Suppression of Cox-2 and TNF-α mRNA in endotoxin tolerance:: Effect of cycloheximide, antinomycin D, and oakadaic acid

Lipopolysaccharide (LPS)-tolerant human promonocytic THP-1 cells produce decreased levels of inflammatory mediators such as eicosanoids and tumor necrosis factor alpha (TNF alpha) 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 mug/mL) and subsequently challenged with LPS (10 mug/mL). In cells rendered tolerant by exposure to LPS for 20 h, LPS-induced expression of cyclooxygenase (Cox)-2 and TNF alpha mRNA was suppressed. Cycloheximide (10 muM) prevented the transcriptional down-regulation of Cox-2 mRNA and to a lesser extent, TNF alpha 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 TNFa: mRNA expression in LPS-tolerant cells. The observation that in naive cells Cox-2 and TNF alpha mRNA levels subside at 3 to 4 h after LPS (10 mug/mL or 1 mug/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-e mRNA expression but had a lesser effect on TNF alpha mRNA expression. However, cycloheximide augmented both Cox-2 and TNF alpha mRNA expression in this group. Also, the serine/threonine phosphatase inhibitor okadaic acid augmented Cox-2 and TNFa mRNA expression in the LPS-tolerant cells. Although LPS-induced TNF alpha production in LPS-tolerant cells was suppressed relative to the naive cells, okadaic acid induced comparable levels of TNF alpha 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-e mRNA appears to be particularly sensitive to down-regulation and, to a lesser extent, TNF alpha mRNA. However, this seems to vary depending on the LPS pretreatment regimen. The ability of a phosphatase inhibitor to induce TNF alpha and expression of Cox-2 and TNF alpha mRNA in LPS tolerance suggests that there may be alterations in phosphorylation status of signaling pathways, transcriptional mechanisms, or post-transcriptional mRNA stability.