摘要：

LPS is well recognized for its potent capacity to activate mouse macrophages to produce TNF-α, an important inflammatory mediator in bacterial infection-related diseases such as septic shock. We demonstrate here that while inducing only low levels of TNF-α alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce TNF-α in the RAW 264.7 macrophage-like cell line. The bacterial DNA effects are mimicked by synthetic CpG-containing oligodeoxynucleotides, but not non-CpG-containing oligodeoxynucleotides. Pretreatment of macrophages with either DNA for 2–8 h inhibits macrophage TNF-α production in responses to DNA/LPS. However, when pretreatment was extended to 24 h, DNA/LPS synergy on TNF-α is further enhanced. RT-PCR analysis indicates that mRNA levels of the TNF-α gene, however, are not synergistically induced by bacterial DNA and LPS. Analyses of the half-life of TNF-α mRNA indicate that TNF-α message has a longer half-life in bacterial DNA- and LPS-treated macrophages than that in bacterial DNA- or LPS-treated macrophages. These findings indicate that the temporally controlled, synergistic induction of TNF-α by bacterial DNA and LPS is not mediated at the transcriptional level. Instead, this synergy may occur via a post-transcriptional mechanism. Recent data indicate that purified bacterial DNA can activate macrophages and other inflammatory cells (reviewed in Refs. 1 and 2 ). Macrophages stimulated with bacterial DNA reportedly produce proinflammatory cytokines such as TNF-α ( 3 , 4 ), IL- 1 ( 4 ), IL-6 ( 5 ), IL-12 ( 6 , 7 , 8 ), IFN-αβ ( 9 , 10 ), IFN-γ ( 9 ), and the reactive nitrogen intermediate, NO ( 11 , 12 ). Subtle structural differences between bacterial and eukaryotic DNA apparently account for the ability of bacterial DNA to serve as an immune-activating agent. Specifically, bacterial DNA is thought to activate inflammatory cells because of its high content of short sequences with unmethylated CpG dinucleotides ( 13 ). In mammalian DNA, CpG-containing sequences occur at a much lower frequency than in bacterial DNA, and the cytosine present in CpG dinucleotides of mammalian DNA is usually methylated ( 14 , 15 ). In vivo studies support the concept that bacterial DNA is an important proinflammatory stimulus, as bacterial DNA has been shown to trigger septic shock in d -galactosamine treated mice. ( 16 ). Also of interest is the finding that bacterial DNA acts synergistically with LPS to induce TNF-α production in vivo, resulting in lethal shock in mice ( 11 , 17 ). The molecular mechanism(s) by which bacterial DNA acts synergistically with LPS to induce TNF-α production remains to be determined. LPS, a constituent of the Gram-negative bacterial cell wall, is well known as a potent inducer of mouse macrophage activation, resulting in production of many inflammatory mediators, including TNF-α, that play a key role in the development of septic shock (reviewed in Refs. 18 and 19 ). The induction of TNF-α secretion from mouse macrophages in response to LPS stimulation is controlled at transcriptional, post-transcriptional, and translational levels ( 17 , 20 , 21 ). In this communication we explore the mechanisms by which LPS and bacterial DNA act synergistically to activate macrophages. We present in vitro data demonstrating that bacterial DNA acts synergistically with substimulatory concentrations of LPS to enhance TNF-α secretion by the murine RAW 264.7 macrophage-like cell line. The observed synergy depends upon the presence of unmethylated CpG residues in DNA and is also dependent upon the temporal order of treatment by LPS and bacterial DNA. Enhanced TNF-α secretion by RAW 264.7 cells simultaneously exposed to bacterial DNA and LPS is not accompanied by enhanced transcription of the TNF-α gene. Analyses of the half-life of TNF-α in differentially treated macrophages suggest that bacterial DNA and LPS act synergistically to enhance TNF-α production through a post-transcriptional event.

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