Fernando L P, Fernando A N, Ferlito M, Halushka P V, Cook J A
Department of Physiology/Neuroscience, Medical University of South Carolina, Charleston 29425, USA.
Shock. 2000 Aug;14(2):128-33. doi: 10.1097/00024382-200014020-00009.
Lipopolysaccharide (LPS)-tolerant human promonocytic THP-1 cells produce decreased levels of inflammatory mediators such as eicosanoids and tumor necrosis factor alpha (TNFalpha) in response to LPS. We hypothesized that transcriptional repression by newly synthesized proteins may be a mechanism for the reduced cellular response to a secondary challenge with LPS. THP-1 cells were desensitized after a 3.5 h or 20 h pre-exposure to LPS (1 microg/mL) and subsequently challenged with LPS (10 microg/mL). In cells rendered tolerant by exposure to LPS for 20 h, LPS-induced expression of cyclooxygenase (Cox)-2 and TNFalpha mRNA was suppressed. Cycloheximide (10 microM) prevented the transcriptional down-regulation of Cox-2 mRNA and to a lesser extent, TNFalpha mRNA, in LPS-tolerant cells. Transcriptional arrest with actinomycin D stabilized steady-state expression of Cox-2 mRNA in naive and tolerant cells but destabilized TNFalpha mRNA expression in LPS-tolerant cells. The observation that in naive cells Cox-2 and TNFalpha mRNA levels subside at 3 to 4 h after LPS (10 microg/mL or 1 microg/mL) suggested that LPS tolerance may occur earlier. Therefore, in subsequent experiments, the effect of LPS pretreatment for only 3.5 h was examined. This abbreviated tolerance regimen diminished secondary LPS-induced Cox-2 mRNA expression but had a lesser effect on TNFalpha mRNA expression. However, cycloheximide augmented both Cox-2 and TNFalpha mRNA expression in this group. Also, the serine/threonine phosphatase inhibitor okadaic acid augmented Cox-2 and TNFalpha mRNA expression in the LPS-tolerant cells. Although LPS-induced TNFalpha production in LPS-tolerant cells was suppressed relative to the naive cells, okadaic acid induced comparable levels of TNFalpha in tolerant and naive cells. These findings support the concept that LPS tolerance is associated with induction of proteins that alter expression of certain genes. Expression of Cox-2 mRNA appears to be particularly sensitive to down-regulation and, to a lesser extent, TNFalpha mRNA. However, this seems to vary depending on the LPS pretreatment regimen. The ability of a phosphatase inhibitor to induce TNFalpha and expression of Cox-2 and TNFalpha mRNA in LPS tolerance suggests that there may be alterations in phosphorylation status of signaling pathways, transcriptional mechanisms, or post-transcriptional mRNA stability.
脂多糖（LPS）耐受的人原单核细胞THP-1细胞在对LPS作出反应时，产生的类花生酸和肿瘤坏死因子α（TNFα）等炎症介质水平降低。我们推测，新合成蛋白质的转录抑制可能是细胞对LPS二次刺激反应降低的一种机制。将THP-1细胞在3.5小时或20小时预先暴露于LPS（1微克/毫升）后使其脱敏，随后用LPS（10微克/毫升）进行刺激。在通过暴露于LPS 20小时而产生耐受的细胞中，LPS诱导的环氧化酶（Cox）-2和TNFα mRNA表达受到抑制。放线菌酮（10微摩尔）可防止LPS耐受细胞中Cox-2 mRNA的转录下调，并在较小程度上防止TNFα mRNA的转录下调。用放线菌素D进行转录阻滞可稳定未致敏细胞和耐受细胞中Cox-2 mRNA的稳态表达，但会使LPS耐受细胞中TNFα mRNA的表达不稳定。在未致敏细胞中，LPS（10微克/毫升或1微克/毫升）处理后3至4小时Cox-2和TNFα mRNA水平下降的观察结果表明，LPS耐受可能更早发生。因此，在随后的实验中，研究了仅3.5小时LPS预处理的效果。这种缩短的耐受方案减少了二次LPS诱导的Cox-2 mRNA表达，但对TNFα mRNA表达的影响较小。然而，放线菌酮增加了该组中Cox-2和TNFα mRNA的表达。此外，丝氨酸/苏氨酸磷酸酶抑制剂冈田酸增加了LPS耐受细胞中Cox-2和TNFα mRNA的表达。尽管与未致敏细胞相比，LPS耐受细胞中LPS诱导的TNFα产生受到抑制，但冈田酸在耐受细胞和未致敏细胞中诱导出相当水平的TNFα。这些发现支持了LPS耐受与诱导改变某些基因表达的蛋白质有关的概念。Cox-2 mRNA的表达似乎对下调特别敏感，TNFα mRNA在较小程度上也是如此。然而，这似乎因LPS预处理方案而异。磷酸酶抑制剂在LPS耐受中诱导TNFα以及Cox-2和TNFα mRNA表达的能力表明，信号通路的磷酸化状态、转录机制或转录后mRNA稳定性可能存在改变。
