role of the macrophage proteasome in response to a second microbial product CpG DNA (unmethylated bacterial DNA). For these studies, we applied Affymetrix microarray analysis of RNA derived from murine macrophages stimulated with CpG DNA in the presence or absence of proteasome inhibitor, lactacystin. The results of these studies revealed that similar to LPS, most of those macrophage genes regulated by CpG DNA are also under the control of the proteasome at 4 h. In contrast to LPS stimulation, however, many of these genes were induced much later than 4 h, at 18 h, in response to CpG DNA. Lactacystin treatment of macrophages completely blocked the CpG DNA-induced gene expression of TNF-α and other genes involved in the production of inflammatory mediators. These data strongly support the conclusion that similar to LPS, the macrophage proteasome is a key regulator of CpG DNA-induced signaling pathways.

ABSTRACT Our previous work demonstrated that the proteasome is central to most of genes induced by lipopolysaccharide. In this study, we evaluated the role of the proteasome in response to two other microbial stimuli, CpG DNA (bacterial DNA) and peptidoglycan (PG), by measuring the effect of proteasome inhibition on cytokine secretion, induction of inflammatory gene expression, and activation of mitogen-activated protein kinases (MAPK) in murine macrophages. Pretreatment of macrophage cultures with lactacystin, a well-established proteasome inhibitor, significantly repressed tumor necrosis factor &agr; secretion and tumor necrosis factor &agr; and interleukin 1&bgr; gene expression, blocked the degradation of I&kgr;B, and dysregulated phosphorylation of MAPK induced by CpG DNA or PG. With respect to MAPK, lactacystin blocked expression of PG- or CpG-induced phosphorylated ERK1 and ERK2 and increased expression of phosphorylated c-Jun amino-terminal kinase but had no significant effect on phosphorylated p38. Increased expression of phoshorylated c-Jun amino-terminal kinase did not lead to an increase in AP-1 binding activity. Collectively, these data strongly support the conclusion that the proteasome is a key regulator of the CpG DNA- and PG-induced signaling pathways.

The treatment of sepsis and septic shock is an important clinical problem. While effective antibiotic intervention and strong supportive care have improved survival, mortality remains at unacceptable levels. The induction of systemic inflammation appears to be clearly mediated through a variety of microbial products, not all of which have uniform ability to induce gene expression in host inflammatory mediator cells. The available evidence would support the conclusion that microbial mediators can function synergistically in the induction of host inflammation, providing a potential explanation that anti-endotoxin and anti-inflammatory agents have not been particularly successful in clinical trials to treat septic shock. The identification of specific recognition molecules on the surface of inflammatory mediator cells responsible for initiation of signal transduction, as well as the elucidation of the specific molecular pathways leading to gene expression, provide new opportunities for the development of effective intervention strategies for treatment of septic shock.

The HLA-G gene is highly expressed at the maternal-fetal interface, where it is believed to participate in the generation and maintenance of maternal tolerance to the fetal semiallograft. This gene has two elements through which interferon-gamma (IFN-gamma) could act to enhance its rate of transcription, an Enhancer A/ICS region and a candidate IFN-gamma activated site (GAS). In this study we investigated functionality of this candidate HLA-G GAS. Two HLA-G-expressing cell lines were tested, the human myelomonocytic cell line, U937, and a mouse fibroblast cell line, S14/8, which is stably transfected with the full length HLA-G gene. Nuclear proteins from IFN-gamma-treated U937 and S14/8 cells bound the interferon regulatory factor-1 (IRF-1) gene GAS sequence (TTC CCCGAA) but not the HLA-G gene's candidate GAS sequence (TTTCGAGAA). Excess unlabeled HLA-G-GAS oligonucleotide failed to inhibit binding of the IRF-1-GAS using the same nuclear extracts. These data indicate that a sequence in the HLA-G gene which would normally permit cytokine enhancement of gene expression, the GAS element, is nonfunctional. This is also true of another regulatory sequence, the Enhancer A/ICS element, suggesting that defects in IFN-gamma response elements prevent inappropriate up-regulation of HLA-G gene expression at the maternal-fetal interface.

LPS is well recognized for its potent capacity to activate mouse macrophages to produce NO, an important inflammatory mediator in innate host defense. We demonstrate here that, although inducing little NO alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce NO in cultures of RAW 264.7 macrophages. The effects of the DNA are mimicked by synthetic CpG-containing oligodeoxynucleotides but not by non-CpG-containing oligodeoxynucleotides. This synergistic activity is not inhibited by neutralizing Abs against IFN. Preincubation of macrophages with DNA for 8-24 h suppresses subsequent synergistic macrophage responses to DNA/LPS, whereas prolonged pretreatment with LPS enhances synergy. RT-PCR analysis indicates that the mRNA levels of the inducible NO synthase gene are also coordinately suppressed or induced. These findings indicate that temporally controlled, synergistic interactions exist between microbial DNA and LPS in the induction of macrophage NO via enhanced inducible NO synthase gene expression.

A new rearranged abietane, incanone, together with a known abietane diterpenoid, sugiol, were isolated from the whole plant of Caryopteris incana. On the basis of the HRMS- and NMR data incanone was determined as 11,12,14,16-tetrahydroxyl-17(15 → 16)-abeo-abieta-8,11,13-trien-one. Incanone showed cytotoxic activity against human leukaemia cells.

Mouse macrophages can be stimulated by interferon (IFN)-γ and bacterial lipopolysaccharide (LPS) to produce nitric oxide (NO) as the result of expression of the inducible NO synthase (iNOS; EC 1.14.13.39) gene. The iNOS gene promoter contains a candidate γ-interferon- activated site (GAS). In transfection studies reported here, it was demonstrated that a luciferase reporter-gene construct, containing four synthetic copies of the iNOS GAS, was inducible when transfected macrophages were stimulated with either IFN-γ, LPS, or a combination of the two. Consistent with this finding were other transfection analyses, which showed that responsiveness of the intact iNOS promoter to these same agents was significantly reduced when two conserved nucleotide positions within the GAS were mutated. Oligonucleotide probes, which mimicked the iNOS GAS, formed a complex with proteins that appeared in the nuclei of IFN-γ or IFN-γ+ LPS-treated macrophages within 30 min of stimulation, as shown by electrophoretic mobility shift assay. LPS alone also caused the the appearance of a nuclear protein capable of binding the iNOS GAS-containing oligonucleotide; however, in contrast to binding induced by IFN-γ, approximately 2 h of stimulation with LPS were required. The protein bound to the iNOS GAS-containing oligonucleotide reacted specifically with an antibody raised against Stat1α, regardless of the stimulus used. These data collectively support the conclusion that binding of Stat1α to the iNOS promoter's GAS is required for optimal induction of the iNOS gene by IFN-γ and LPS.